Disease model

Abstract

The present invention provides a method for producing an arrhythmia disease model. It also provides a corresponding arrhythmia disease model or arrhythmia disease model system. The arrhythmia disease model or arrhythmia disease model system may be used to screen for compounds that have the ability to treat arrhythmia. Methods of screening one or more test compounds for treating arrhythmia using the model or system are therefore also provided.

Description

[0001] Disease Model

[0002] The present invention provides a method for producing an arrhythmia disease model. It also provides a corresponding arrhythmia disease model or arrhythmia disease model system. The arrhythmia disease model or arrhythmia disease model system may be used to screen for compounds that have the ability to treat arrhythmia. Methods of screening one or more test compounds for treating arrhythmia using the model or system are therefore also provided.

[0003] Background

[0004] Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are increasingly used in drug discovery and development. Their regulatory impact has been acknowledged by FDA acceptance of hPSC-CMs in integrated nonclinical risk-assessment for establishing cardiac safety, and more recently, the inclusion of hPSCs in the FDA Modernization Act (2022) for testing drug effectiveness. Nevertheless, the ability of hPSC-CMs to reveal disease phenotypes or assess how compounds affect the heart could be improved if their phenotype were more adult-like than fetal. hPSC-based 3D models, such as organoids or cardioids and engineered heart tissues (EHTs) increase cardiomyocyte maturity but most of these methods need specialized expertise, generally large cell numbers or specific equipment. These requirements not only render them costly, but also pose challenges in their routine implementation in high-throughput screenings (HTS) within the pharmaceutical industry. Scalability of 3D models to accommodate multi-well microplates, compatibility with established assay methods, and the assurance of reproducibility remain substantial hurdles in leveraging 3D models effectively for drug discovery programs.

[0005] A particular issue is that, to date, there are no reliable disease models directed to cardiac arrhythmias. Thus, there are no reliable methods for screening drugs that can treat cardiac arrhythmias. Previous studies demonstrate the potential of utilizing cardiomyocytes to model catecholaminergic polymorphic ventricular tachycardia-1 (CPVT1), a cardiac disease, revealing disease-associated characteristics including elevated diastolic Ca2+concentrations, decreased Ca2+content in the sarcoplasmic reticulum (SR), and increased susceptibility to (stress-induced) arrhythmias. CVPT 1 is caused by mutations in the gene encoding the calcium-handling ryanodine receptor, RyR2. A RyR2 mutation can alter the sensitivity of the RyR2 channel to Ca2+activation (for example by stress via the p-adrenergic receptor / PKA / phospholamban signaling pathway), leading to enhanced spontaneous Ca2+release during sarcoplasmic reticulum (SR) Ca2+overload and subsequent delayed after depolarization (DAD)-mediated arrhythmias. However, these studies have often been limited by low reproducibility, utilization of low-throughput technologies, or a primary focus on qualitative data. Crucially, the lack of optimization for high-throughput screening methods has hindered the use of CPVT 1 human induced pluripotent stem cell derived- cardiomyocyte (hiPSC-CM) models for identifying new or repurposed drugs for CPVT1. Several earlier studies have reported cardiac toxicity assays and screens using primary- or stem cell-derived cardiomyocytes with predictivity between 50% and 87% of compounds tested. In comparison, animal models are 78% accurate in determining whether drugs will affect cardiac contractility.

[0006] There is a need to generate scalable, affordable, scaffold-free, 3D cardiac microtissues (MTs) that require low cell numbers and no specialized equipment for easy-to-use assays in cardiac safety and efficacy assessment.

[0007] Brief summary of the disclosure

[0008] The inventors have developed a cardiac arrhythmia disease model involving 3D cardiac MTs that are scalable, amenable to automation and rapid screening, and are highly predictive of drug responses. The invention is in part based on observations of MTs containing RyR2-mutated cardiomyocytes which showed disease relevant arrhythmia that could be quantified using a semiautomated scoring method and rescued using a clinically-relevant drug. This may advantageously be used to generate MTs at scale to use them in simple and reliable assays for high throughput screening (HTS) of >1000 compounds, ultimately identifying therapeutic targets for inherited cardiac arrhythmic diseases, like CPVT1. High reproducibility, proper validation and standardization as shown herein are essential steps towards pharmaceutical industry adoption and regulatory authority acceptance of these models.

[0009] The inventors demonstrate, for the first time, a robust CPVT 1 arrhythmic phenotype in a high- throughput manner (i.e. 384 well plates) using MTs primed with dibutyryl cAMP (dbcAMP). These MTs mimic the arrhythmic phenotype of CPVT1 and remarkably, these arrhythmias could be effectively rescued through (acute) treatment with the sodium- and RyR2-channel blocker, Flecainide, which has emerged as an effective secondary agent for suppressing arrhythmias in patients suffering from CPVT-1. Thus, the MTs also respond to rescue (therapeutic) compounds as expected for that disease, demonstrating that the inventors have generated a reliable disease model.

[0010] By incorporating robotics, these assays have been upscaled, enabling HTS that is suitable for screening tens of thousands of compounds. This was in part demonstrated in MTs generated from hiPSC-CMs, hiPSC-cardiac endothelial cells (cECs), and hiPSC-cardiac fibroblasts (cFbs) using small molecules in the first induction step for all three cell types to reduce batch-to-batch variability, improve scalability and reduce material costs. Fluorescence-based assays to assess calcium transients or action potential metrics were developed for 3D MTs, scaled up from 96- to 384-well plates, and were reproducible across multiple (cryopreserved) cell batches and accurately reported cardiac functionality. The methods provided herein were used to automate MT formation and screen for arrhythmia in several 384 well plates simultaneously, demonstrating the utility of the invention. Using the automated system developed here, blind screens of 12-13 reference drugs with known positive, negative, or neutral inotropic effects were performed, which resulted in an accuracy rate of more than 81 % and a reproducibility rate between three independent screens (several months apart) of more than 90%. For some drugs, action potential analysis was used to guide the prediction of positive or negative inotropic effects. Drugs such as the calcium sensitizer / PDE3 inhibitor, Levosimendan, that has been incorrectly classified using 2D cardiac models, was correctly identified by the invention.

[0011] Accordingly, in one aspect, the invention provides a method for producing an arrhythmia disease model, comprising: contacting cells with an exogenous agent under conditions that induce arrhythmia in the cells, wherein the exogenous agent penetrates the cell membrane and activates the intracellular cAMP signalling pathway. Suitably, the arrhythmia disease model may be a human arrhythmia disease model.

[0012] In a similar aspect, the invention provides a method for producing an arrhythmia disease model, comprising: contacting electrically excitable cells with an exogenous agent under conditions that induce arrhythmia in the cells, wherein the exogenous agent is a cell-membrane penetrating activator of the intracellular cAMP signalling pathway.

[0013] In one aspect, the invention provides an arrhythmia disease model system comprising:

[0014] (i) an exogenous agent that penetrates the cell membrane and activates the intracellular cAMP signalling pathway; and

[0015] (ii) cells having arrhythmia, wherein the cells have been contacted with the exogenous agent of (i) under conditions that induce arrhythmia. Suitably, the arrhythmia disease model may be a human arrhythmia disease model.

[0016] In a similar aspect, the invention provides an arrhythmia disease model system comprising:

[0017] (i) an exogenous agent that is a cell-membrane penetrating activator of the intracellular cAMP signalling pathway; and

[0018] (ii) electrically excitable cells; wherein (i) and (ii) are in contact with each other. Suitably, the arrhythmia disease model system may be a human arrhythmia disease model system.

[0019] Suitably, in the method or the system, the exogenous agent may bind to protein kinase (PKA) and / or induce PKA activity in the cells. In other words, the exogenous agent may be a PKA binder and / or a PKA activator. Suitably, in the method or the system, the exogenous agent may bind to exchange proteins activated by cyclic AMP (EPAC) and / or induce EPAC activity in the cells. In other words, the exogenous agent may be an EPAC binder and / or a EPAC activator.

[0020] Suitably, in the method or the system, the exogenous agent may be an agent selected from the group consisting of: db-cAMP, 8-Br-cAMP-AM, Sp-cAMPS, 8-CPT-cAMP, 2'-O-MS-cAMP, 8-CL- cAMP, 6-Bnz-cAMP, 8-pCPT-2-O-Me-cAMP-AM; and analogs and derivatives thereof.

[0021] Suitably, in the method or the system, the arrhythmia disease may be selected from the group consisting of: cardiac arrhythmia disease, a calcium handling disease, channelopathy, supraventricular arrhythmia disease, ventricular arrhythmia disease, structural heart disease, cardiomyopathy, atrial fibrillation, metabolic arrhythmia disease, mitochondrial arrhythmia disease, optionally wherein the channelopathy is long QT syndrome type 2 (LQT2) or catecholaminergic polymorphic ventricular tachycardia (CPVT), further optionally wherein the CPVT is CVPT1 or CVPT2.

[0022] Suitably, in the method or the system, the cells may carry one or more mutations associated with arrhythmia disease, optionally wherein the cells comprise one or more mutations in a gene selected from the group consisting of: ryanodine receptor 2 (RYR2), calsequestrin 2 (CASQ2), potassium voltage-gated channel subfamily H member 2 (KCNH2), potassium inwardly rectifying channel subfamily J member 2 (KCNJ2), Calcium Voltage-Gated Channel Subunit Alphal C (CACNA1C), Calcium Voltage-Gated Channel Auxiliary Subunit Beta 2 (CACNB2), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 1 (CACNA2D1), Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A), Calmodulin 1 (CALM1), Calmodulin 2 (CALM2), Calmodulin 3 (CALM3), Phospholamban (PLN), A-Kinase Anchoring Protein 9 (AKAP9), Potassium Voltage- Gated Channel Subfamily E Regulatory Subunit 1 (KCNE1), Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 2 (KCNE2), Myosin Binding Protein C3 (MYBPC3), Titin (TTN), Ankyrin 2 (ANK2), Lamin A / C (LMNA), Alanyl-TRNA Synthetase 2 (AARS2), and Plakophilin 2 (PKP2), further optionally wherein the one or more mutations is in human RYR2, CASQ2, KCNH2, KCNQ1 , SCN5A, KCNJ2, CACNA1C, CALM1 , CALM2, PKP2, AARS2, LMNA, TTN or MYBPC3, and the one or more mutations is selected from: a) M4109R - RYR2; b) F2483I - RYR2; c) P2328S - RYR2; d) S406L - RYR2; e) E2311 D - RYR2; f) D307H - CASQ2; g) R420Q - RYR2; h) exon 3 del, P2328S, T2538R, L4115F, Q4201R and V4653F- RYR2; i) I4587V - RYR2; j) L3741P-RYR2; k) c.339-354 del (CASQ2G112+5X) - CASQ2; l) D3638A - RYR2; m) D358N and R4651I - RYR2; n) R4147S-RYR2; o) Y4962C-RYR2; p) G1681A- KCNH2; q) R176W-KCNH2; r) A561T-KCNH2; s) A561V-KCNH2; t) A561P-KCNH2; u) N996I - KCNH2; v) IVS9-28A / G - KCNH2 ; w) A422T-KCNH2; x) G603D-KCNH2; y) G604S - KCNH2; z) A614V-KCNH2; aa) G601S- KCNH2; bb) R534C- KCNH2; cc) N633S - KCNH2; dd) R685P- KCNH2; ee)V822M - KCNH2; ff) R366X-KCNH2; gg) S428X- KCNH2; hh)R752W - KCNH2; ii) T983I - KCNH2; jj) p.S1112Pfs*171 - KCNH2; kk) N588K- KCNH2;

[0023] II) R594Q-KCNQ1; mm) 1795insD - SCN5A; nn) R535Q - SCN5A; oo) R218W-KCNJ2; pp) G406R-CACNA1C; qq) N639T-CACNA1C; rr) F142L- CALM1; ss) D130G-CALM2; tt) R1629X - SCN5A; uu) C.1643delG - PKP2; vv) K672Rfs - PKP2; ww) R958X - AARS2; xx) R592W- AARS2; yy) V730M - AARS2; zz) R216C - LMNA; aaa) K219T - LMNA; bbb) H222P - LMNA; ccc) R225X - LMNA; ddd) D461Y - LMNA; eee) W467X - LMNA; fff) R471 H - LMNA; ggg) R482W- LMNA; hhh) Q493X -LMNA; iii) A22352fs - TTN; jjj) W792Vfs+40X - MYBPC3.

[0024] Suitably, in the method or the system, the cells may comprise electrically excitable cells, optionally wherein the electrically excitable cells comprise:

[0025] (a) human pluripotent stem cell (hPSC) derivatives, optionally wherein the hPSC derivatives are cardiac cells, and / or

[0026] (b) cardiac cells, optionally wherein the cardiac cells are hPSC-derived cardiomyocytes.

[0027] Suitably, in the method or the system, the cells may be in a 2D or 3D cell culture, or a mixture thereof.

[0028] Suitably, in the method or the system, the exogenous agent may be contacted with the cells for up to 7 days, optionally up to 72 hours, up to 48 hours, or up to 24 hours.

[0029] Suitably, in the method or the system, the cells may be provided in a cell culture vessel, optionally wherein the cell culture vessel is a multi-well cell culture plate, further optionally wherein the multiwell cell culture plate is at least a 96-well cell culture plate.

[0030] Suitably, in the method or the system, the exogeneous agent may be present at a concentration between 0.005 mM and 250 mM, optionally wherein the concentration is about 0.5 mM. Suitably, the method may further comprise a step of identifying cells having arrhythmia after the contacting step.

[0031] Suitably, in the method, the step of identifying cells having arrhythmia may comprise: a) labelling the cells with an arrhythmia detection reagent, optionally wherein the arrhythmia detection reagent is a chemical calcium dye, a chemical membrane potential dye, a genetically- encoded calcium indicator, or a genetically-encoded voltage indicator; and b) detecting the arrhythmia.

[0032] Suitably, the method may further comprise: c) contacting the cells with a rescue compound; and d) detecting if there is a change in arrhythmia in the cells contacted with the rescue compound, wherein a reduction or an absence of arrhythmia is indicative of an effective arrhythmia model.

[0033] Suitably, in the method, the detecting the arrhythmia may comprise detecting changes in intracellular calcium levels, electrical activity, and / or contraction activity of the cells, optionally wherein the detecting changes in intracellular calcium levels, electrical activity, and / or contraction activity of the cells comprises recording the changes.

[0034] In one aspect, the invention provides a method of screening one or more test compounds for treating arrhythmia, said method comprising: a) contacting the one or more test compounds with an arrhythmia disease model produced by the method of the invention, or with an arrhythmia disease model system of the invention; and b) determining the effect of the one or more test compounds on arrhythmia in the arrhythmia disease model or arrhythmia disease model system, wherein if the test compound reduced arrhythmia in the arrhythmia disease model or arrhythmia disease model system as compared to a control, it is indicative of the compound being suitable for treating arrhythmia. Suitably, the arrhythmia disease model may be a human arrhythmia disease model.

[0035] Suitably, determining the effect of the one or more test compounds may comprise labelling the cells of the arrhythmia disease model or the arrhythmia disease model system with an arrhythmia detection reagent, optionally wherein the arrhythmia detection reagent is a chemical calcium dye, a chemical membrane potential dye, a genetically-encoded calcium indicator, or a genetically- encoded voltage indicator.

[0036] Suitably, the method may further comprise combining cells in suspension to form a 2D cell culture or a 3D cell culture. Suitably, the method may be an automated method and / or may be a high throughput assay.

[0037] Suitably, the one or more test compounds may be contacted with the arrhythmia disease model produced by the method of the invention, or with the arrhythmia disease model system of the invention, at a concentration of about 0.05 to about 200 pM, preferably about 0.1 to about 100 pM.

[0038] The invention also provides an arrhythmia disease model produced by the method of the invention.

