Modeling atrial fibrillation through intermittent tachypacing-induced electrical remodeling in matured cardiomyocytes

Abstract

Provided herein are compositions, systems, and methods employing in vitro models for atrial fibrillation and compositions and methods for generating the models. In some embodiments, the models are generated via intermittent tachypacing-induced electrical remodeling in human iPSC-derived matured atrial cardiomyocytes and atrial fibroblasts.

Description

[0001] UM-44787.601

[0002] MODELING ATRIAL FIBRILLATION THROUGH INTERMITTENT TACHYPACING-INDUCED ELECTRICAL REMODELING IN MATURED CARDIOMYOCYTES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 761,636, filed February 21, 2025, the disclosure of which is herein incorporated by reference in its entirety.

[0005] FIELD

[0006] Provided herein are compositions, systems, and methods employing in vitro models for atrial fibrillation and compositions and methods for generating the models. In some embodiments, the models are generated via intermittent tachypacing-induced electrical remodeling in human iPSC-derived matured atrial cardiomyocytes and atrial fibroblasts. BACKGROUND

[0007] Atrial fibrillation (AF) is a prevalent cardiac arrhythmia characterized by irregular heart rhythm and rapid atrial contractions. It arises from abnormal electrical activity within the atria, which disrupts normal cardiac conduction. AF is associated with a significantly increased risk of thromboembolic events, particularly stroke, as well as other cardiovascular complications (1-5). These health-related consequences contribute to substantial morbidity, mortality, and an elevated socioeconomic burden (6-8). The CAPTAF (Catheter Ablation Compared with Pharmacological Therapy for Atrial Fibrillation) clinical trial demonstrated that patients with symptomatic AF who were refractory to anti arrhythmic drug therapy experienced significant improvements in quality of life 12 months after undergoing catheter ablation (9). Similarly, the CABANA (Catheter Ablation Versus Anti arrhythmic Drug Therapy for Atrial Fibrillation) trial found that catheter ablation reduced the recurrence of symptomatic AF by 51% compared to anti arrhythmic drug therapy, in addition to decreasing the overall AF burden in treated patients (10). Furthermore, early rhythm control has been shown to improve clinical outcomes in AF patients, although individuals with newly diagnosed AF are at a heightened risk of hospitalization (11).

[0008] Despite these positive findings, recurrence of AF after catheter ablation remains a major concern. Factors contributing to recurrence include recovery of ablated tissue, theUM-44787.601

[0009] presence of non-pulmonary vein triggers, and substrate remodeling (2,12). These challenges underscore the need for more effective and sustained therapeutic approaches for managing AF.

[0010] Translating mechanistic insights gained from basic AF research into clinical applications remains difficult. Key barriers include the high costs associated with drug development, regulatory hurdles in the approval process, and the frequent failure of clinical trials. Additionally, challenges in extrapolating findings from animal models to humans, compounded by limitations in current model organisms and experimental methods that do not fully capture the complexities of human AF mechanisms, further complicate the translation of scientific discoveries into viable clinical treatments (13).

[0011] Human pluripotent stem cells (hPSCs) have emerged as a valuable tool for personalized medicine and drug discovery (14-19). Somatic cells obtained from patients can be reprogrammed into human induced pluripotent stem cells (hiPSCs), which can then be differentiated into various cell types, including cardiac cells. These iPSC-derived cells closely mimic the physiological characteristics of native cells and offer advantages over primary human cells, such as the ability to be cultured in larger quantities and maintained for extended periods. Recent advancements in technology and refined protocols have permitted the generation of hiPSC-derived cardiomyocytes (hiPSC-CMs) and their directed differentiation into chamber-specific cardiac cell types, providing a model for studying cardiac development and disease (20). Genetic engineering approaches in hiPSCs have facilitated the study of cardiac genetic mutations and their underlying mechanisms in vitro, positioning hiPSCs as a valuable human model system for studying cardiac genetics and diseases (21-25).