[0039] Advantageously, the arrhythmia observed in the arrhythmia disease models and arrhythmia disease model systems described herein are very reproducible. The methods used herein to generate the models and systems are methods that reproducibly induce arrhythmia. The models, systems and methods described herein are therefore scalable, amenable to automation and rapid screening, and are highly predictive of drug responses.

[0040] Advantageously, arrhythmia disease models and arrhythmia disease model systems described herein are human models and systems. The models and systems described herein may be referred to as in vitro models or systems. Surprisingly, the in vitro human arrhythmia disease models and arrhythmia disease model systems described herein are very reproducible, scalable, amenable to automation and rapid screening, and are highly predictive of drug responses.

[0041] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0042] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0043] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0044] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0045] Various aspects of the invention are described in further detail below. Brief description of the drawings

[0046] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0047] Figure 1 shows differentiation of hiPSC-derived endothelial cells, cardiomyocytes and cardiac fibroblasts. A) representative immunofluorescence images of DAPI, CD31 , cTnT and Vimentin of one cardiac microtissue made with the ImageXpress Micro Confocal High-Content Imaging System (HCI, Molecular Devices) using a 20x magnification objective and Z stack acquisition. Scale bar 100 pm. B) average values of the beat rate, peak amplitude, peak width duration at 50% of decay (PWD50), and the peak-to-peak (p-p) intra-well variability of calcium and action potential traces from cardiac microtissues. Data represents averages ± SEM. N=3 independent experiments per read-out, n=288 (calcium), n=174 (voltage). C) calcium transients recorded from one cardiac microtissue before (top panel) and after (lower panel) addition of 0.1 pM Isoproterenol. RFU, Relative Fluorescent Units; 0.02 sec. exposure time. D) effect of 0.1 pM Isoproterenol on the beat rate and amplitude of cardiac microtissues (N=2 independent experiments, n=8 microtissues). Data represents percentage change from baseline, corrected for vehicle control (0.1 % DMSO). E) action potential metrics from one cardiac microtissue before (top panel) and after (lower panel) addition of 0.03 pM E4031. 0.016 sec. exposure time. F) Effect of 0.03 pM E4031 on the action potential duration at 50% (APD50) and 70% (APD70) of repolarization (N=2 independent experiments, n=7 microtissues). Data presents percentage change from baseline of averages ± SEM, corrected for vehicle control (0.1 % DMSO).

[0048] Figure 2 shows dbcAMP-induced arrhythmia in CPVT cardiac microtissues. A) calcium transients recorded from untreated CPVT 1 microtissues (top panel) and treated (lower two panels) with dbcAMP. The arrhythmia triggered by dbcAMP is rescued after treatment with flecainide (lower panel). The middle and lower panels show traces from the same microtissue. B) bar graph showing the effect of triggering arrhythmia by treating CPVT1 microtissues with dbcAMP compared to vehicle control (0.005% DMSO). C) bar graph showing the rescuing effect of flecainide (Flee., 3 pM) compared to vehicle control (0.1% DMSO) in dbcAMP-treated CPVT1 microtissues.

[0049] Figure 3 shows automating formation, maintenance and labeling of cardiac microtissues using robotics. A) schematic overview of the automation process using the TECAN Fluent® Workstation, including timeline and optional downstream readouts of the cardiac microtissues. B) & C) intra-plate variability in microtissue size measured at different days in culture from 1 plate including manually seeded microtissues (Manual, M) and 2 plates including automatically seeded microtissues using the Fluent® Workstation (Auto 1 , A1 and Auto 2, A2). Microtissues size was measured using the Cytation Imaging Multi-Mode Reader. Each datapoint represents an individual microtissue (n=59-74 (manual), n=363-383 (Auto 1), n=360-378 (Auto 2)). D) Peak-to- Peak intra-well variability of calcium transients recorded from automated and manual microtissues made at the same day, from the same batch of cells. Data represents averages ± SEM (n=15 (auto), n=18 (manual)). E) effect of 0.75 pM nifedipine on the calcium peak amplitude of automated and manual microtissues. Data shows percentage change from baseline of averages ± SEM and is corrected for vehicle control (n=6).

[0050] Figure 4 shows blinded calcium screen on cardiac microtissues. A) heat plot showing the results of a blind screen of 13 reference compounds on cardiac microtissues. The formation, maintenance and labeling of the microtissues was fully automated. 6 different calcium-related parameters were used to classify (“Blinded Assignment”) the compound as either a positive inotrope (PI), negative inotrope (Nl) or non-inotropic effect compound (NE). The figure represents 1 screen, n=5 microtissues per concentration at baseline. Data represents averages, showing percentage change from baseline, corrected for vehicle control (0.1% DMSO); Q=quiescent. B) effect of two positive inotropes, Isoproterenol and Levosimendan, on the amplitude and beat rate of calcium transients recorded from 3D microtissues or 2D monolayers at the same day, including the same cardiac cells, in the same ratio. Data represent averages ± SEM, showing percentage change from baseline, corrected for the vehicle control (0.1% DMSO). n=3 (monolayers), n=4 (microtissues) per concentration. * indicate significance compared to baseline values.

[0051] Figure 5 shows the characterization of hiPSC-derived endothelial cells, cardiomyocytes and cardiac fibroblasts. A) protocol for hiPSC differentiation into cardiac endothelial cells (cECs), cardiomyocytes (CMs) and cardiac fibroblasts (cFbs) showing small molecule-based mesoderm induction including timepoints of passaging (triangle) and freezing (snowflake). B) representative bright-field images of hiPSCs at day 0 followed by the different cardiac cell types at the end of their differentiation (D, days of differentiation from hiPSCs) for CTRL1-hiPSCs (LUMC0020iCTRL-06). RA, retinoic acid. Scale bar: 100 pm. C) flow cytometry plots indicating the expression of CD31 in cardiac endothelial cells (directly after isolation (80.5%) and before cryopreservation (98.2%), left), cTnT in cardiomyocytes (middle) and Vimentin in cardiac fibroblasts (right). Counts are normalized to mode D) immunofluorescence images showing the expression of WT1 , GATA4, and CX-43 in epicardial cells (EPC, top panels) and cardiac fibroblasts (cFb, lower panels). WT-1 expression is lost when the epicardial cells become cardiac fibroblasts. EPCs and cFbs are derived from the same hiPSC differentiation. Scale bar 100 pm.

[0052] Figure 6 shows a Spearman's correlation heatmap showing clustering of the cardiomyocytes, endothelial cells, epicardial cells and fibroblasts from hiPSCs differentiated using the cytokine protocol (CP) or small molecule protocol (SP) or from primary origin based on RNA-seq profiles using genes filtered based on their expression across all the samples according to Chen et al, 2016.

[0053] Figure 7 shows a method for classifying arrhythmias. A) example traces showing regular, type A and type B arrhythmia and noise signals in calcium recordings. Key features to aid in their classification are indicated on the trace. Type A is an example of an arrhythmic trace; while regular is a non-arrhythmic trace. B) Poincare plot showing the rhythmicity of a Type A arrhythmia signal compared to a regular signal.

[0054] Figure 8 shows the accuracy and precision of the automated process. A) accuracy in total volume per well showed by the intra- and inter-plate coefficient of variation (N=3 plates). Volume is determined by measuring the absorbance of the tartrazine dye and using the standard curve (right). Data presented are averages ± standard deviation. B) precision in pipetting small volumes in a total volume of 25 pl / well showed by the intra- and inter-plate coefficient of variation. Data presented are averages including individual datapoints (N=3 plates).

[0055] Figure 9 shows a snapshot of Calcium-6-loaded microtissues. Images captured from automatically formed microtissues in a 384 wells plate, loaded with the Calcium-6 dye. Pictures were taken post-compound addition using the Cytation Imaging Multi-Mode Reader.

[0056] Figure 10 shows contraction, calcium and voltage parameters. A) representation of a contraction transient showing increases and decreases in contraction time (clinotropy), amplitude (inotropy) and relaxation time (lusitropy). B) calcium and voltage transients showing the relevant parameters used as surrogate readouts to classify compounds as positive inotrope, negative inotrope, or non- inotropic effect compounds.

[0057] Figure 11 shows a summary table of the blind high-throughput screens. Summary of three independent blinded screens of 12-13 reference compounds per screen. 6 different calcium- related parameters were used to classify (“Blinded Assignment”) the compound as either a positive inotrope (PI), negative inotrope (Nl) or non-inotropic effect compound (NE). Accuracy rate is determined by dividing the total correctly classified compounds (31) by the total compounds tested (38). The myosin-modulators, Omecamtiv Mecarbil (“Omecamtiv”) and Blebbistatin, are expected to be classified as “NE” in a calcium assay.

[0058] Figure 12 shows that isoproterenol does not trigger arrhythmias in the disclosed model. A) bar graph quantifying the percentage of CPVT1 microtissues showing arrhythmias or no arrhythmias after treatment with either vehicle (DMSO), dbcAMP, isoprotenerol or untreated. Data based on the CPVT 1 disease line and treatment for 7 days. B) bar graph quantifying the percentage of CPVT1 human iPSC-derived cardiomyocytes cultured as a 2D monolayer showing arrhythmias or no arrhythmias after treatment with either vehicle (DMSO) or dbcAMP.

[0059] Figure 13 shows dbcAMP induces arrhythmias also in LQT2 disease models. A) calcium transients recorded from LQT2 microtissues either without the addition of any exogenous agent (untreated), or treatment with vehicle (DMSO) or dbcAMP. Treatment was for 7 days. B) bar graph quantifying the percentage of LQT2 microtissues showing arrhythmias or no arrhythmias either without the addition of any exogenous agent (untreated), or treatment with vehicle (DMSO) or dbcAMP. Treatment was for 7 days.

[0060] Figure 14 shows rescue of arrhythmia is specific to compounds used in the clinic or in clinical trials. A) Calcium transients recorded from dbcAMP-treated LQT2 microtissues that have been co-treated with vehicle or with lumacaftor. Treatment was for 7 days. B) Bar graph quantifying the percentage of dbcAMP-treated LQT2 microtissues showing arrhythmias or no arrhythmias that were co-treated with vehicle (0.005% DMSO) or with lumacaftor (10 pM). Treatment was for 7 days. C) Calcium transients recorded from dbcAMP-treated CPVT1 microtissues that have been co-treated with vehicle or with lumacaftor. Treatment was for 7 days. D) Bar graph quantifying the percentage of dbcAMP-treated CPVT1 microtissues showing arrhythmias or no arrhythmias that were co-treated with vehicle (0.005% DMSO) or with lumacaftor (10 pM). Treatment was for 7 days.

[0061] Figure 15 shows that acute treatment with flecainide of hiPSC-derived disease models with dbcAMP-induced arrhythmias only rescues the disease phenotype for CPVT1 and not LQT2, reflecting clinical observations. A) calcium transients recorded showing response of dbcAMP- treated CPVT1 or LQT2 microtissues following acute treatment with flecainide (3 pM). B) Bar graph quantifying the percentage of dbcAMP-treated CPVT1 or LQT2 microtissues showing arrhythmias or no arrhythmias following acute treatment with flecainide.

[0062] Figure 16 shows A) a bar graph indicating percentage of microtissues in 384 well plates at different days after their automated formation with an arrhythmic phenotype following treatment with dbcAMP for 72 hrs. Numbers above the bars indicate the percentage for each plate. Note that arrhythmias can be consistently detected at timepoints of day 14 and day 21 , but the percentage drops at day 31. B) Bar graph showing percentage of dbcAMP-treated arrhythmic microtissues formed by automation that became non-arrhythmic following acute treatment with either flecainide (3 pM) or vehicle (0.1 % DMSO). Automated systems were used to form, maintain, add the exogenous element, label with Calcium-6 dye, and apply compounds to the CPVT1 disease model. Numbers in brackets are the total number of wells treated per condition on each plate. Figure 17 shows an example non-arrhythmic and arrhythmic traces obtained from recordings made using a microelectrode array (MEA) in CPVT1 hiPSC-derived cardiomyocytes.

[0063] Figure 18 shows that various cAMP analogs can reproducibly trigger arrhythmias in CPVT1 hiPSC-derived cardiomyocytes following treatment with the analog for at least 48 hrs, and subsequently labelling with Calcium-6 for 1 hr. Presence of arrhythmias was determined from recordings made using a microelectrode array (MEA), with all cAMP analogs inducing arrhythmias in more than 60% of the wells. Notably, dbcAMP, 2-O-MS-cAMP, 8-CPT-cAMP, 8-Br-cAMP-AM, Bnz-cAMP and 8-pCPT-cAMP-AM all induced arrhythmias in 100% of the wells. Numbers above the bars indicate the percentage of wells showing an arrhythmic phenotype.

[0064] Figure 19 shows that while dbcAMP alone can induce arrhythmias in CPVT1 hiPSC-derived cardiomyocytes following 72 hr of treatment, the proportion of wells showing arrhythmias increases to 100% following labelling of dbcAMP-treated cells with either of the chemical calcium dyes, Calcium-6 and Fluo-4, for 1 hr. Labelling cells with just the dyes and not treating with dbcAMP (-dbcAMP) does not cause an arrhythmic phenotype. Presence of arrhythmias was determined from recordings made using a microelectrode array (MEA). Numbers above the bars indicate the percentage of wells showing an arrhythmic phenotype.

[0065] Figure 20 shows dbcAMP induces arrhythmias also in patient-derived CPVT1 disease models. A) Calcium transients recorded from microtissues containing hiPSC-CMs with the RYR2 mutation R4147S following 72 hour treatment with dbcAMP. B) Calcium transients recorded from microtissues containing hiPSC-CMs with the RYR2 mutation Y4962C following 72 hour treatment with dbcAMP. C) Bar graph quantifying the percentage of samples showing arrhythmias upon treatment with dbcAMP for 72 hours for 2 additional CPVT1 disease models (R4147S-RYR2: 8.51 % Normal, 91.49% Arrhythmic; Y4962C-RYR2: 0% Normal, 100% Arrhythmic). N refers to number of samples.

[0066] Figure 21 shows consistent 100% triggering of arrhythmias by db-cAMP in CPVT-CMs in a screen with three 384-well plates. Assay was based on recording Ca2+signals using calcium-6 dye. In all wells with a valid signal (i.e. excluding wells labelled noise in black), arrhythmias (dark grey) were detected.

[0067] Figure 22 shows the same 384-well plates as in Figure 21 but following addition of 92 potential rescue compounds at 3 different concentrations. Assay was based on recording Ca2+signals using calcium-6 dye. Wells remaining arrhythmic are in dark grey, while wells in which addition of the compound returned the CPVT1-CMs to a normal rhythmic beat (i.e. rescued the arrhythmia) are labelled light grey. Figure 23 shows a Sankey plot depicting the 17 hit compounds and their modes of action identified from an efficacy screen of 92 FDA-approved compounds performed on dbcAMP-triggered arrhythmic CPVT 1 microtissues. A hit was defined as a compound that achieved 100% arrhythmia rescue of the arrhythmic microtissue at 1 or more test concentrations. Test concentrations were 0.1 mM, 1 mM, 10 mM. Compounds preceded with a dark line are compounds previously tested for preventing arrhythmic phenotypes in other CPVT1 models or clinical studies, validating the ability of the assay to identify relevant rescue compounds.

[0068] Detailed description

[0069] The present invention provides methods for producing arrhythmia disease models, arrhythmia disease model systems, and other methods involving such arrhythmia disease model and arrhythmia disease model systems.

[0070] “Arrhythmia” as used herein refers to irregular or abnormal rhythm in the contraction of cells compared to the base-line rhythm in contraction of said cells, or when compared to the rhythm in the contraction of wild-type or healthy cells, wherein the contraction may be caused by electrical activity. In the context of the model of the present disclosure, an arrhythmia may be determined by an abnormal pattern on a graph showing calcium transients in the model and / or an abnormal pattern on a graph showing the electrical activity of the cells in the model and / or an abnormal pattern on a graph showing contraction transients of the cells. An exemplary normal (i.e. non- arrhythmic) pattern of calcium transients and action potential is shown in Figure 1 C (baseline) and Figure 1 E (baseline). An example of an abnormal (i.e. arrhythmic) pattern is shown in Figure 1 C (+isoproterenol) and Figure 1 E (+E4031). Of note, isoproterenol does not penetrate the cell membrane and therefore is not encompassed by the methods, models, and systems described herein.