[0012] Recent studies have utilized hiPSC-CMs to model AF. One study employed optogenetic intermittent tachypacing in engineered heart tissue over a period of 3 weeks and observed limited electrical remodeling associated with AF (26). Another study using atrial engineered human myocardium (aEHM) and iPSC-atrial CMs subjected to 24 hours of pacing revealed AF remodeling characteristics (27). Despite demonstrating certain remodeling effects under both acute and chronic pacing regimens, these studies did not provide models that fully recapitulate the complex electrophysiological properties observed in mature atrial tissue during AF. Improved models are needed.UM-44787.601

[0013] SUMMARY

[0014] Provided herein are compositions, systems, and methods employing in vitro models for atrial fibrillation and compositions and methods for generating the models. In some embodiments, the models are generated via intermittent tachypacing-induced electrical remodeling in human iPSC-derived matured atrial cardiomyocytes and atrial fibroblasts.

[0015] For example, in some embodiments, methods are provided for generating such in vitro models (e.g., in vitro atrial fibrillation (AF) cell culture systems). In some embodiments, the methods comprise: a) co-culturing human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) and human atrial cardiac fibroblast (haCFs); and b) remodeling co-cultured cells using an intermittent tachypacing protocol (ITPP). In some embodiments, the remodeled cells have one or more (e.g., one, two, three, four) or each of the characteristics of AF cardiomyocytes relative to non-AF cardiomyocytes selected from the group consisting of: reduced action potential (AP) frequency, prolonged action potential duration (APD), changes in calcium handling (e.g., prolonged calcium transient duration 80% (CaTD80%), calcium upstroke slope, and / or amplitude of calcium release), upregulation of collagen III and TGFβ1, and increase in frequency of spontaneous activations.

[0016] In some embodiments, the co-culturing comprises generating a hiPSC-aCM / haCF ratio of 50 / 50 to 80 / 20 or approximately 50 / 50 to approximately 80 / 20. In some embodiments, the ratio is 70 / 30 or approximately 70 / 30. As used herein, “approximately,” in reference to percentages includes values + / - 5%.

[0017] In some embodiments, the haCFs are derived from stem cells (e.g., induced pluripotent stem cells). In some embodiments, the haCFs are obtained from a human donor. In some embodiments, the hiPSC-aCMs and haCFs are obtained from or derived from a single individual.

[0018] In some embodiments, the hiPSC-aCMs and / or haCFs are modified to express a heterologous factor. In some embodiments, the heterologous factor is a genetically encoded indicator. In some embodiments, the genetically encoded indicator is a genetically encoded calcium indicator or voltage indicator.

[0019] In some embodiments, the ITPP comprises subjecting a cultured monolayer of the cocultured cells to chronic electrical field pacing. In some embodiments, the chronic electrical field pacing comprises applying an electrical field stimulation at first frequency cycles for aUM-44787.601

[0020] first time period followed by second frequency cycles for a second time period, repeated at a time frequency for a duration. In some embodiments, the electrical field stimulation comprises 20 V / cm with 0.5 ms pulse duration, the first frequency cycles are at 1.5 Hz, the first time period is 30-60 minutes (e.g., 45 minutes), the second frequency cycles are at 3.5-5 Hz (e.g., 4 Hz, 5 Hz), the second time period is 10-30 minutes (e.g., 15 minutes), the time frequency is hourly, and / or the duration is 5-10 days (e.g., 7 days).

[0021] In some embodiments, prior to the co-culturing, the cardiomyocytes (hiPSC-aCMs) and / or human atrial cardiac fibroblast (haCFs) are purified, for example, to reduce cellular heterogeneity (e.g., to enrich the percentage of cells that are the desired cells).

[0022] Also provided herein are in vitro atrial fibrillation (AF) cell culture systems generated by any of the above methods or methods described elsewhere herein.

[0023] In some embodiments, provided herein are in vitro atrial fibrillation (AF) cell culture systems comprising: a co-culture of human -induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) and human atrial cardiac fibroblast (haCFs). In some embodiments, the system comprises cells having one or more (e.g., one, two, three, four) or each of the characteristics of AF cardiomyocytes relative to non-AF cardiomyocytes selected from the group consisting of reduced action potential (AP) frequency, prolonged action potential duration (APD), changes in calcium handling (e.g., prolonged calcium transient duration 80% (CaTD80%), calcium upstroke slope, amplitude of calcium release), upregulation of collagen III and TGFβ1, and increase in frequency of spontaneous activations.

[0024] In some embodiments, the co-culture comprises a hiPSC-aCM / haCF ratio of 50 / 50 to 80 / 20 or approximately 50 / 50 to approximately 80 / 20. In some embodiments, the ratio is 70 / 30 or approximately 70 / 30.

[0025] In some embodiments, the haCFs are derived from stem cells (e.g., induced pluripotent stem cells). In some embodiments, the haCFs are obtained from a human donor. In some embodiments, the hiPSC-aCMs and haCFs are obtained from or derived from a single individual.

[0026] In some embodiments, the hiPSC-aCMs and / or haCFs are modified to express a heterologous factor. In some embodiments, the heterologous factor is a genetically encodedUM-44787.601

[0027] indicator. In some embodiments, the genetically encoded indicator is a genetically encoded calcium indicator or voltage indicator.

[0028] In some embodiments, the system comprises a culture medium. In some embodiments, the culture medium comprises is test compound (e.g., a drug). In some embodiments, the system comprises a culture surface (e.g., a surface of a culture dish or one or more wells of a multi-well plate) upon which a monolayer of the co-culture is attached. In some embodiments, the system further comprises an electrode in contact with the monolayer. In some embodiments, the system further comprises an instrument that measures one or more electrical, chemical, or optical properties of the co-culture.

[0029] Also provided herein are methods of using the in vitro systems. In some embodiments, the method comprises determining a change in the co-culture in response to a stimulus (e.g., a change in an AF-related characteristic in response to a stimulus). In some embodiments, the stimulus comprises administration of a test compound (e.g., a drug).

[0030] Definitions

[0031] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0032] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the disclosure.

[0033] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.” As used herein, the term “human induced pluripotent stem cell” (hiPSC) refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adultUM-44787.601

[0034] somatic cell, via inducing the expression of certain de-differentiation genes in the non-pluripotent cell, e.g., Oct3 / 4, Sox2, Klf4 and / or c-Myc genes.

[0035] As used herein, “immature cardiomyocytes” refers to a cardiomyocyte derived from pluripotent embryonic stem cells (PESC) and / or induced pluripotent stem cells (iPSC) in in vitro culture, which do not possess the properties of an adult or adult-like cardiomyocyte.

[0036] As used herein, the term “test compound” refers to a chemical to be tested by one or more screening method(s). A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof. Usually, various predetermined concentrations of test compounds are used for screening, such as 0.01 micromolar, 1 micromolar, and 10 micromolar. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound having a known effect.

[0037] The term “subject” as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of cell isolation, treatment, observation, or experiment.

[0038] Description of Figures

[0039] FIG. 1. Functional electrical remodeling of hiPSC-ACM syncytia submitted to intermittent tachypacing. (A) Time-sequence plot of representative action potential traces obtained from optical mapping of control hiPSC-aCM syncytia (Control) and hiPSC-aCM syncytia subjected to ITPP for 7 days (ITPP) are shown. Voltage recordings of action potentials were obtained using FluoVolt. Following 7 days of ITPP, paced hiPSC-aCM syncytia exhibited an increased frequency of spontaneous APs relative to control (A = +31±6%, p<0.0001) (B) alongside reductions in Fridericia-corrected AP duration (80%) (A = -15±3%, p=0.0012) (C) and AP triangulation (A = -13±3%, p=0.0104) (D), with preserved upstroke slope (E) and significantly faster conduction velocity (A = +40±6%, p=0.0002) (F). Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed using Kolmogorov- Smirnov test for normality and subsequent unpaired parametric t-tests for all panels (B-F). (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, ITPP = intermittent tachypacing protocol, AP = action potential).