[0071] “Arrhythmia disease” as used herein therefore refers to a disease or condition associated with or caused by arrhythmia. Examples of such diseases are provided elsewhere herein.

[0072] In the context of the invention, a “model” refers to one or more cells in vitro that display some or all of the pathological processes and / or phenotypes (e.g., a characteristic or trait) that are observed in a disease in vivo. In this context, a “model” may also be referred to as an “in vitro model”. By way of example, an arrhythmia disease model (which may also be referred to as an in vitro arrhythmia disease model) may refer to one or more cells in vitro that display all or some phenotypes observed in arrhythmia disease in vivo. In other words, an arrhythmia disease model may recapitulate in vitro the arrhythmia disease observed in vivo. The terms “arrhythmia disease model”, “in vitro arrhythmia disease model” and “model” are used interchangeably herein, unless the context specifically indicates otherwise. By the same token, a cardiac arrhythmia disease model may recapitulate in vitro the cardiac arrhythmia disease observed in vivo. The terms “cardiac arrhythmia disease model”, “in vitro cardiac arrhythmia disease model” and “cardiac model” may be used interchangeably herein, unless the context specifically indicates otherwise.

[0073] As used herein a “system” may refer to a group of related parts that may be dedicated to a particular function. By way of example an arrhythmia disease model system may refer to a group comprising an arrhythmia disease model and other parts such as the exogenous agent, cell culture media, an arrhythmia detection reagent, and / or any other reagent. In line with the arrhythmia disease model provided herein, an arrhythmia disease model system also recapitulates in vitro the arrhythmia disease observed in vivo. The terms “arrhythmia disease model system”, “in vitro arrhythmia disease model system”, “model system” and “system” are used interchangeably herein, unless the context specifically indicates otherwise. Similarly, the terms “cardiac arrhythmia disease model system”, “in vitro cardiac arrhythmia disease model system”, “cardiac model system” and “cardiac system” may be used interchangeably herein, unless the context specifically indicates otherwise.

[0074] The model and / or system provided herein comprises cells that display arrhythmia (for example cardiac arrhythmia). In other words, the model and / or system comprises cells that are arrhythmic. The arrhythmia may be present in any cells disclosed herein, such as any electrically excitable cells (i.e. cells that are capable of displaying arrhythmia). Cells that are electrically excitable may exhibit electrical activity as a consequence of the movement of ions across the cell membrane between the extracellular and intracellular spaces. Methods of measuring electrical activity are well known in the art and include by way of example measuring electrical activity directly (e.g. by measuring cellular membrane potential), or measuring electrical activity indirectly (e.g. by measuring intracellular calcium levels, and / or contraction activity of the cells). Non-limiting examples of electrically excitable cells include cardiac cells (i.e. cardiomyocytes), neuronal cells, muscle cells (such as smooth muscle cells or cardiomyocytes), and / or pancreatic beta cells.

[0075] For the avoidance of doubt, in addition to comprising cells that display arrhythmia, the model and / or system provided may also comprise cells that are not arrhythmic. For example, in addition to comprising cells that display arrhythmia, the model and / or system provided may also comprise cells that are non-electrically excitable cells. An example of this is provided in the examples section below, wherein microtissues (MT) are described, which comprise cardiac cells (e.g. cardiomyocytes) that display arrhythmia (i.e. are arrhythmic) as well as other cells that are non- electrically excitable cells (such as fibroblasts, and / or endothelial cells). Such non-electrically excitable cells may be described as “supporting cells” or “ancillary cells”. As would be clear to a person of skill in the art, the disease of interest affects the type of cell used in the model and / or system. For example, when a cardiac arrhythmia disease model and / or system is desired, cardiac cells such as cardiomyocytes (optionally together with one or more supporting cells such as cardiac fibroblasts, and / or cardiac endothelial cells may be used). Likewise, when a neuronal arrhythmia disease model and / or system is desired, neuronal cells may be used. Similarly, when a muscle arrhythmia disease model and / or system is desired, muscle cells (e.g. smooth muscle cells or cardiomyocytes) may be used. In another example, when a pancreatic beta cell arrhythmia disease model and / or system is desired, pancreatic beta cells may be used.

[0076] In one example, the model and / or system is a cardiac arrhythmia disease model and / or system. In this example, the cells in the model and / or system may comprise cardiomyocytes (optionally together with one or more supporting cells selected from the group consisting of cardiac fibroblasts, and cardiac endothelial cells). Suitably, a cardiac arrhythmia disease model and / or system may comprise cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells.

[0077] In another example, the model and / or system is a neuronal arrhythmia disease model and / or system, in this example, the cells in the model and / or system may comprise neuronal cells, either alone or in combination with other supporting cells.

[0078] In another example, the model and / or system is a muscle arrhythmia disease model and / or system. In this example, the cells in the model and / or system may be muscle cells (e.g. smooth muscle cells) either alone or in combination with other supporting cells.

[0079] In another example, the model and / or system is a pancreatic beta cell arrhythmia disease model and / or system. In this example, the cells in the model may be pancreatic beta cells either alone or in combination with other supporting cells.

[0080] Various methods, arrhythmia disease models, and arrhythmia disease model systems disclosed herein involve contacting cells with a compound, such as an exogenous agent, rescue agent, and / or test compound. Contacting cells with an exogenous agent may be typically under conditions that induce arrhythmia (for example cardiac arrhythmia) in the cells.

[0081] “Exogenous agent” as used herein may refer to an agent that has a source of origin different to the cell that it is to be contacted with. In some examples, the exogenous agent is capable of being produced naturally by the cell that it is to be contacted with, however, it is being added to the cell as an exogenous source rather than being produced by the cell. In this example, the exogenous agent may be added to the cell in a concentration or amount that is in excess to that which the cell would naturally produce it. In other examples, the exogenous agent is not naturally produced by the cell at all. For example, the exogenous agent may be a synthetic agent.

[0082] The exogenous agent may be one or a number (e.g. a mixture) of different compounds.

[0083] The exogenous agent may mimic endogenous cAMP and / or may be an analog of cAMP. The exogenous agent may thus be an agent selected from the group consisting of: db-cAMP (dibutyryl cAMP, also known as N6, 2'-0-dibutyryl adenosine 3', 5'-monophosphate), 8-Br-cAMP-AM (also known as Bromoadenosine- 3’, 5’-cyclic monophosphate, acetoxymethyl ester), Sp-cAMPS (also known as Sp-Adenosine- 3', 5'-cyclic monophosphorothioate), 8-CPT-cAMP (also known as 8-(4- Chlorophenylthio)adenosine- 3’, 5’-cyclic monophosphate), 2'-O-MS-cAMP (also known as 2'-O- Monosuccinyladenosine- 3', 5'-monophosphate), 8-CL-cAMP (also known as 8-Chloroadenosine- 3', 5'-cyclic monophosphate); 8-pCPT-2'-O-Me-cAMP (also known as 8-(4-Chlorophenylthio)-2'- O-methyladenosine-3', 5'-cyclic monophosphate); 6-Bnz-cAMP (also known as N6- Benzoyladenosine-3',5'-cyclic monophosphate sodium salt); and analogs and derivatives thereof. The analogs and derivatives thereof may be synthetic.

[0084] In one example, the exogenous agent may thus be an agent selected from the group consisting of: db-cAMP (dibutyryl cAMP, also known as N6, 2'-O-di butyryl adenosine 3', 5'-monophosphate), 8-Br-cAMP-AM (also known as Bromoadenosine- 3’, 5’-cyclic monophosphate, acetoxymethyl ester), 8-CPT-cAMP (also known as 8-(4-Chlorophenylthio)adenosine- 3’, 5’-cyclic monophosphate), 2'-O-MS-cAMP (also known as 2'-O-Monosuccinyladenosine- 3', 5'- monophosphate), 8-pCPT-2'-O-Me-cAMP (also known as 8-(4-Chlorophenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate); 6-Bnz-cAMP (also known as N6- Benzoyladenosine-3',5'-cyclic monophosphate sodium salt); and analogs and derivatives thereof. The analogs and derivatives thereof may be synthetic.

[0085] The exogenous agent used in connection with the invention may penetrate the cell membrane. In other words, the exogenous agents may be cell-permeable and / or cell-membrane permeable. The phrases “exogenous agent that penetrates the cell membrane” and “cell membrane penetrating exogenous agent” are used interchangeably herein. Exogenous agents disclosed herein such as db-cAMP (dibutyryl cAMP), 8-Br-cAMP-AM, Sp-cAMPS, 8-CPT-cAMP, 2'-O-MS- cAMP, 8-CL-cAMP; and analogs and derivatives thereof, can penetrate the cell membrane and are cell-permeable. Such exogenous agents may therefore cross the cell membrane to enter the cell where it may have downstream effects such as activation of various signalling pathways such as activating the cAMP signalling pathway.

[0086] In some examples, an exogenous agent that mimics endogenous cAMP and / or is an analog of cAMP may be contacted with electrically excitable cells selected from the group consisting of: cardiac cells (such as cardiomyocytes), neuronal cells, muscle cells (such as smooth muscle cells or cardiomyocytes), and / or pancreatic beta cells. For example, an exogenous agent that mimics endogenous cAMP and / or is an analog of cAMP may be contacted with the cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system). In other example, an exogenous agent that mimics endogenous cAMP and / or is an analog of cAMP may be contacted with neuronal cells (e.g. in the context of a neuronal arrhythmia disease model and / or system). In other example, an exogenous agent that mimics endogenous cAMP and / or is an analog of cAMP may be contacted with muscle cells (e.g. smooth much cells) (e.g. in the context of a muscle arrhythmia disease model and / or system). In other example, an exogenous agent that mimics endogenous cAMP and / or is an analog of cAMP may be contacted with pancreatic beta cells (e.g. in the context of a pancreatic beta arrhythmia disease model and / or system).

[0087] In some examples, db-cAMP may be contacted with electrically excitable cells selected from the group consisting of: cardiac cells (such as cardiomyocytes), neuronal cells, muscle cells (such as smooth muscle cells or cardiomyocytes), and / or pancreatic beta cells. For example, db-cAMP may be contacted with the cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system). In other example, db-cAMP may be contacted with neuronal cells (e.g. in the context of a neuronal arrhythmia disease model and / or system). In other example, db-cAMP may be contacted with muscle cells (e.g. smooth much cells) (e.g. in the context of a muscle arrhythmia disease model and / or system). In other example, db-cAMP may be contacted with pancreatic beta cells (e.g. in the context of a pancreatic beta arrhythmia disease model and / or system).

[0088] The exogenous agent used in connection with the invention may penetrate the cell membrane, where it enters the cell and may activate the intracellular cAMP signalling pathway. An exogenous agent that penetrates the cell membrane and activates the intracellular cAMP signalling pathway may also be described as a “cell membrane penetrating activator of the intracellular cAMP signalling pathway” herein.

[0089] The “intracellular cAMP signalling pathway” as used herein refers to the signalling pathway within cells that is regulated by cAMP. cAMP is known to directly activate for example, exchange proteins directly activated by cAMP (Epac), hyperpolarization-activated cyclic nucleotide-gated channels (HCN) and Popeye domain containing (POPDC) proteins, as well as protein kinase A (PKA) and other cAMP-dependent kinases. The intracellular cAMP signalling pathway is well known to a person of skill in the art, see for example, Patra C, et al., Biochemistry, cAMP. In: StatPearls. StatPearls Publishing, Treasure Island (FL); 2022.

[0090] In one example, the exogenous agent binds to protein kinase (PKA) and / or induces PKA activity in the cells. In other words, the exogenous agent may be a PKA binder and / or PKA activator. In one example, the exogenous agent binds to exchange proteins activated by cyclic AMP (EPAC) and / or induces EPAC activity in the cells. In other words, the exogenous agent may be an EPAC binder and / or EPAC activator.

[0091] A major component of calcium regulation and homeostasis is Ca2+ buffering in both the SR and cytoplasm. Ca2+ cycling plays a critical role in regulating excitation-contraction coupling in cells such as cardiomyocytes thus its disruption can result in arrhythmic events. In this regard, an exogenous agent may cause an increase in intracellular cAMP to high levels, (even higher than those achieved after p-adrenergic stimulation alone). This in turn may increase activation of the cAMP signalling pathway which may lead to dysregulation of Ca2+, for example, augmentation of Ca2+ influx and sarcoplasmic reticular (SR) Ca2+ uptake, SR Ca2+ overload and subsequent Ca2+ release that triggers arrhythmia.

[0092] Methods for determining whether an exogenous agent penetrates the cell membrane and activates the intracellular cAMP signalling pathway are well known in the art. For example, by transducing or transfecting the cells with a genetically encoded cAMP FRET sensor (e.g. the EPAC-SH187FRET sensor or the AKAP79-CUTie FRET sensor), performing real-time cAMP FRET measurements with this sensor and analysing the generated imaging data. See for example, Klarenbeek J, et al (2015) Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PLoS One 10:e0122513; and Surdo N.C., et al. FRET biosensor uncovers cAMP nano-domains at beta-adrenergic targets that dictate precise tuning of cardiac contractility. Nat. Commun. 2017;8: 15031. doi: 10.1038 / ncomms15031.

[0093] The exogenous agent used in connection with the invention may activate the intracellular cAMP signalling pathway by binding to PKA and / or inducing PKA activity. A non-limiting example of such an exogenous agent is 6-Bnz-cAMP.

[0094] “Binding” as used herein may refer to a physical interaction between two or more molecules. For instance, binding of the exogenous agent to PKA may refer to a direct physical interaction between the exogenous agent and PKA. Alternatively, binding of the exogenous agent to PKA may be indirect. For instance, the exogenous agent may bind to one or more molecules that bind directly to PKA.

[0095] Binding of the exogenous agent to PKA (directly or indirectly) may in turn induce PKA activity. For example, wherein the exogenous agent is db-cAMP or the like, two molecules of db-cAMP or the like may bind to the regulatory subunits of pKA triggering conformational changes that dissociate the complex, resulting in activation of the catalytic subunits of PKA (inducing PKA activity) for subsequent phosphorylation of substrates in various subcellular compartments. Activated PKA may phosphorylate a number of other proteins including enzymes that convert glycogen into glucose, AM PA receptors, proteins that promote muscle contraction in the heart leading to an increase in heart rate, and transcription factors, which regulate gene expression.

[0096] Alternatively, inducing PKA activity may occur without binding of the exogenous agent to PKA. In such case, the exogenous agent may bind to one or more molecules other than PKA resulting in downstream activation of PKA. For example, these one of more molecules may in turn activate PKA by binding to PKA directly or indirectly.

[0097] The exogenous agent used in connection with the invention may activate the intracellular cAMP signalling pathway by binding to EPAC and / or inducing EPAC activity. A non-limiting example of such an exogenous agent is 8-pCPT-2-O-Me-cAMP-AM.

[0098] The term “EPAC” refers to “exchange proteins activated directly by cyclic AMP”. EPAC refers to a family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs) that mediate protein kinase A (PKA)-independent signal transduction properties of the second messenger cAMP. Two variants of EPAC exist (EPAC1 and EPAC2), both of which couple cAMP production to the activation of Rap, a small molecular weight GTPase of the Ras family.

[0099] “Binding” as used herein may refer to a physical interaction between two or more molecules. For instance, binding of the exogenous agent to EPAC may refer to a direct physical interaction between the exogenous agent and EPAC. Alternatively, binding of the exogenous agent to EPAC may be indirect. For instance, the exogenous agent may bind to one or more molecules that bind directly to EPAC.

[0100] Binding of the exogenous agent to EPAC (directly or indirectly) may in turn induce EPAC activity. For example, wherein the exogenous agent is db-cAMP or the like, two molecules of db-cAMP or the like may bind to one of the isoforms of EPAC. This binding induces conformational changes that activate guanine nucleotide exchange factor (GEF) activity of EPAC and promotes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on small GTPases, such as Rap1 and Rap2, thereby inducing their activation. In this active state, they can interact with and regulate downstream effectors involved in various cellular processes such as integrins involved in cell adhesion, as well as proteins involved in regulating calcium handling, such as ryanodine receptors (RyR2), thereby contributing to calcium release in cells such as cardiomyocytes and facilitating excitation-contraction coupling in the heart. Alternatively, inducing EPAC activity may occur without binding of the exogenous agent to EPAC. In such case, the exogenous agent may bind to one or more molecules other than EPAC resulting in downstream activation of EPAC. For example, these one of more molecules may in turn activate EPAC by binding to EPAC directly or indirectly.

[0101] The exogenous agent used in connection with the invention, may be contacted or have been contacted with cells under conditions that induce arrhythmia in the cells.

[0102] Conditions that induce arrhythmia may be dependent on one or more parameters. One parameter may be the concentration of the exogenous agent. The concentration of the exogenous agent may be any concentration disclosed herein. Another parameter may be the length of time the exogenous agent is contacted with the cells for. These parameters may be determined using methods known to the skilled person and non-limiting examples are provided elsewhere herein.

[0103] When the cell cultures (2D or 3D) are initially contacted with the exogenous agent, the cell culture may be up to 7 days, up to 14 days, up to 21 days, or up to 31 days after the cell culture was first formed. Accordingly, the cell culture may be contacted with the exogenous agent when the cell culture is up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, up to 16 day, up to 17 days, up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days, up to 31 days, after the cell culture was first formed. The cell culture may also be contacted with the exogenous agent when the cell culture is formed (i.e. , as soon as the cell culture has formed).

[0104] In a further example, the cell cultures (2D or 3D) may be cultured for a period of at least 1 day, at least 4 days, at least 7 days, at least 11 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days, or at least 31 days before contact with the exogenous agent. Accordingly, the cell cultures may be cultured for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 day, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 31 days, before contact with the exogenous agent. The cell culture may also be contacted with the exogenous agent when the cell culture is formed (i.e., as soon as the cell culture has formed). In one example, the exogenous agent may be initially contacted with the cell culture at least 11 days after initial formation of the MT, wherein the exogenous agent is then in contact with the MT for 72 hours. In other words, the MT may be cultured for a period of at least 11 days before contact with the exogenous agent for a further 72 hours. In another example, the exogenous agent may be initially contacted with the cell culture at least 18 days after initial formation of the MT, wherein the exogenous agent is then in contact with the MT for 72 hours. In other words, the MT may be cultured for a period of at least 18 days before contact with the exogenous agent for a further 72 hours. In another example, the exogenous agent may be initially contacted with the cell culture at least 29 days after initial formation of the MT, wherein the exogenous agent is then in contact with the MT for 72 hours. In other words, the MT may be cultured for a period of at least 29 days before contact with the exogenous agent for a further 72 hours. These examples are particularly relevant when 3D cultures are used.

[0105] In one example, the cells may be cultured for a period of at least 7 days before contact with the exogenous agent for a further 24-72 hours. In one example, the cells may be cultured for a period of at least 11 days before contact with the exogenous agent for a further 24-72 hours. In one example, the cells may be cultured for a period of at least 15 days before contact with the exogenous agent for a further 24-72 hours. In one example, the cells may be cultured for a period of at least 21 days before contact with the exogenous agent for a further 24-72 hours. These examples are particularly relevant when 2D cultures are used.

[0106] As used herein, "contacting" includes directly exposing cells to an exogenous agent (for example by immersion of the cells in a solution comprising the exogenous agent) and / or introducing (for example by pipetting) the exogenous agent onto the cells. Pipetting may occur with an automated liquid handling device such as a TECAN Fluent® automated workstation. The exogenous agent may be administered to the cells by dissolving the exogenous agent in a solution (e.g. media) containing the cells. Alternatively, the exogenous agent may first be dissolved in a solution and the solution comprising the exogenous agent is provided onto the cells, for example, by submerging the cells in the solution. Alternatively, the exogenous agent may first be dissolved in the solution and the cells submerged in the solution subsequently. In another example, the cells may be provided within a vessel, such as a multi-well plate, and a solution containing exogenous agent may be introduced into wells containing the cells by pipetting.

[0107] As it will be appreciated, in the context of the present disclosure, when reference is made to “contacting the cells” this may also encompass contacting the models and / or systems described herein, and vice versa.

[0108] The exogenous agent may be brought into contact with the cells alone, in conjunction with a solvent (e.g., dimethylsulfoxide (DMSO) or the like) or a carrier (including, e.g., a peptide, lipid or solvent carrier), or water, or in conjunction with another compound. The exogenous agent may be dissolved in any suitable solvent. The solvent may be DMSO, cell culture media, or water.

[0109] Any appropriate amount of the exogenous agent may be contacted with cells. An appropriate amount of the exogenous agent may be an amount that is sufficient to induce arrhythmia in cells.

[0110] The arrhythmia disease (being recapitulated in the arrhythmia disease model and / or arrhythmia disease model system) may be a disease selected from the group consisting of: a cardiac arrhythmia disease, a calcium handling disease, a channelopathy, a supraventricular arrhythmia disease, a ventricular arrhythmia disease, a structural heart disease, a cardiomyopathy, an atrial fibrillation, a metabolic arrhythmia disease, and a mitochondrial arrhythmia disease.

[0111] In one example, the arrhythmia disease is a cardiac arrhythmia disease, optionally wherein cardiac arrhythmia disease is selected from the group consisting of: a cardiac arrhythmia disease, a calcium handling disease, a channelopathy, a supraventricular arrhythmia disease, a ventricular arrhythmia disease, a structural heart disease, a cardiomyopathy, an atrial fibrillation, a metabolic arrhythmia disease, and a mitochondrial arrhythmia disease.

[0112] “Channelopathy” as used herein may refer to any disease that develops due to one or more defect in ion channels caused by genetic factors. Mutations in genes encoding ion channels, which impair channel function, are the most common cause of channelopathies. Several channelopathies are known in the art. In a non-limiting example, the channelopathy may be long QT syndrome type 2 (LQT2) or catecholaminergic polymorphic ventricular tachycardia (CPVT). The CPVT may be CVPT1 or CVPT2.

[0113] In one example, the arrhythmia disease is CVPT1. Optionally, in this example, the exogenous agent is selected from the group consisting of: db-cAMP (dibutyryl cAMP), 8-Br-cAMP-AM, Sp- cAMPS, 8-CPT-cAMP, 2'-O-MS-cAMP, 8-CL-cAMP; and analogs and derivatives thereof. Typically, in this example, the cells are cardiac cells.

[0114] In one example, the arrhythmia disease is CVPT1 and the exogenous agent is db-cAMP. Typically, in this example, the cells are cardiac cells.

[0115] In one example, the arrhythmia disease is LQT2 and the exogenous agent is db-cAMP. Typically, in this example, the cells are cardiac cells

[0116] “Calcium handling disease” as used herein may refer to any disease that develops where Ca2+ stores within the cell and or subcellular organelles are dysregulated. For instance, Ca2+ influx may be augmented, and sarcoplasmic reticulum (SR) Ca2+ uptake may be augmented, resulting in subsequent Ca2+ overload, which can lead to store-overload calcium release. Overall Ca2+ cycling plays a critical role in regulating excitation-contraction coupling in cardiomyocytes. A major component of calcium regulation and homeostasis is Ca2+ buffering in both the SR and cytoplasm. A calcium handling disease may encompass channelopathy, cardiomyopathy, arrhythmia diseases and mitochondrial arrhythmia disease. For instance, genetic mutations in a range of genes linked to channelopathy or cardiomyopathy phenotypes can affect the ability of the cardiomyocyte to respond to sudden changes in calcium in either of these two compartments, leading to dysregulated Ca2+and arrhythmias.

[0117] In one example, the exogenous agent is db-cAMP. Without being bound to the following theory, the mechanism by which db-cAMP could lead to arrhythmias in channelopathies may be due to cAMP binding directly to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. HCN channels may be referred to as pacemaker channels as they help to generate rhythmic activity within groups of heart and brain cells. At least two of these isoforms are known to be expressed in cardiomyocytes e.g. hPSC-derived CMs. These channels are shown to be normally activated at hyperpolarized voltages of the cell membrane. However, binding of cAMP to HCN may also activate these channels with a depolarizing current derived from the influx of Na+ and efflux of K+ ions through the HCN channels. Functional analysis has shown that dysregulated activation of HCN channels in cardiomyocytes e.g. hPSC-derived CMs can result in DADs (delayed afterdepolarization - arrhythmias) and more ectopic beats under stressful conditions (isoproterenol stimulation) compared with healthy control CMs. Alternatively, arrhythmias observed in some channelopathies may be due to cAMP binding directly to the potassium ion channel, hERG, which plays a key role in the repolarization phase of the action potential in cardiomyocytes. Direct binding of cAMP to hERG may result in a hyperpolarizing shift in voltagedependent activation. The summation of cAMP-mediated effects is a net diminution of the effective current. However, if HERG is complexed with the K+ channel accessory proteins MiRP1 or minK, the stimulatory effects of cAMP may be favoured. These findings provide a direct link between stress and arrhythmia by a unique mechanism where a single second messenger exerts complex regulation of an ion channel via two distinct pathways. In any case, the arrhythmia disease model and system thereof as disclosed herein can be used to model all channelopathies through the effects of the exogenous agent on the cAMP signalling pathway.

[0118] The cells that are used in the methods, arrhythmia disease models, and arrhythmia disease model systems disclosed may carry one or more mutations associated with the arrhythmia disease. For example, the cells may comprise one or more mutations in a human gene including but not limited to those in the group consisting of ryanodine receptor 2 (RYR2) - Ensembl gene ref: ENSG00000198626, calsequestrin 2 (CASQ2) - Ensembl gene ref: ENSG00000118729, potassium voltage-gated channel subfamily H member 2 (KCNH2) - Ensembl gene ref: ENSG00000055118, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) - Ensembl gene ref: ENSG00000123700, Calcium Voltage-Gated Channel Subunit Alphal C (CACNA1C) - Ensembl ref: ENSG00000151067, Calcium Voltage-Gated Channel Auxiliary Subunit Beta 2 (CACNB2) - Ensembl ref: ENSG00000165995, Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 1 (CACNA2D1) - Ensembl ref: ENSG00000153956, Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A) - Ensembl ref: ENSG00000183873, Calmodulin 1 (CALM1) - Ensembl ref: ENSG00000198668, Calmodulin 2 (CALM2) - Ensembl ref: ENSG00000143933, Calmodulin 3 (CALM3) - Ensembl ref: ENSG00000160014, Phospholamban (PLN) - Ensembl ref: ENSG00000198523, A-Kinase Anchoring Protein 9 (AKAP9) - Ensembl ref: ENSG00000127914, Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 1 (KCNE1) - Ensembl ref: ENSG00000180509, Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 2 (KCNE2) - Ensembl ref: ENSG00000159197, Myosin Binding Protein C3 (MYBPC3) - Ensembl ref: ENSG00000134571 , Titin (TTN) - Ensembl ref: ENSG00000155657, Ankyrin 2 (ANK2) - Ensembl ref: ENSG00000145362, Lamin A / C (LMNA) - Ensembl ref: ENSG00000160789, Alanyl-TRNA Synthetase 2 (AARS2) - Ensembl ref: ENSG00000124608, and Plakophilin 2 (PKP2) - Ensembl ref: ENSG00000057294.

[0119] Mutations in one or more of these genes are associated with the development of arrhythmia diseases.

[0120] In examples where the one or more mutations is in human RYR2, CASQ2, KCNH2, KCNQ1 , SCN5A, KCNJ2, CACNA1C, CALM1 , CALM2, PKP2, AARS2, LMNA, TTN or MYBPC3, the one or more mutations may be selected from: a) M4109R - RYR2; b) F2483I - RYR2; c) P2328S - RYR2; d) S406L - RYR2; e) E2311 D - RYR2; f) D307H - CASQ2; g) R420Q - RYR2; h) exon 3 del, P2328S, T2538R, L4115F, Q4201 R and V4653F - RYR2; i) I4587V - RYR2; j) L3741 P - RYR2; k) c.339-354 del (CASQ2G112+5X) - CASQ2; l) D3638A - RYR2; m) D358N and R4651 I - RYR2; n) R4147S - RYR2; o) Y4962C - RYR2; p) G1681A- KCNH2; q) R176W-KCNH2; r) A561T-KCNH2; s) A561V-KCNH2; t) A561P-KCNH2; u) N996I - KCNH2; v) IVS9-28A / G - KCNH2; w) A422T-KCNH2; x) G603D-KCNH2; y) G604S - KCNH2; z) A614V-KCNH2; aa)G601S- KCNH2; bb) R534C- KCNH2; cc) N633S - KCNH2; dd) R685P- KCNH2; ee)V822M - KCNH2; ff) R366X-KCNH2; gg)S428X- KCNH2; hh)R752W - KCNH2; ii) T983I - KCNH2; jj) p.S1112Pfs*171 - KCNH2; kk) N588K- KCNH2;

[0121] II) R594Q-KCNQ1; mm) 1795insD - SCN5A; nn) R535Q - SCN5A; oo) R218W-KCNJ2; pp) G406R-CACNA1C; qq) N639T - CACNA1C; rr) F142L- CALM1; ss) D130G-CALM2; tt) R1629X-SCN5A; uu)C.1643delG - PKP2; vv) K672Rfs - PKP2; ww) R958X - AARS2; xx) R592W-AARS2; yy) V730M - AARS2; zz) R216C-LMNA; 1 aaa) K219T - LMNA; bbb) H222P - LMNA; ccc) R225X - LMNA; ddd) D461Y - LMNA; eee) W467X - LMNA; fff) R471 H - LMNA; ggg) R482W- LMNA; hhh) Q493X -LMNA; iii) A22352fs - TTN; jjj) W792Vfs+40X - MYBPC3.

[0122] One or more mutations in KCNH2 are associated with the development of LQT2. KCNH2 has been linked to more than 500 mutations resulting in disease. One or more mutations in RYR2 or CASQ2 are associated with the development of CPVT 1 or CPVT2. RYR2 has been linked to more than 170 mutations resulting in disease. CASQ2 has been linked to more than 30 mutations resulting in disease.

[0123] Additional arrhythmia disease-causing mutations are known in the art. Cells containing one or more mutations resulting in arrhythmia disease may be used in the arrhythmia disease model and / or arrhythmia disease model system disclosed herein.

[0124] Various methods, arrhythmia disease models and / or systems disclosed herein involve contacting cells with an exogenous agent.

[0125] The cells may be electrically excitable cells. Such cells may exhibit electrical activity as a consequence of the movement of ions across the cell membrane between the extracellular and intracellular spaces. Electrically excitable cells may thus include cardiac cells, neurons, muscle cells (such as smooth muscle cells) and pancreatic beta cells. Suitably, the cardiac cells may be cardiomyocytes. Suitably, the cardiac cells may be derived from human-induced pluripotent stem cells (i.e. they may be cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC- CMs). Such cells may be capable of exhibiting rhythm in contraction caused by electrical activity and accordingly, be capable of exhibiting abnormal / irregular rhythm (arrhythmia).

[0126] Suitably, the cells may be in cell culture. The term "cell culture" refers to a process which allows the growth, the differentiation, and / or the maintenance of cells under controlled conditions, generally outside of their natural environment.

[0127] Suitably, the cells may be in a 3D cell culture. By 3D cell culture it is meant that the cells are not cultured as a monolayer on a flat surface, such as a cell culture flask. Instead, the cells may form clumps (also known as aggregates or microtissues (MTs)). Suitably the clumps may be scaffold-free.

[0128] Suitably, the MTs may comprise CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs.