[0040] FIG. 2. Intracellular calcium handling alterations of hiPSC-aCM syncytia submitted to intermittent tachypacing. (A) Representative time-sequence plot of calcium transientsUM-44787.601

[0041] obtained from optical mapping of control hiPSC-aCM syncytia (Control) and hiPSC-aCM syncytia subjected to ITPP for 7 days (ITPP) is shown. ITPP markedly increased the variability of calcium release events (A = +332±88%, p=0.0006) (C) without significantly altering the frequency of spontaneous calcium release (B). Similarly, the beat-to-beat variability in calcium transient duration increased following ITPP (A = +83±8%, p<0.0001) (E) despite no significant change in CaTD80%(D). ITPP decreased both the upstroke slope (A = -41±7%, p=0.0012) (F) and amplitude of intracellular calcium release (A = -51±4%, p=0.0005) (G); furthermore, the propagation speed of the resulting calcium wave was significantly slowed (A = -81±1%, p<0.0001) (H). The speed of SR calcium reuptake was not affected by ITPP (I), however it became significantly more variable between transients (A = +103±3%, p<0.0001) (J). The diastolic calcium level following the CaT remained unchanged following ITPP (K). Statistical analysis was performed using Kolmogorov-Smirnov test for normality and subsequent unpaired parametric t-test (B, D-K) or Mann-Whitney test (C). (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, ITPP = intermittent tachypacing protocol, CaT = calcium transient, CaTD80%= calcium transient duration 80%, SR = sarcoplasmic reticulum).

[0042] FIG 3. Arrhythmia inducibility test reveals calcium overload susceptibility in hiPSC-aCM syncytia following intermittent tachypacing. (A) Representative traces of AIT stimulation in Control and ITPP hiPSC-aCM syncytia demonstrate transition between AIT pacing (3 Hz) and rest. The post-rest potentiation calcium transient (PRP-CaT) marking the transition from pacing to rest is labeled with a blue arrow; example of a premature calcium release event is labeled with a red arrow. Diastolic calcium levels during AIT pacing (FopPaci.ng;’ red line)7and rest (vF 0„,pRest’ blue line)7are indicated; ’ (vB)7ITPP -treated hiPSC-aCM syncytia exhibited reduced recovery of baseline calcium levels after AIT cessation (A = -50±12%, p=0.0352). The PRP-CaT, marking calcium flux during transition from AIT pacing to rest, presented decreased upstroke slope of intracellular calcium release (A = -65±5%; p=0.0004) (C) and decreased intracellular calcium release amplitude (A = -67±5%; p=0.0008) (D) in iPSC-aCM syncytia treated with ITPP. Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed by Kolmogorov-Smirnov Test for normality followed by Mann-Whitney Test (B, C, D). (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, AIT = arrhythmia inducibility test, ITPP = intermittent tachypacing protocol).UM-44787.601

[0043] FIG. 4. Stable co-culture of hiPSC-aCMs and haCFs induces atrial fibrosis markers associated with structural remodeling AF. (A) Representative micrographs of cTnT / TE7 immunostaining in control (100 / 0) and co-culture (90 / 10, 70 / 30) preparations. (B) Quantification of cTnT and TE7 fluorescence area proportional to total cell area confirms the maintenance of co-culture ratio after 7 days, with cTnT: TE7 fluorescence proportions closely approximating initial seeding ratios of 100 / 0 (0.96±0.03 to 0.01±0.001), 90 / 10 (0.84±0.03 to 0.15±0.01) and 70 / 30 (0.72±0.03 to 0.34±0.03) preparations. (C, E) Representative micrograph panels of immunostaining for Collagen III (C) and TGFβ1 (E). (D, F) Quantification shows a proportional increase in fibrosis markers with co-culture ratio: 90 / 10 preparations expressed a ~36-fold (p=0.0008) increase in Collagen III (D) and ~ 1.3-fold (p=0.0364) increase in TGFβ1 (F), while 70 / 30 preparations expressed a ~103-fold (p<0.0001) increase in Collagen III (D) and ~1.6-fold (p=0.0016) increase in TGFβ1 (F). Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed using One-Way ANOVA with Bonferroni correction. (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, haCF = human atrial Cardiac Fibroblast).