[0129] Suitably, the MTs may comprise about 1000 to about 10,000 cells. Suitably, the MTs may comprise about 2,500 to about 7,500 cells, more suitably the MTs may comprise about 5000 cells. In this context the term “about” means + / - 5%, or less. For example + / - 4%, + / - 3%, or less.

[0130] Alternatively, the MTs may be supported by a 3D scaffold. In this context, supported by the scaffold means that the cells, or a portion of the cells, grow on a 3D scaffold. Appropriate 3D scaffolds are known in the art, for example, collagen scaffolds.

[0131] MTs can be generated from hiPSC-CMs, hiPSC-cardiac endothelial cells (cECs), and hiPSC- cardiac fibroblasts (cFbs) as previously described by Campostrini, G. et al. Nature Protocols vol. 16 2213-2256 (2021).

[0132] The 3D cell culture may alternatively comprise organoids, cardioids and / or engineered cardiac tissues (ECTs).

[0133] Alternatively, the cells may be in a 2D culture. The term “2D culture” means that the cells are cultured as a monolayer on a substantially flat surface. The flat surface, such as the surface of a cell culture flat may be coated with a substrate. Many examples of substrates are known in the art. Merely by way of example, the substrate may be Matrigel, laminin, type I collagen, PDMS, fibronectin, vitronectin and the like.

[0134] Suitably, the cells may be cultured in suspension. By cultured in suspension it is meant that the cells, or portion of the cells, do not adhere to a surface, for example a scaffold or a flat surface of a cell culture vessel (such as a cell culture flask). It will be appreciated that when cells form MTs (or organoids, cardioids, or engineered cardiac tissues (ECTs)) the cells may be in suspension. Whilst the MTs (or organoids, cardioids, or engineered cardiac tissues (ECTs)) may settle at the bottom of a cell culture vessel they need not adhere to it.

[0135] As it will be appreciated by the person of skill in the art, cells are typically cultured in culture vessels. As used herein, "cell culture vessel" refers to a container suitable for culturing cells. Many types of cell culture vessels are known in the art. Suitably, the vessel may be a multi-well cell culture plate, a cell culture flask, a roller bottle, a cell culture dishes or plates, a microtiter plate, or a multi-layered cell growth flask. The vessel may be made from for example plastic or glass. As it will be appreciated a person of skill in the art, a multi-well cell culture plate may be particularly useful in the context of high throughput screening. Suitably, the multi-well cell culture plate may be a 96-well plate, or for example a 384-well plate.

[0136] Suitably, when cells are cultured in 2D (e.g. as adherent cells), about 10,000 to about 100,000 cells may be cultured in a single cell culture vessel (e.g. in a well in a multi-well cell culture plate, such as a 0.1 cm2well or a 0.34 cm2well). Suitably, a single cell culture vessel (e.g. a well in a multi-well cell culture plate such as a 0.1 cm2well or a 0.34 cm2well) may comprise about 10,000 to 50,000 cells. See data provided in figures 17 to 20, where such CM cell numbers were used.

[0137] In the method for producing an arrhythmia disease model, the cells are contacted with an exogenous agent under conditions that induce arrhythmia in the cells. Suitably, such conditions comprise contacting the cells with an exogenous agent at a concentration capable of inducing arrhythmia. The concentration may depend on the nature of the exogenous agent used. Suitably, the concentration of the exogenous agent (such as dibutyryl cAMP (db-cAMP)) may be from about 0.0005mM to about 250mM. Suitably, the concentration of the exogenous agent (such as db- cAMP) may be from about 0.005mM to about 50mM. Suitably, the concentration of the exogenous agent (such as db-cAMP) may be from about 0.05mM to about 5mM. Suitably, the concentration of the exogenous agent (such as db-cAMP) may be from 0.1 mM to about 1 mM. Suitably, the concentration of the exogenous agent (such as db-cAMP) may be from about 0.25mM to about 0.75mM. Suitably, the concentration of the exogenous agent (such as db-cAMP) may be from about 0.04mM to about 0.6mM. More suitably the concentration of the exogenous agent (such as db-cAMP) may be about 0.5mM.

[0138] Suitably, conditions that induce arrhythmia in the cells may include contacting the cells with an exogenous agent (such as db-cAMP) at a concentration capable of inducing arrhythmia for a time period sufficient to induce arrhythmia. Merely by way of example, such a time period may be up to 7 days, optionally up to 72 hours, up to 48 hours, up to 24 hours, up to 60 minutes, or up to 30 minutes. Merely by way of example, such a time period may be at least 30 minutes and up to 7 days, optionally at least 30 minutes and up to 72 hours, at least 30 minutes and up to 48 hours, at least 30 minutes and up to 24 hours, or at least 30 minutes and up to 60 minutes. Suitably, such a time period may be from about 30 minutes to about 7 days, for example from about 60 minutes to about 72 hours, for example from about 24 hours to about 72 hours, or from about 48 hours to about 72 hours. Suitably, when the exogenous agent is db-cAMP the conditions that induce arrhythmia in the cells may comprise contacting the cells with db-cAMP at a concentration from about 0.1 mM to about 1 mM for a time period of from about 24 hours to about 72 hours. Suitably, when the exogenous agent is db-cAMP the conditions that induce arrhythmia in the cells may comprise contacting the cells with db-cAMP at a concentration from about 0.25 mM to about 0.75 mM for a time period of from about 24 hours to about 72 hours. Suitably, when the exogenous agent is db-cAMP the conditions that induce arrhythmia in the cells may comprise contacting the cells with db-cAMP at a concentration of about 0.5mM for a time period of from about 24 hours to about 72 hours, for example, for about 72 hours.

[0139] In a suitable embodiment of the method for producing an arrhythmia disease model, the cells may be contacted with an arrhythmia detection agent and an exogenous agent under conditions that induce arrhythmia in the cells.

[0140] Suitably, the arrhythmia detection agent, such as a calcium dye (for example calcium-6 dye) may be comprised within in loading dye (Calcium 6 Assay Reagent, otherwise known as Component A (Molecular Devices), dissolved in Component B (Molecular Devices) or 1X HBSS Buffer plus 20 mM HEPES), which is diluted between about 1.5 fold to about 6 fold, optionally between about 1.8 fold to about 2.0 fold, in cell culture media. Without wishing to be bound by this hypothesis, the present inventors believe that, surprisingly, contacting the cells the arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye) may improve the arrhythmic phenotype of the disease model. In other words, the arrhythmia detection reagent (such as a calcium dye, for example calcium-6 dye) may make the model of the invention an improved model of arrhythmia. As such, it can be said that the conditions that induce arrhythmia may comprise contacting the cells with the arrhythmia detection reagent (such as a calcium dye, for example calcium-6 dye) as well as the exogenous agent. Suitably, cells may be contacted with the arrhythmia detection reagent (such as a calcium dye, for example calcium-6 dye) for a time period of from about 1 minute to about 24 hours, for example from about 10 minutes to about 12 hours, for example from about 20 minutes to about 6 hours, or for example from about 1 hour to about 2 hours (for example, 1.5 hours (90 minutes)). Suitably, cells may be contacted with the arrhythmia detection reagent (such as a genetically-encoded calcium / voltage indicator) for a time period of from about 24 hours to about 72 hours.

[0141] Suitably, the arrhythmia detection agent, such Fluo-4 dye, may be dissolved in DMSO (e.g. at a concentration of about 1 to 5 mM, preferably at 4 mM), which is diluted between about 500 fold to about 1250 fold, optionally about 1000 fold, in cell culture media. Optionally, before diluting in cell culture media the Fluo-4 can be diluted about 9-fold in 100x PowerLoad concentrate (Molecular Devices). Also optionally, 1x Probenecid solution can be added to prevent extrusion of dye by anion pumps in some cell types. Without wishing to be bound by this hypothesis, the present inventors believe that, surprisingly, contacting the cells the arrhythmia detection agent (such as a calcium dye, for example fluo-4 dye) may improve the arrhythmic phenotype of the disease model. In other words, the arrhythmia detection reagent (such as a calcium dye, for example fluo-4 dye) may make the model of the invention an improved model of arrhythmia. As such, it can be said that the conditions that induce arrhythmia may comprise contacting the cells with the arrhythmia detection reagent (such as a calcium dye, for example fluo-4 dye) as well as the exogenous agent. Suitably, cells may be contacted with the arrhythmia detection reagent (such as a calcium dye, for example fluo-4 dye) for a time period of from about 1 minute to about 24 hours, for example from about 10 minutes to about 12 hours, for example from about 20 minutes to about 6 hours, or for example from about 1 hour to about 2 hours (for example, 1.5 hours (90 minutes)). Suitably, the arrhythmia detection reagent (such as a calcium dye, for example fluo-4 dye) may be washed out from the cells before arrhythmia detection is performed. For example, after incubation of cells with fluo-4 dye (e.g. at 37°C for about an hour), media may removed and cells may be washed (e.g. with PBS). New culture media containing the exogenous agent may be readded to the cells before detection.

[0142] It will be appreciated that in the context of the arrhythmia disease model or system described herein, the exogenous agent and / or arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be present at a concentration that induces arrhythmia, as described hereinabove.

[0143] In some embodiments, the method for producing an arrhythmia disease model may comprise the step of contacting the cells with one or more rescue compounds. A “rescue compound” as used herein refers to a compound that is known to have the ability to reduce arrhythmia. Suitably, the rescue compound may reduce arrhythmia by restoring the rhythm of cell contraction back to that observed in the cell model or system prior to addition of the exogenous agent such as dbcAMP (and optionally the addition of a calcium dye, such as calcium-6 dye or fluo-4 dye). As it would be appreciated, the rescue compound may restore the rhythm of cell contraction to the rhythm of a wild-type cell line.

[0144] A compound known to have the ability to reduce arrhythmia may be useful for example as a control in a screening assay for identifying a test compound for treating arrhythmia. Alternatively or additionally, a compound known to have the ability to reduce arrhythmia may be useful for confirming that the model or system described herein is an effective arrhythmia model. By effective, it is meant that it at least partially recapitulates the arrhythmic phenotype that is observed in vivo for the arrhythmic disease of interest. In the context of the present disclosure, the term “reduce arrhythmia” or “reduction of arrhythmia” means reducing the incidence of arrhythmia, reducing the severity of arrhythmia, and / or completely stopping arrhythmia. In the context of the present disclosure a rescue compound may also be referred to as an anti-arrhythmic compound.

[0145] Suitably, a rescue compound may be selected from the group consisting of flecainide, thapsigargin, propranolol, forskolin, cAMP, dantrolene, KN-93, RyR-stabilizing compound S107, nadolol, AAV9-mediated hCASQ2 replacement, efsevin, kaempferol, carvedilol, metoprolol, RyR- stabilizing compound Rycal, and myristoylated autocamtide-2-related inhibitory peptide (AIP), ryanodine, S107, lumacaftor, minoxidil, nicorandil, ginsenoside, Rg3, telmistartan, LUF7346, ICA-105574, NS1643, PD118057, quinidine, vernakalant, perhexiline, verapamil, nifedipine, diltiazem, phenytoin, dexniguidipine, suramin, dapoxetine, aliskerin-hemifumarate, aprindine, amyleine, mepivacaine, A-887826, bepridil, pincainide, NS-8593, KB-R943, zaldaride or a combination thereof.

[0146] In one example, the rescue compound may be selected from the group consisting of flecainide, thapsigargin, propranolol, forskolin, cAMP, dantrolene, KN-93, RyR-stabilizing compound S107, nadolol, AAV9-mediated hCASQ2 replacement, efsevin, kaempferol, carvedilol, metoprolol, RyR- stabilizing compound Rycal, and myristoylated autocamtide-2-related inhibitory peptide (AIP), ryanodine, S107, phenytoin, dexniguidipine, suramin, dapoxetine, aliskerin-hemifumarate, aprindine, amyleine, mepivacaine, A-887826, bepridil, pincainide, NS-8593, KB-R943, zaldaride or a combination thereof. These compounds may be particularly relevant as rescue compounds for catecholaminergic polymorphic ventricular tachycardia-1 (CPVT1) (see also the screens provided herein). Accordingly, these compounds may reduce the incidence, reduce the severity of, and / or completely stop arrhythmia due to CPVT1.

[0147] In one example, the rescue compound may be selected from the group consisting of lumacaftor, minoxidil, nicorandil, ginsenoside, Rg3, telmistartan, LUF7346, ICA-105574, NS1643, PD118057, vernakalant, verapamil, nifedipine, diltiazem or a combination thereof. These compounds may be particularly relevant as rescue compounds for LQT2. Accordingly, these compounds may reduce the incidence, reduce the severity of, and / or completely stop arrhythmia due to LQT2.

[0148] Suitably, the rescue compound may be flecainide. This rescue compound may be particularly relevant in the context of CPVT 1 disease models, systems and methods. Suitably, flecainide may be contacted with the cells at a concentration of from about 1 to about 3 pM, suitably at about 3 pM. Suitably, the rescue compound may be lumacaftor. This rescue compound may be particularly relevant in the context of LQT2 disease models, systems and methods. Suitably, lumacaftor may be contacted with the cells at a concentration of from about 5 to about 10 pM, suitably at about 10 pM.

[0149] Suitably, the cells may be contacted with a rescue compound (for example flecainide) from about 5 to about 30 minutes. However, as it will be appreciated by the person skilled in the art, the time required for the rescue compound to reduce arrhythmia may depend upon the severity and / or cause of arrhythmia (for example mutation). For example, for LQT2 disease models, systems and methods this rescue period has to be longer.

[0150] The method for producing an arrhythmia disease model may further comprise the step of identifying cells having arrhythmia. It will be appreciated that this step may be performed after the cells have been contacted with an exogenous agent (and optionally arrhythmia detection agent) and / or after the cells have been contacted with a rescue compound. Identifying cells having arrhythmia after the cells have been contacted with an exogenous agent (and optionally arrhythmia detection agent) may be useful to confirm whether the exogenous agent and / or conditions used in fact induce arrhythmia in the cells.

[0151] Methods for identifying cells having arrhythmia are known in the art. Suitably, the cells may be identified by an assay that detects changes in intracellular calcium levels, electrical activity and / or contraction activity of the cells. Each of the parameters listed above (voltage, calcium, contraction) can be evaluated independently. Suitably, such an assay may be an optical assay (sometimes referred to as the “calcium flux assay”, “action potential assay”, or “contraction kinetics assay”). In addition, electrical activity can be assessed using electrophysiological assays such as a multi electrode assay, and contraction kinetics, can also be measured by impedance assays.

[0152] Suitably, in the context of an optical assay, the step of identifying cells having arrhythmia may comprise obtaining optical records for at least 1 minute, at least 2 minutes, preferably at least 3 or about 3 minutes. Suitably, optical records during the voltage / contraction assay may comprise obtaining between 30 to 100, or more frames per second. Suitably, optical records obtained during the voltage / contraction assay may be obtained at about 60 frames per second.

[0153] Suitably, in the context of an electrophysiological assay such as multi electrode assays or a contraction assay such as impedance assays, recordings may be made at a set frequency (10 kHz - 50 kHz); for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, preferably at least 3 or about 3 minutes, more preferably at least 4 minutes or about 4 minutes, most preferably at least 5 minutes or about 5 minutes. Identifying cells having arrhythmia may involve the use of an arrhythmia detection reagent. An arrhythmia detection reagent is any agent that allows the detection of the presence or absence, and optionally severity, of arrhythmia. The type of arrhythmia detection reagent may depend upon the type of cells. Suitably, the arrhythmia detection reagent may be selected from the group consisting of a chemical calcium dye, a chemical membrane potential dye, a genetically-encoded calcium indicator and a genetically-encoded voltage indicator. An example of a chemical calcium dye is calcium-6 dye (Molecular Devices). Suitably, the dye may be a fluorescent dye. A chemical calcium dye is a dye that allows measuring of calcium (Ca2+) flux during an assay. A chemical membrane potential dye is a dye that allows measuring increases and decreases in membrane potential, for example during muscle contraction, nerve-impulse propagation, or cell signaling. A genetically-encoded calcium indicator (GECI) is a protein that allows measuring of Ca2+ levels in a cell and relates changes in Ca2+ levels to a form of output that is often fluorescence- or luminescence-based. A genetically-encoded voltage indicator (GEVI) is a protein that senses membrane potential in a cell and relates the change in voltage to a form of output that is often fluorescence-based.

[0154] Examples of arrhythmia detection agents useful in the context of the present disclosure include calcium-6, Fluo-3, Fluo-4, Fluo-8, Cal520, Calbryte-520, Calcium Green-1 , Calcium Green 5N, Oregon Green 488 BAPTA-1 , Rhod-2 AM, FluoVolt, Merocyanine 540, RH1692, ANNINE6, Annine6+, VoltageFluor 2.1. Cl, BeRSTI, di-2-ANEPEQ, dsRVF5, di-2-ANEP-VoLDeMo, GCaMP3, GCaMP5G, GCaMP6, GCaMP8, R-GECO, jRCaMPI b, Pericam, TN-XXL, Case12, YC-Nano, Arch, VoltagEGFP, Ace-mNeon, VSFP-CR, ASAP1 , ASAPIf, ASAP3, Mermaid, QuasAr. In one example, the arrhythmia detection agent may be calcium-6 or Fluo-4.

[0155] In the context of the methods provided herein, use of an arrhythmia detection reagent is particularly advantageous, as the arrhythmia detection reagent may place additional stress on the cells, thereby facilitating the reproducibility of arrhythmia generation. See the examples section below. Such detection agents can affect the electrophysiological properties of cardiomyocytes in general (see Kopljar et al., 2018, Novakova et al., 2008; Yiangou et al., 2022).

[0156] In some examples, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system). For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT 1 arrhythmia disease model and / or system.

[0157] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs.

[0158] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70: 15: 15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells.

[0159] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70: 15: 15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells.

[0160] In some examples, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) at a concentration of about 0.5mM. For example, db- cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system at a concentration of about 0.5mM.

[0161] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT 1 arrhythmia disease model and / or system at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70: 15: 15 of CMs:cECs:cCFs.

[0162] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells.

[0163] In a specific some example, db-cAMP may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells. For example, db-cAMP may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells.

[0164] In some examples, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system). For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT 1 arrhythmia disease model and / or system.

[0165] In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs.

[0166] In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70: 15: 15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells. In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system), wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70: 15: 15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells.

[0167] In some examples, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) wherein the db-cAMP is at a concentration of about 0.5mM. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system, wherein the db-cAMP is at a concentration of about 0.5mM.

[0168] In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT 1 arrhythmia disease model and / or system wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs.

[0169] In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 5000 cells.

[0170] In a specific some example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells (e.g. in the context of a cardiac arrhythmia disease model and / or system) wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells. For example, db-cAMP and an arrhythmia detection agent (such as a calcium dye, for example calcium-6 dye or fluo-4 dye) may be contacted with cardiac cells in the context of a CPVT1 arrhythmia disease model and / or system wherein the db-cAMP is at a concentration of about 0.5mM, wherein the model or system comprises CMs, cECs and cFbs at a ratio of about 70:15:15 of CMs:cECs:cCFs, and the model or system comprises at least, about, or up to 7000 cells.

[0171] In one aspect, the present invention provides a method of screening a test compound for treating arrhythmia. Suitably, the arrhythmia may be a cardiac arrhythmia. However, other suitable types of arrhythmia are disclosed elsewhere herein.

[0172] The term “method of screening” means that the method is suitable for testing for a particular property or effect of one or more compounds. Suitably, the method may be suitable for testing a large number of test compounds. For example, the method may be suitable for testing at least 30 compounds in a single assay. For example, the method may be suitable for testing at least 40 compounds in a single assay. For example, the method may be suitable for testing at least 50 compounds in a single assay. For example, the method may be suitable for testing at least 60 compounds in a single assay. For example, the method may be suitable for testing at least 70 compounds in a single assay. For example, the method may be suitable for testing at least 80 compounds in a single assay. For example, the method may be suitable for testing at least 90 compounds in a single assay.

[0173] For example, the method may be suitable for testing at least 100 compounds in a single assay.

[0174] In one example, the method may be suitable for testing at least 200 compounds in a single assay.

[0175] In one example, the method may be suitable for testing at least 300 (e.g. at least 384) compounds in a single assay. In one example, the method may be suitable for testing at least 500 or 1000 compounds in a single assay.

[0176] Accordingly, the methods described herein can be useful for screening compounds (e.g. test compounds and / or rescue compounds) that may reduce arrhythmia, in a high throughput screening context.

[0177] Suitability, portions of the screening procedure can be automated.

[0178] “Method of screening” also refers to the determination of a set of different properties or effects of one compound and / or combination of compounds in a single assay.

[0179] As used herein, the term “test compound” includes a compound that has or has not previously been identified as a compound capable of treating arrhythmia. A compound that has been previously identified as a compound capable of treating arrhythmia may have been identified as treating a specific type of arrhythmia, and may now be screened to determine whether it has therapeutic utility in a different type of arrhythmia, different patient population, and / or arrhythmia caused by or associated with a different genetic mutation.

[0180] In some embodiments, the test compound may be the rescue compound as described hereinabove.

[0181] The test compound may be for example a small compound, or a biologic (such as a peptide, protein (for example antibody), nucleic acid, etc).

[0182] The term “treating” as used herein refers to the alleviation or prevention of symptoms of a particular disorder in a patient, or the improvement of an ascertainable measurement associated with a particular disorder. Suitably, the alleviation of symptoms may be a reduction of arrhythmia (for example reducing the severity of arrhythmia, and / or completely stopping arrhythmia). Suitably, in the context of prevention, the compound may reduce the chances and / or severity of arrhythmia developing in cells that are at risk of arrhythmia. Celis that are at risk of developing arrhythmia may be cells that have predisposing genetic mutations.

[0183] The method of screening a test compound comprises the step of contacting the test compound with an arrhythmia disease model produced by the method described herein, or with an arrhythmia disease model system described herein.

[0184] The term “contacting” is described elsewhere herein. However, for avoidance of doubt, in the context of contacting a test compound with an arrhythmia disease model or system, this term includes directly exposing the model or system (i.e. some or all of the cells in the model or system) to the test compound (for example by immersion of the cells in a solution comprising the test compound) and / or introducing (for example by pipetting) the test compound to the cells.

[0185] The test compound may be brought into contact with the cells alone, in conjunction with a solvent (e.g., dimethylsulfoxide (DMSO) or the like) or a carrier (including, e.g., a peptide, lipid or solvent carrier), or water, or in conjunction with another test compound.

[0186] Any appropriate amount of the test compound may be contacted with the cells. Typically, the test compound may be contacted with the cells at varying concentrations to determine therapeutic utility.

[0187] The method of screening a test compound further comprises the step of determining the effect of the test compound on arrhythmia in the arrhythmia disease model or arrhythmia disease model system. The term “determining the effect” as used herein refers to determining whether the compound reduces or does not reduce arrhythmia in the model or system. It will be appreciated that whether a compound reduces or not arrhythmia may be determined by measuring the arrhythmia. Methods for measuring arrhythmia are described elsewhere herein in the context of identifying cells having arrhythmia. Merely by way of example, these methods include assays that detect changes in intracellular calcium levels, electrical activity and / or contraction activity of the cells. Suitably, such assays may be selected from the group consisting of an optical assay (sometimes referred to as the “calcium flux assay”, “action potential assay”, or “contraction kinetics assay”), a multi electrode assay or an impedance assay.

[0188] It will be appreciated that test compounds that have been identified as being able to reduce (for example completely eliminate) arrhythmia in the cell model or system may have the ability to treat arrhythmia in vivo. Methods for determining if a test compound is able to reduce (for example completely eliminate) arrhythmia are well known. Such methods may include comparing the rhythm to a control, wherein the control may be the rhythm observed in a wildtype MT, or the rhythm observed in a model or system contacted with a rescue compound as described elsewhere herein.

[0189] Suitably, determining the effect of the one or more test compounds on the arrhythmia may comprises labelling the cells of the arrhythmia disease model or the arrhythmia disease model system with an arrhythmia detection reagent. As described elsewhere herein, the arrhythmia detection reagent may be a chemical calcium dye, a chemical membrane potential dye, a genetically-encoded calcium indicator, or a genetically-encoded voltage indicator.

[0190] Aspects of the invention are demonstrated by the following non-limiting examples. EXAMPLES

[0191] RESULTS

[0192] Differentiation of hiPSC-derived cardiac cells using small molecules

[0193] A protocol to differentiate hiPSC-CMs, -cECs and -cFbs from hiPSCs and subsequent coaggregation of 5000 of these cells in total to form tri-cellular cardiac MTs has been previously described by Campostrini, G. et al.. Cytokines were replaced in the first step towards cardiac mesoderm differentiation with the Wnt-signalling pathway activator CHIR99021 for the first 48h (Fig. 5). This yielded homogeneous cardiac mesoderm populations that served as precursors for all three of the other cell types. This adaption standardized the early differentiation by reducing batch-to-batch variability and reduced cost, which is important for upscaling MT production for screening applications. From day 2, differentiation to CMs, cECs, or cFbs was induced, as shown in Fig. 5A-B. The three different cardiac cell types were characterized by flow cytometry (Fig. 5C). This showed high purities (>85%) for the specific cardiomyocyte-, endothelial- and fibroblast markers (cTnT, CD31 , and vimentin, respectively). For the cFbs, an intermediate differentiation step to epicardial cells (EPCs) was included. EPCs had typical epithelial cobblestone-like morphology (Fig. 5B) and nuclear expression of WT1 , GATA-4, and CX-43 by immunofluorescence (Fig. 5D). cFbs from the same EPC batch lost WT-1 expression and retained GATA-4 at the same time. Whole-transcriptome RNA-sequencing (RNA-seq) on hiPSC- CMs, - cECs, -EPCs, and -cFbs using the small molecule protocol (SP) were compared with the cytokine-protocol (CP) published previously by Giacomelli. E et al. and with their respective primary human tissue equivalents. Previous principal component analysis (PCA) of the RNA-seq on the CP-samples confirmed striking genome-wide expression correspondence between primary human cardiac cells and their hiPSC-derived equivalents. Here, the correlation matrix in Fig. 6 was based on Spearman's rank correlation and showed that each hiPSC-derived cell type from either protocol clustered closely. Both SP- and CP- hiPSC-derived cells could be cryopreserved and thawed for use in subsequent MT formations and / or downstream functional assays.

[0194] All subsequent data were generated using CMs or MT s from SP-derived cardiac cell types, unless otherwise stated.

[0195] Functional assessment of cardiac microtissues in calcium and voltage applications

[0196] Cryopreserved stocks of hiPSC-CMs, -cECs, and -Fbs were used to form cardiac MTs. The cardiac cells were thawed and cultured for 7 (hiPSC-CMs) or 3-4 (hiPSC-cECs and -cFbs) days before being combined and seeded to undergo self-assembly as spheroids of 5,000 cells / well in V-bottom 96-well microplates. The resulting MTs were refreshed every 3-4 days with mBEL medium supplemented with VEGF and FGF2 and cultured for 16-21 days. Within 48 hours of seeding, MTs formed round, contracting spheroids. After 2 weeks in culture, MTs had diameters of 425.3 ± 74.4 pm and had morphologies and cellular architecture by immunofluorescence as expected from our previous studies. Specifically, the presence of cardiomyocytes, cardiac endothelial cells and cardiac fibroblasts was confirmed by the expression of cTnT, CD31 and vimentin respectively (Fig 1A). On the day of assay, MTs were transferred to a ULA 384-well black / clear round bottom spheroid plate to conduct functional assays. To assess MTs functionality in high-throughput format, we developed fluorescence-based calcium and voltage assays to measure and analyze calcium transients or action potential metrics of individually beating MTs in 384 well plates. Using the Hamamatsu Functional Drug Screening System (FDSS / pCell) for both calcium and voltage read-outs, MTs showed robust signals with low variability in baseline parameters and an overall intra-well peak-to-peak variability of 5% (calcium, n=288, N=3) or 14% (voltage, n=174, N=3) (Fig. 1 B). Representative calcium and action potential recordings are shown in Fig. 1C and Fig. 1 E, respectively. To validate the assays, MTs were treated with two reference compounds, each with a specific mechanism of action affecting calcium handling (isoproterenol, a p-adrenergic agonist) or action potential duration (E4031 , a hERG-channel blocker). Analysis of calcium transients showed an increase in calcium peak amplitude and beat rate 30 min. after 0.1 pM isoproterenol treatment (Fig. 1C-D), whereas 30 min. of 0.03 pM E4031 treatment caused an increase in action potential duration (APD) and subsequent early afterdepolarization (EAD)-like arrhythmic events in action potential metrics (Fig. 1E-F). These data confirmed that MTs are functional and would therefore be suitable for use at larger scale to assess calcium handling as well as voltage kinetics.

[0197] Arrhythmogenic disease phenotype revealed and rescued in CPVT-1 cardiac microtissues We next investigated the suitability of MTs to model arrhythmogenic diseases, specifically assessing whether the MTs could exhibit clear enough disease phenotypes for use in large-scale screens designed to identify new therapeutic agents. Our choice as an example was CPVT 1. We generated MTs containing CMs from hiPSCs that were genetically engineered using CRISPR / Cas9 to express in heterozygosity the RYR2M4019Rmutation associated with CPVT1. At baseline, regular calcium oscillations were observed in more than 85% of the CPVT1-MTs (Fig. 2A-B). Since p-adrenergic stimulation alone is not always sufficient to reveal arrhythmogenic phenotypes robustly, whether it was possible to bypass p-adrenergic receptor stimulation and manipulate the PKA pathway by treating CPVT1-MTs directly with dbcAMP was investigated. dbcAMP increases intracellular cAMP levels, stimulating the PKA pathway which results in the phosphorylation of the L-type Ca2+channel, RyR2 and phospholamban, and causing excessive increases in Ca2+influx and SR Ca2+uptake. Diastolic calcium leak from defective RYR2 channels can cause arrhythmic events and in particular delayed afterdepolarizations (DADs) in CPVT1. After treatment with dbcAMP for 3 days, an arrhythmogenic phenotype was triggered reproducibly in 80% of the CPVT1-MTs (Fig. 2A-B). To investigate whether these dbcAMP-induced arrhythmic events were indeed related to mutated RyR2 channels, we treated CPVT1-MTs with Flecainide. Acute addition of Flecainide rescued 100% of the dbcAMP-induced arrhythmic events in CPVT- 1-MTs, while 70% of the CPVT1-MTs treated with the vehicle control still showed arrhythmias (Figure 2A, C). It was thus demonstrated that the 3D cardiac MT model of CPVT1 showed dbcAMP-induced arrhythmias that could be rescued by pharmacological treatment as measured by calcium transients and that both diseased phenotype and rescue can be efficiently detected in scalable high-throughput formats.