[0044] FIG. 5. Fibrosis alters electrophysiology of hiPSC-aCM preparations. (A) Representative time-sequence plots of action potential traces summarizing electrophysiology characteristics of control (100 / 0) and hiPSC-aCM / haCF co-culture preparations at 90 / 10 and 70 / 30 ratios. Optical mapping of action potentials were performed using FluoVolt. (B) Spontaneous activation frequency decreased with increasing fibroblast proportion (90 / 10: A = -16±6%, p=0.0184; 70 / 30: A = -61±1%, p<0.0001). (C-F) Co-culture with 30% haCFs further achieved prolongation of the Fridericia-corrected AP duration (80%) (70 / 30: A = +130%±l%, p<0.0001) (C), prolongation of the Fridericia-corrected AP triangulation (70 / 30: A = +143±9%, pO. OOOl) (D), and decrease in the upstroke slope of the AP (70 / 30: A = -66±2%, p<0.0001) (E). Notably, coculture at 30% achieved significant slowing of the conduction velocity of the AP (70 / 30: A = -52±6%, p=0.0260). Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed using One-Way ANOVA with Bonferroni correction. (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, haCF = human atrial cardiac fibroblast, AP = action potential).

[0045] FIG. 6. Fibrosis disrupts intracellular calcium handling of hiPSC-aCM preparations. (A) Representative calcium transients assessed by optical mapping indicate that hiPSC-UM-44787.601

[0046] aCM / haCF co-culture at a 70 / 30 ratio alters intracellular calcium handling. (B, C) Co-culture at 70 / 30 ratio significantly increased calcium release event variability (70:30: A = +169±44%, p=0.0039) (C), while spontaneous calcium release frequency remained unchanged (B). Calcium transient duration (80%) was significantly prolonged by 70 / 30 coculture (70 / 30: A = +53±9%, p=0.0002) (D). Co-culture with 30% haCFs also prolonged the SR reuptake of calcium (70 / 30: A = +39±9%, p=0.0022) (E) while also reducing the upstroke slope (70:30: A = -50±3%, pO. OOOl) (F) and amplitude (70 / 30: A = -34±6%, p=0.0003) (G) of intracellular calcium release without change to diastolic calcium levels (H). Furthermore, the intracellular propagation of the calcium wave was decreased by 70 / 30 co-culture (70 / 30: A = -66±5%, p<0.0001) (I). Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed using One-Way ANOVA with Bonferroni correction. (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, haCF = human atrial cardiac fibroblast, SR = sarcoplasmic reticulum).

[0047] FIG. 7. Fibrosis introduces a conduction block to intermittent tachypacing of hiPSC-aCMs. (A-C) Representative time-sequence plots of action potentials from control (100 / 0) hiPSC-aCM preparations (A), and ITPP -treated (7 days) 100 / 0 (B) or 70 / 30 hiPSC-aCM / haCF co-culture preparations(C) (F-ITPP). Both 100 / 0 and 70 / 30 preparations responded to ITPP with increased frequency of spontaneous activations (F-ITPP 100 / 0: A = +36±7%, p=0.0011; F-ITPP 70 / 30: A = +25±7%, p=0.0217) and shortened action potential duration (80%) (F-ITPP 100 / 0: A = -13±3%, p=0.0389; F-ITPP 70 / 30: A = -16±3%, p=0.0122); further, the addition of fibroblasts did not significantly alter the magnitude of increase in spontaneous beat frequency (E) or shortening of the APD80% (G). Unlike F-ITPP 100 / 0 preparations, which exhibited an increase to conduction velocity post-7 days ITPP (F-ITPP 100 / 0: A = +45±9%, p=0.0089) (H), F-ITPP 70 / 30 preparations did not demonstrate increased conduction velocity; rather, the addition of 30% fibroblasts notably caused a decrease in conduction velocity of the action potential (F-ITPP 70 / 30: A = -47±9%) (I). Bar graphs depict mean ± standard deviation, with individual data points shown. Statistical analysis was performed using One-Way ANOVA with Bonferroni correction (D, F, H) and by Kolmogorov-Smirnov Test for normality followed by either Unpaired t-Test (E, I) or Mann-Whitney Test (G). (hiPSC-aCM = human induced pluripotent stem cell-derived atrial cardiomyocyte, haCF = human atrial cardiac fibroblast, ITPP = intermittent tachypacing protocol, F-ITPP = fibrosis ITPP, APD80% = action potential duration (80%)).UM-44787.601

[0048] FIG. 8. Molecular remodeling under intermittent tachypacing with fibrosis. (A) Representative western blot results for phospholamban (PLN) phospho-phospholamban (Serine-16 and Threonine-17; pPLN), and SERCA2a in control (100 / 0) and fibrosis (70 / 30) samples subjected to ITPP. Quantification of western blot results demonstrates significant downregulation of PLN across all 70 / 30 samples and 100 / 0 samples subjected to ITPP (100 / 0 ITPP: A=-57.88±8.53%, p=0.0008; 70 / 30 Control: A=-35.58±8.79%, p=0.0356; 70 / 30 ITPP: A=-59.29±l 1.08%, p=0.0006) (B), without significant change to pPLN (C) and SERCA2a (D) expression, or the ratio of inactivated PLN (pPLN / PLN) (E). However, the summative ratio of inactivated PLN to SERCA2a was significantly increased when ITPP was applied to 70 / 30 fibrosis co-cultures (70 / 30 ITPP: A=+300.8±53.46%, p=0.0022) (F). Quantitative PCR (qPCR) analysis of calcium-handling and atrial fibrillation (AF)-related genes in hiPSC-aCM monocultures (100 / 0) and fibroblast co-cultures (70 / 30) under control and intermittent tachypacing (ITPP) conditions. Expression levels of RYR2 (ryanodine receptor 2), ATP2A2 (SERCA2a), PLN (phospholamban), NPPA (atrial natriuretic peptide A), NPPB (atrial natriuretic peptide B), and TGFB1 (transforming growth factor β1) were normalized to housekeeping gene expression and expressed as fold change relative to control. In the 70 / 30 ITPP group, PLN, NPPA, NPPB, and TGFB1 transcripts were significantly upregulated compared with their respective controls, whereas RYR2 and ATP2A2 remained unchanged.

[0049] DETAILED DESCRIPTION

[0050] Provided herein are compositions, systems, and methods employing in vitro models for atrial fibrillation and compositions and methods for generating the models. In some embodiments, the models are generated via intermittent tachypacing-induced electrical remodeling in human iPSC-derived matured atrial cardiomyocytes and atrial fibroblasts. For example, in some embodiments, the technology uses an intermittent tachypacing protocol (ITPP) in matured hiPSC-aCMs co-cultured with human atrial cardiac fibroblast (haCF) to induce and recapitulate AF-associated characteristics. The technology provides in vitro models, and uses thereof, having unprecedented features and properties that allow more accurate atrial fibrillation-related therapeutic target validation, drug repurposing, drug safety testing, therapy selection, and other methodologies.

[0051] hiPSC-CMs represent a powerful model for investigating patient-specific genetic variations implicated in various cardiovascular diseases. Numerous studies have employed hiPSC-CMs to explore the molecular mechanisms underlying AF. Notable examples includeUM-44787.601

[0052] investigations of the PITX2 (rs138163892) gain-of-function mutation (37), the TTN-T32756I variant in the titin gene (38), the Pkdl R3227C mutation in polycystin-1 (39), the RyR2 c.14638G > A (p. V4880I) variant in the cardiac ryanodine receptor (40), lamin A / C (LMNA) gene shift mutation (c,1304_1307dup) (41), and mutations in the SCN5A gene (E428K and N470K) (42) associated with the cardiac sodium channel. However, to date, hiPSC-CMs have not provided a sufficiently accurate AF model.