[0198] Computational scoring for rapid and precise quantification of cardiac arrhythmia

[0199] Conventional methods for monitoring drug- or disease-induced arrhythmic events in vitro are prone to subjective bias and are technically challenging at high throughput. To address this, a computational scoring method was developed able to classify and score arrhythmic events in a semi-automated manner using rules defined by the operator (Fig. 7A). In this computational scoring method arrhythmias were defined as Type A when there were reoccurring events whereby following a contraction the calcium trace fails to return to baseline before a second contraction occurs. Alternatively, these arrhythmias were identified as irregular rhythm, either due to a spontaneous ectopic beat or missed contraction against the average rhythm of the calcium trace. Type B arrhythmias were classified as fibrillation whereby the peak-to-peak time was below 1.25 seconds, or as tachycardia, whereby the peak-to-peak time was below 1 .25 seconds and high amplitude contractions are detected. Peak-and-trough detection was performed using a nearest neighbour principle, where each point x is compared to its n closest points. If x is either the highest or lowest value among those n points it is marked (xi-1 :n<xi>xi+1 :n for peak detection and -xi- 1 :n<-xi>-xi+1 :n for troughs). After peak detection, each complete trace was classified as rhythmic, arrhythmic-like (Type A), fibrillation-like (Type B) or noise. Noise was defined as traces with amplitudes smaller than 1290 (ranging from 1000 - 2000) relative fluorescent units (RFU). Type A arrhythmic detection was made by looking at the variation in the fluorescent units of the troughs and setting a threshold on the maximum value (between 1000-1500 RFU) as well as the standard deviation (between 150-400 RFU) of all troughs. Additionally, traces were considered arrhythmic if the ratio between Poincare ellipse statistics length and width reached a certain ratio, corrected for the mean RR-interval (Fig. 7B). Additional traces were classified as arrhythmic if they were not detected using the above parameters but had irregular rhythms identified by high standard deviations of frequency (between 0.25 - 1) and a mean frequency below 30.

[0200] Automated scoring resulted in an overall arrhythmia incidence score of 29% and 75% in dbcAMP- treated MTs from CTRL and CPVT1 cardiomyocytes, respectively. This was 0% and 23% in untreated MTs, respectively. Subsequent acute treatment of dbcAMP-treated MTs with Flecainide reduced the automated arrhythmia score from 83.3% to 16.7% in CPVT1-MTs, whereas in CTRL- MTs, an increase in arrhythmia score from 16.7% to 83.3% was observed. This highlighted the protective effect of Flecainide in CPVT 1 , as well as the potential proarrhythmic effect of flecainide in healthy subjects. Furthermore, this scoring method could be readily adapted to any data from the FDSS / pCell scoring the proarrhythmic effects of drugs or diseases in HTS. Using robotics to automate the formation, maintenance and labeling of cardiac microtissues.

[0201] We next addressed whether MT production and screening were amenable to automation for large screens within drug discovery programs. For this, the Fluent® Automated Workstation was used to streamline MT formation, culture medium refreshment, dye labeling, and (optionally) transfer of MTs to assay plates for subsequent functional analysis (Fig. 3A). Scripts were written for the Fluent® Workstation to automate these processes. Initial dry tests confirmed that the scripts ran without hardware or software errors. Subsequent liquid tests included two validation steps using an absorbance-based volumetric assay to assess pipetting performance. This performance is characterized by its accuracy (trueness of the delivered volume to that required), and precision (spread or distribution of dispensed volumes at a required volume). Pipetting 25 pl, the volume used for seeding cells, resulted in an inaccuracy of <5% and a coefficient of variation (CV) of <3% (N=3 plates, Fig. 8A). The inter- and intra-plate variability in precision showed CV <5% (N=3 plates, Fig. 8B). The script was then validated using hiPSC-derived cardiac cell types pre-cultured as described earlier. The cell suspension was transferred to the Fluent® Workstation for automated seeding in ULA 384-well black / clear round-bottomed spheroid plates. Optimization of variables such as cell density, mixing speed, aspiration speed, and dispensing height and speed resulted in MTs derived from 6000 cells with similar morphologies and functionality to manually seeded MTs cultured in 96 V-shaped plates. Overall, the MTs were smaller (373.9±37.0 pm vs. 425.3 ±74.4 pm on average at day 14, Fig. 3B-C), but showed less variability in size (CV < 10% vs. CV < 18%). Seeding of three 384 well plates was complete in 6:15 minutes (cf. ~15 minutes for 4x3 96 well plates by manual seeding and another ~60 min for transferring MTs from 4x96 well plates to 1x384 well plate at the assay day). Automated MTs showed a lower peak-to-peak variability in calcium transients than manually seeded MTs (6.5 ±1.3% vs. 8.3 ±2.6%, Fig. 3D) but similar negative inotropic response to the L-type calcium channel blocker, Nifedipine (-49.6 ± 6.7% vs -47.5% ± 7.4, Fig. 3E). For subsequent functional assays, such as (immuno)fluorescence for high-content imaging (HCI) or HTS, MTs require labeling with antibodies or fluorescent dyes. However, manual execution of labeling protocols, which may include multiple washing steps, is time-consuming and can lead to loss of MTs, high background, and variability in the recorded signal, especially when multiple plates are needed for screens. Scripts were therefore developed to automate the labeling and washing steps for the Calcium-6 and FluoVolt dyes used here. Fig. 9 shows a representative image of a 384 well plate filled with automatically generated MTs, labelled with the Calcium-6 dye, after a compound screen.

[0202] This automated approach streamlined the process, avoided losing MTs, and reduced variability in the recorded signal.

[0203] Cardiac microtissues show improved drug response prediction in blinded HTS Giacomelli, E. et al. previously demonstrated improved structure and metabolism of cardiomyocytes in MTs as they matured. It was therefore asked whether cardiomyocytes produced at scale in MTs as here showed improved ability to predict inotropic drug responses in HTS. Detection of positive inotropy (i.e. increased contraction force, Fig. 9A) requires a higher degree of maturation via, for example, enhanced myofilament organization and calcium handling. Most inotropic compounds work by affecting intracellular calcium levels, either directly (e.g. via L- type Ca2+channels) or indirectly (e.g. via the p-receptor-cAMP-PKA pathway). Depending on the specific mode of action (MoA), positive inotropy can therefore be captured by an increase in calcium peak amplitude, an increase in calcium transient kinetics (rising and falling slope), an increase in beat rate (positive chronotropy) and / or an increase in intracellular calcium load, typically mirrored by an increase in the area under the curve (AUG, Fig. 10B). Using our novel robotics approach, we conducted a blinded screen were conducted using a set of 12-13 inotropic reference drugs. The drugs included ranged from conventional, relatively easily detectable drugs (such as L-type calcium channel modulators) to more challenging drugs like PDE3 inhibitors. The blind screen was repeated twice (once with manually seeded MTs) and classification was performed by two, independent, blinded operators and was based on 6 different calcium-related parameters. Live recordings of the calcium flux from 1 screen were carried out before and after compound addition using the FDSS / pCELL system. Figure 4 shows the results of one screen with heat plots indicating the effects of each compound and each concentration on the 6 parameters. The three independent screens together resulted in an accuracy rate of > 81% for classifying the drugs as positive, negative, or neutral inotropes (Fig. 11). The two screens using automatically generated MTs (Screen II and Screen III) showed reproducibility of 100%, meaning that all the compounds were classified exactly the same in the two screens. The outcome of these two screens was 90.9% similar to the manual screen (Screen I). In some cases, action potential analysis was used to enhance the prediction of inotropic effects of some drugs.

[0204] Furthermore, in an independent experiment, positive inotropes isoproterenol and Levosimendan increased the calcium peak amplitude in MTs (33% and 96%, respectively). IN contrast, in 2D monolayers these compounds only induced positive chronotropic responses (16% and 13%, respectively) without affecting the amplitude (Fig. 4B). This is in line with previous findings where 2D models failed to capture positive inotropic responses, especially where the mode of action involved cAMP signaling. Overall, these results highlight the potential of MTs to capture both negative and positive inotropic responses accurately and demonstrate that the 3D environment supports inotropic responsiveness. dbcAMP triggering arrhythmias and compound-specific rescue of the phenotype

[0205] The inventors demonstrated that the addition of dbcAMP triggers arrhythmias in the CPVT1 3D cell model and that this phenotype is rescued upon addition of Flecainide (Fig. 2A). Fig. 2B shows the percentage of microtissues displaying either a non-arrhythmic or arrhythmic response. It was further clear that other exogenous agents distinct from db-cAMP, such as isoprotenerol (compound acting upstream in the signalling pathway) does not trigger arrhythmias in the disclosed disease model (Fig. 12A). Isoproterenol is most commonly used to try to trigger arrhythmias in cardiac disease models known in the art. It was further established that db-cAMP could trigger arrhythmias in CPVT1 cardiomyocytes cultured in 2D (Fig. 12B), and that dbcAMP also induces arrhythmias in LQT2 disease models (Fig. 13). It was also established that db-cAMP could trigger arrhythmias in additional patient-derived CPVT1 models that contained hiPSC- derived cardiomyocytes generated using either the CP or SP method and that contained either the R4147S-RYR2 mutation (Fig 20B) or Y4962C-RYR2 mutation (Fig 20B). This was also very reproducible between individual samples (Fig 20C).

[0206] The cardiac arrhythmia disease model was also verified as being reflective of the disease being modelled. Addition of the compound lumacaftor prevented dbcAMP induced arrhythmias in the hPSC-derived model for LQT2 (Fig. 14A and 14B) but not for CPVT1 (Fig. 14C and 14D). Currently, a clinical trial (ClinicalTrials.gov ID #NCT04581408) has been initiated to determine if an FDA-approved drug that contains lumacaftor can be used to therapeutically treat certain LQT2 patients. Likewise, acute treatment with 3 iM flecainide of human pluripotent stem cell (hPSC)- derived disease models with dbcAMP-induced arrhythmias only rescued the disease phenotype for CPVTI and not LQT2(Fig. 15). In the clinic, flecainide is considered as compound suitable for treating arrhythmias in CPVT 1 patients, but not LQT2 patients. Thus, the disease model disclosed herein demonstrates that compounds used in the clinic or in clinical trials are capable of rescuing of arrhythmia in the disease model. The automated process disclosed herein (as shown in Fig. 3A) was tested with microtissues whereby it was shown that MT sizes across wells of a 384-well plate were consistent. Disease models involving microtissues treated with db-cAMP were readily produced by the automated process. Further, dbcAMP-treated arrhythmic microtissues formed by automation were capable of rescue by flecainide indicating that the automated procedure produced a disease model reflective of CPVT1 (Fig.16).

[0207] Efficacy screen identifies compounds that rescue arrhythmias in CPVT1 cardiac microtissues

[0208] Beyond cardiac safety assessment, predictive and automated cMT assays provide opportunities for large-scale efficacy screenings to identify compounds with therapeutic potential. The inventors demonstrated this by performing a screen of 92 FDA-approved drugs on the CPVT 1 3D cell model that had been exposed to dbcAMP for 72 hrs. The dbcAMP induced arrhythmias in 100% of CPVT1 cMTs at baseline (Fig. 21), with 17 compounds identified that completely rescued the arrhythmogenic phenotype at one or more test concentration (Fig 22. and Fig. 23). Of these hits, 8 had been previously evaluated in CPVT1 studies, while 9 demonstrated novel antiarrhythmic effects in this model. Notably, 11 compounds were antiarrhythmic drugs classified as ion channel blockers, including sodium (flecainide, phenytoin, aprindine, amyleine, mepivacaine, A-887826) and calcium channel agonists (dexniguldipine, bepridil, pincainide), a SK4 potassium blocker (NS- 8593), and a NXC channel blocker (KB-R7943). The remaining hits identified had unique modes of action, such as calmodulin or serotonin transport inhibition. For this screen, the entire process was automated with the formation, maintenance, treatment with db-cAMP and labelling with calcium-6 of the 3D cell model, as well as addition of rescue compounds performed using a liquid handling robot.

[0209] Discussion

[0210] Developing scalable HTS approaches for 3D human stem cell models remains a major challenge for disease modeling and drug discovery. In this study, we industrialized 3D cardiac MTs for use in HTS intended to identify new and safe therapeutics. Firstly, the earliest differentiation steps were optimized by replacing cytokines with small molecules. This adaption reduced cost and can lead to improved reproducibility between differentiations. It was subsequently shown that these cardiac MTs were fully functional in high-throughput using fluorescence-assays that measured calcium and voltage transients in 384 well plates. MTs exhibited low variability in calcium and action potential baseline parameters and captured drug-induced effects on calcium peak amplitude and action potential duration (APD). MTs containing hiPSC-CMs carrying in heterozygosity the RYR2M4019Rmutation also captured the phenotype and expected therapeutic drug response of CPVT1 , a highly lethal form of inherited arrhythmogenic disease characterized by adrenergically-mediated polymorphic ventricular tachycardia. Although previous models of CPVT1 using hiPSC-CMs have demonstrated disease-associated features such as calcium abnormalities and stress-induced arrhythmia, they have predominantly relied on 2D models with low-throughput methods and outputs that exhibit relatively low reproducibility. For example, both Fatima et al. and Kujala et al. observed arrhythmic activity in CPVT-CMs after adrenergic stimulation, but with rates of 32% (using a multi-electrode array (MEA) single well chip, 60 electrodes) and 30% (using patch clamp recordings), respectively. In addition, Novak et al. showed that only 16% of the CPVT1 hiPSC-CMs showed arrhythmic behavior in calcium transients after isoproterenol treatment. Itzhaki et al., 2012 demonstrated that forskolin, a compound that can stimulate the production of endogenous cAMP in cells by directly activating the enzyme adenylyl cyclase, triggered arrhythmias in 75% of CPVT1 hiPSC-CMs. However, this was based on very low-throughput (8 wells in total with 6 wells arrhythmic). In addition, in a subsequent study (Maizels etal. 2017) by the same group using another CPVT hiPSC-CM model, arrhythmias were only observed in 55% of samples treated with forskolin. This variability and low reproducibility suggests that forskolin would not be a suitable arrhythmogenic trigger for high- throughput screens. In contrast, the inventors have reproducibly triggered arrhythmias in CPVT1- MTs by treating them with dbcAMP for three days. This was consistent across multiple 384-well plates, with arrhythmias detected in 100% of wells (total 1081 wells) with a valid signal. It was hypothesized that in this case, dbcAMP actually mimics excessive adrenergic stimulation, augmenting Ca2+influx and SR Ca2+uptake, SR Ca2+overload and subsequent Ca2+release that triggers arrhythmic events through the leaky RYR2 receptors in CPVT1-MTs. Thus, dbcAMP most likely increases intracellular cAMP to much higher levels than typically achieved after p- adrenergic stimulation alone, which may be due to generally low basal receptor tone and low cAMP levels in hiPSC-CMs. Subsequent acute treatment with Flecainide rescued arrhythmic events in CPVT1-MTs, most likely following a dual mode of action: suppression of spontaneous SR Ca2+release events via RyR2 inhibition and suppression of triggered beats via Na+channel block. Flecainide has already shown promise in suppressing CPVT arrhythmias in patients not responsive to p-blockers (with the addition of Ca2+antagonists).

[0211] Of interest, the inventors made use of a multielectrode array (MEA) as an additional assay that can be used to detect arrhythmias in electrically active cells such as hiPSC-CMs. In this assay a non-arrhythmic signal is recognised by regular, rhythmic field potential peaks with consistent amplitudes and beat intervals (Fig. 17-left), while arrhythmic traces are recognised by irregular, erratic field potential peaks with inconsistent amplitudes and beat intervals (Fig. 17-middle). Tachycardia-like arrhythmias can also be detected with fibrillation-like arrhythmic contractions (Fig. 17-Right), shown by rapid uninterrupted beating and prolonged rest between phases.

[0212] Incubation of hiPSC-CMs with a number of different cAMP analogs (all 0.5 .M) for 48-72 hours and calcium-6 dye for a further 1 hour showed all analogs, except 8-CI-cAMP, produced 100% arrhythmias in the MEA assay (Fig. 18). With 8-CI-cAMP, 62.5% of the wells were arrhythmic. It is likely that altering the concentration of this compound could also result in 100% of wells showing arrhythmias. Interestingly, while dbcAMP alone could cause arrhythmias (18.8% of MEA recordings; Fig. 19), it appeared the combination of dbcAMP and calcium dye led to more consistent arrhythmic signals with incubation in either Calcium-6 or Fluo-4 dye resulting in 100% of wells becoming arrhythmic (Fig. 19). However, incubating the CPVT1-CMs with the calcium dye alone (-dbcAMP) was not sufficient to trigger arrhythmias (Fig. 19), mirroring what was observed in the optical Ca2+assay and confirming the presence of dbcAMP in combination with a dye facilitates reproducible induction of arrhythmias.

[0213] It is known that commonly used synthetic voltage and Ca2+dyes, as well as genetically-encoded voltage or calcium indicators, can alter the electrophysiological properties of cardiomyocytes (see Kopljar et al., 2018, Novakova et al., 2008; Yiangou et al., 2022 for examples). The combination of this, along with the high intracellular cAMP levels achieved by culturing the CMs with dbcAMP may place additional stress on the cells, thereby facilitating greater consistency for generating arrhythmias. As a final industrialization step, robotics were used for MT formation, maintenance, and assays with high accuracy and precision in pipetting (CV <3% and <5%, respectively). Each 384-well plate could be filled with functional MTs showing low variability within 2 minutes. Blind screens of inotropic test compounds using these plates showed high accuracy and reproducibility, demonstrating the benefits of: 1) reduced variation between individual MTs as well as between plates due to the use of robotics; 2) enhanced cardiomyocyte maturation in MTs; and 3) the reliability of MTs for drug screening where a high reproducibility is required. Crucially, for several drugs known to affect patients but misclassified in hiPSC-CM 2D models, such as Levosimendan, the inventors found accurate prediction in the MT model, likely due to the predominance of PDE4 over PDE3. Nevertheless, the SR calcium-handling properties of hiPSC-CMs may still be relatively immature in MTs, since CDN1163, a SERCA activator, was not detected as a positive inotrope. In addition, aspirin was misclassified as a positive inotrope based on the effect at the highest two concentrations (300 and 1000 pM) on the calcium peak amplitude and rising slope. However, these test concentrations fell outside of the commonly tested concentration range (0.1- 100 pM) in hiPSC-cardiac models. Within the commonly tested range the inventors did not observe an effect. Furthermore, direct contraction force can not be measured in MTs, which caused drugs like the myosin II inhibitor Blebbistatin to be missed. Of note, Blebbistatin can become inactive upon illumination with blue light, leading to loss of pharmacological activity. Thus, careful illumination control is necessary when using Blebbistatin in fluorescence-based assays.

[0214] As a proof-of-concept the inventors performed a larger screen of 92 FDA-approved drugs at 3 different concentrations (0.1 pM; 1 pM, 10 pM). These were selected based on their potential antiarrhythmic effects (e.g. ion channel blockers), lack of expected antiarrhythmic effects (e.g. aspirin), potential to exacerbate the arrhythmias (e.g. caffeine), or where the expected effect was unclear but based on their mode of action they could be antiarrhythmic (e.g. suramin). Of the 17 compounds that completely rescued the arrhythmogenic phenotype at one or more test concentrations, the majority (65% of hits) were ion channel blockers, most likely acting by reducing Ca2+overload in the arrhythmogenic hiPSC-CMs. Notably, 9 of the 17 compound hits had not previously been tested for treating CPVT 1 , highlighting the potential of this HTS assay in identifying novel therapeutic avenues. Among these was zaldaride, an antidiarrheal agent that inhibits calmodulin (Aikawa et al., 2000). Given that mutations in calmodulin genes are linked to arrhythmic disorders like CPVT1 through interactions between calmodulin, RyR2 and the L-type calcium channel (Wleklinski et al., 2020), the antiarrhythmic mechanism of zaldaride may involve inhibiting these interactions.

[0215] In conclusion, production and use of cardiac (disease) MTs, based on our method utilizing cryopreserved differentiated hiPSC derivatives, robotics and automation equipment hold great promise for implementation in HTS and potential identification of novel therapeutic targets for inherited cardiac arrhythmic diseases, like CPVT1. Even without HTS facilities, as in most academic laboratories, it was demonstrated that the outcomes of scaled out manual assays were similar to those that were fully automated. The principal differences were in reduced variance in data points in the HTS automated assay and the speed at which compound libraries could be screened.

[0216] REFERENCES

[0217] European Medicines Agency. ICH guideline E14 / S7B: clinical and Nonclinical Evaluation of QT / QTc Interval Prolongation and Proarrhythmic Potential-questions and answers. https: / / www.ema.europa.eu / en / documents / scientific-guideline / ich-guideline-e14 / s7b-clinical- nonclinical-evaluation-qt / qtc-interval-prolongation-proarrhythmic-potential-questions-answers- step-5_en.pdf (2022).

[0218] US Congress. FDA Modernisation Act 2.0. https: / / www.congress.gov / bill / 117th- congress / senate-bill / 5002 (2023).

[0219] Chen Y, Lun ATL and Smyth GK. From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline [version 2; peer review: 5 approved], F1000Research vol. 5, pp. 1438 (2016).

[0220] Campostrini, G. et al. Generation, functional analysis and applications of isogenic three- dimensional self-aggregating cardiac microtissues from human pluripotent stem cells. Nature Protocols vol. 16 pp. 2213-2256 (2021).

[0221] Novak, A., Barad, L., Lorber, A., Gherghiceanu, M. & Rappaport, B. Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations. J. Cell. Mol. Med. 19, 2006-2018 (2015).

[0222] Fatima, Azra, et al. In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells. Cellular Physiology and Biochemistry vol. 28.4 pp. 579-592 (2011). Giacomelli, E. et al. Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease. Cell Stem Cell 26, 862-879. e11 (2020).

[0223] Kujala, K. et al. Cell Model of Catecholaminergic Polymorphic Ventricular Tachycardia Reveals Early and Delayed Afterdepolarizations. PLoS One 7, e44660 (2012).

[0224] Patra C, et al., Biochemistry, cAMP. In: StatPearls. StatPearls Publishing, Treasure Island (FL); 2022.

[0225] Klarenbeek J, et al (2015) Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PLoS One 10:e0122513; Surdo N.C., et al. FRET biosensor uncovers cAMP nano-domains at beta-adrenergic targets that dictate precise tuning of cardiac contractility. Nat. Commun. 2017;8: 15031 . doi: 10.1038 / ncomms15031.

[0226] Aikawa N, Karasawa A, Ohmori K. Effect of zaldaride maleate, an antidiarrheal compound, on intracellular cyclic nucleotide-mediated intestinal ion secretion in rats. Eur J Pharmacol. 2000 Apr 14;394(2-3):233-6. doi: 10.1016 / s0014-2999(00)00148-5. PMID: 10771288.

[0227] Wleklinski MJ, Kannankeril PJ, Knollmann BC. Molecular and tissue mechanisms of catecholaminergic polymorphic ventricular tachycardia. J Physiol. 2020 Jul;598(14):2817-2834. doi: 10.1113 / JP276757. Epub 2020 Apr 27. PMID: 32115705; PMCID: PMC7699301.

[0228] Itzhaki, I. et al. Modeling of catecholaminergic polymorphic ventricular tachycardia with patientspecific human-induced pluripotent stem cells. Journal of the American College of Cardiology 60, 990-1000 (2012).

[0229] Maizels, L. et al. Patient-Specific Drug Screening Using a Human Induced Pluripotent Stem Cell Model of Catecholaminergic Polymorphic Ventricular Tachycardia Type 2. Circ.: Arrhythmia Electrophysiol. 10, e004725 (2017).

[0230] Kopljar et al., Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay; Journal of Pharmacological and Toxicological Methods Volume 91 , Pages 80-86, May-June 2018.

[0231] Novakova et al., Effects of voltage sensitive dye di-4-ANEPPS on guinea pig and rabbit myocardium. General Physiology and Biophysics, 01 Mar 2008, 27(1):45-54

[0232] Yiangou et al., Optogenetic Reporters Delivered as mRNA Facilitate Repeatable Action Potential and Calcium Handling Assessment in Human iPSC-Derived Cardiomyocytes. Stem Cells, Volume 40, Issue 7, Pages 655-668, July 2022.

Claims

Claims1. A method for producing an arrhythmia disease model, comprising: contacting cells with an exogenous agent under conditions that induce arrhythmia in the cells, wherein the exogenous agent penetrates the cell membrane and activates the intracellular cAMP signalling pathway.

2. An arrhythmia disease model system comprising:(i) an exogenous agent that penetrates the cell membrane and activates the intracellular cAMP signalling pathway; and(ii) cells having arrhythmia, wherein the cells have been contacted with the exogenous agent of (i) under conditions that induce arrhythmia.

3. The method of claim 1 or the system of claim 2, wherein the exogenous agent:(a) binds to protein kinase (PKA) and / or induces PKA activity in the cells; and / or(b) binds to Exchange proteins activate by cyclic AMP (EPAC) and / or induces EPAC activity in the cells.

4. The method or system according to any one of the preceding claims, wherein the exogenous agent is an agent selected from the group consisting of: db-cAMP, 8-Br-cAMP-AM, Sp-cAMPS, 8-CPT-cAMP, 2'-O-MS-cAMP, 8-CL-cAMP, 6-Bnz-cAMP, 8-pCPT-2-O-Me-cAMP- AM; and analogs and derivatives thereof.

5. The method or system according to any one of the preceding claims, wherein the arrhythmia disease is selected from the group consisting of: cardiac arrhythmia disease, a calcium handling disease, channelopathy, supraventricular arrhythmia disease, ventricular arrhythmia disease, structural heart disease, cardiomyopathy, atrial fibrillation, metabolic arrhythmia disease, mitochondrial arrhythmia disease, optionally wherein the channelopathy is long QT syndrome type 2 (LQT2) or catecholaminergic polymorphic ventricular tachycardia (CPVT), further optionally wherein the CPVT is CVPT1 or CVPT2.

6. The method or system according to any one of the preceding claims, wherein the cells carry one or more mutations associated with arrhythmia disease, optionally wherein the cells comprise one or more mutations in a gene selected from the group consisting of: ryanodine receptor 2 (RYR2), calsequestrin 2 (CASQ2), potassium voltage-gated channel subfamily H member 2 (KCNH2), potassium inwardly rectifying channel subfamily J member 2 (KCNJ2), Calcium Voltage-Gated Channel Subunit Alphal C (CACNA1C), Calcium Voltage-Gated Channel Auxiliary Subunit Beta 2 (CACNB2), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 1 (CACNA2D1), Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A),Calmodulin 1 (CALM1), Calmodulin 2 (CALM2), Calmodulin 3 (CALM3), Phospholamban (PLN), A-Kinase Anchoring Protein 9 (AKAP9), Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 1 (KCNE1), Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 2 (KCNE2), Myosin Binding Protein C3 (MYBPC3), Titin (TTN), Ankyrin 2 (ANK2), Lamin A / C (LMNA), Alanyl-TRNA Synthetase 2 (AARS2), and Plakophilin 2 (PKP2), further optionally wherein the one or more mutations is in human RYR2, CASQ2, KCNH2, KCNQ1, SCN5A, KCNJ2, CACNA1C, CALM1, CALM2, PKP2, AARS2, LMNA, TTN or MYBPC3, and the one or more mutations is selected from: a) M4109R-RYR2; b) F2483I - RYR2; c) P2328S - RYR2; d) S406L - RYR2; e) E2311D-RYR2; f) D307H - CASQ2; g) R420Q - RYR2; h) exon 3 del, P2328S, T2538R, L4115F, Q4201R and V4653F- RYR2; i) I4587V - RYR2; j) L3741P-RYR2; k) c.339-354 del (CASQ2G112+5X) - CASQ2; l) D3638A - RYR2; m) D358N and R4651I - RYR2; n) R4147S-RYR2; o) Y4962C-RYR2; p) G1681A- KCNH2; q) R176W-KCNH2; r) A561T-KCNH2; s) A561V-KCNH2; t) A561P-KCNH2; u) N996I - KCNH2; v) IVS9-28A / G - KCNH2 ; w) A422T-KCNH2; x) G603D-KCNH2; y) G604S - KCNH2; z) A614V-KCNH2; aa) G601S- KCNH2; bb) R534C- KCNH2; cc) N633S - KCNH2; dd) R685P- KCNH2;ee)V822M - KCNH2; ff) R366X-KCNH2; gg) S428X- KCNH2; hh)R752W - KCNH2; ii) T983I - KCNH2; jj) p.S1112Pfs*171 - KCNH2; kk) N588K- KCNH2;II) R594Q-KCNQ1; mm) 1795insD - SCN5A; nn) R535Q - SCN5A; oo) R218W-KCNJ2; pp) G406R-CACNA1C; qq) N639T-CACNA1C; rr) F142L-CALM1; ss) D130G-CALM2; tt) R1629X-SCN5A; uu) C.1643delG - PKP2; vv) K672Rfs - PKP2; ww) R958X - AARS2; xx) R592W-AARS2; yy) V730M - AARS2; zz) R216C-LMNA; aaa) K219T - LMNA; bbb) H222P- LMNA; ccc) R225X-LMNA; ddd) D461Y-LMNA; eee) W467X-LMNA; fff) R471H-LMNA; ggg) R482W-LMNA; hhh) Q493X-LMNA; iii) A22352fs - TTN; jjj) W792Vfs+40X - MYBPC3.

7. The method or system according to any one of the preceding claims, wherein the cells comprise electrically excitable cells, optionally wherein the electrically excitable cells comprise:(a) human pluripotent stem cell (hPSC) derivatives, optionally wherein the hPSC derivatives are cardiac cells, and / or(b) cardiac cells, optionally wherein the cardiac cells are hPSC-derived cardiomyocytes.

8. The method or system according to any one of the preceding claims, wherein the cells are in a 2D or 3D cell culture, or a mixture thereof.

9. The method or system according to any one of the preceding claims, wherein the exogenous agent is contacted with the cells for up to 7 days, optionally up to 72 hours, up to 48 hours, or up to 24 hours.

10. The method or system according to any one of the preceding claims, wherein the cells are provided in a cell culture vessel, optionally wherein the cell culture vessel is a multi-well cell culture plate, further optionally wherein the multi-well cell culture plate is at least a 96-well cell culture plate.

11. The method or system according to any one of the preceding claims, wherein the exogeneous agent is present at a concentration between 0.005 mM and 250 mM, optionally wherein the concentration is about 0.5 mM.

12. The method according to any one of the preceding claims, wherein the method further comprises a step of identifying cells having arrhythmia after the contacting step.

13. The method according to claim 12, wherein the step of identifying cells having arrhythmia comprises: a) labelling the cells with an arrhythmia detection reagent, optionally wherein the arrhythmia detection reagent is a chemical calcium dye, a chemical membrane potential dye, a genetically- encoded calcium indicator, or a genetically-encoded voltage indicator; and b) detecting the arrhythmia.

14. The method according to claim 13, further comprising: c) contacting the cells with a rescue compound; and d) detecting if there is a change in arrhythmia in the cells contacted with the rescue compound, wherein a reduction or an absence of arrhythmia is indicative of an effective arrhythmia model.

15. The method according to any one of the preceding claims, wherein the detecting the arrhythmia comprises detecting changes in intracellular calcium levels, electrical activity, and / or contraction activity of the cells, optionally wherein the detecting changes in intracellular calcium levels, electrical activity, and / or contraction activity of the cells comprises recording the changes.

16. A method of screening one or more test compounds for treating arrhythmia, said method comprising: a) contacting the one or more test compounds with an arrhythmia disease model produced by the method of any one of claims 1 , 3-15, or with an arrhythmia disease model system according to any one of claims 2-11 ; and b) determining the effect of the one or more test compounds on arrhythmia in the arrhythmia disease model or arrhythmia disease model system, wherein if the test compound reduced arrhythmia in the arrhythmia disease model or arrhythmia disease model system as compared to a control, it is indicative of the compound being suitable for treating arrhythmia.

17. The method according to claim 16, wherein determining the effect of the one or more test compounds comprises labelling the cells of the arrhythmia disease model or the arrhythmia disease model system with an arrhythmia detection reagent, optionally wherein the arrhythmia detection reagent is a chemical calcium dye, a chemical membrane potential dye, a genetically- encoded calcium indicator, or a genetically-encoded voltage indicator.

18. The method according to claim 16 or 17, wherein the method further comprises combining cells in suspension to form a 2D cell culture or a 3D cell culture.

19. The method according to any one of claims 16 to 18, wherein the method is an automated method and / or is a high throughput assay.

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