[0053] Electrical pacing of hiPSC-CMs has been employed to improve the cardiomyocyte maturity and to induce cardiac arrhythmias. Acute and chronic effect of electric pacing was tested at 60 beats / min for 24 hours and 7 days respectively. Although there was no difference observed between the pacing regimens in relation to cell size, reactive oxygen species or apoptosis, the Ca2+transient amplitude and the upstroke velocity improved in 7 days pacing with a decrease in late Na+current (43). Overexpression of the KCNJ2 gene that encodes the inwardly rectifying potassium channel that is low in expression in hiPSC-CMs was shown to enhance its maturation by hyperpolarizing the maximal diastolic potential (MDP), increasing upstroke velocity and shortening APD. It also led to enlarged cell size with elongated phenotype (44). In a self-organizing 3-dimensional tissue ring hiPSC-CM model, rapid pacing up to 4 Hz for 2 weeks induced re-entrant waves that resulted in improved contractility, structural and Ca2+handling maturation (45). In a previous study, optogenetic tachypacing of atrial hiPSC-CMs cultured into engineered heart tissue was employed. The study observed that intermittent tachypacing led to certain electrical remodeling characteristics, including an increase in action potential amplitude and a shift in the spontaneous beating pattern. However, no significant shortening of APD was observed, a phenomenon commonly associated with AF remodeling (26).

[0054] The technology provided herein provides an improved human in vitro model for AF using hiPSC-CMs by, for example, addressing challenges related to cellular maturation and chamber-specific purity observed in previous work. These improved models facilitate a deeper understanding of the pathophysiology of AF and allow for more effective evaluation and selection of drugs and other therapeutic interventions.

[0055] In some embodiments, high performance in vitro models for AF are generating through a combination of two or more, three or more, or each of: 1) ensuring proper maturation of stem cells; 2) ensuring sufficient homogeneity of matured cells; 3) co-culturing human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) withUM-44787.601

[0056] human atrial cardiac fibroblasts (haCFs) (which may be obtained from donor tissue, generated in vitro from stem cells (e.g., iPSCs), or from any other source); and 4) remodeling cells using a pacing protocol.

[0057] Cultured cells

[0058] In some embodiments, the in vitro models employ matured human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) co-cultured with human atrial cardiac fibroblasts (haCFs). In some embodiments, hiPSC-aCM are generated from human iPSC cell lines. iPSC cell lines may be obtained from any suitable source including, but not limited to, WiCell (DF19-9-11T human iPSC line), ATCC, STEMCELL Technologies, The Jackson Laboratory, Carles River Laboratories, or other vendors or sources. In some embodiments, the hiPSC-aCM are generation from human iPSC cells derived from an individual (e.g., a patient). In some embodiments, the haCFs are generated from iPSC cells or other stem cells. In some embodiments, the haCFs are obtained from donors. In some embodiments, the haCFs are obtained from an individual (e.g., a patient). In some such embodiments, the hiPSC-aCMs and haCFs are generated from and / or obtained from the same individual (e.g., patient).

[0059] In some embodiments, culture conditions are selected to promote highly matured cells. For example, in some embodiments, hiPSCs are treated to promote the development of highly organized sarcomeres, elevated levels of cardiac troponin I (cTnl), enhanced mitochondrial distribution, and / or improved electrophysiological function, thereby approximating characteristics of mature CMs. In some embodiments, maturation achieves chamber-specific cardiomyocyte differentiation, for example, generating atrial, ventricular, and / or pacemaker cells that are distinct in function and structure.

[0060] In some embodiments, the in vitro models comprise a co-culture of hiPSC-aCMs and haCFs. In some embodiments, the ratio of hiPSC to aCM in the co-culture is 70 / 30 or approximately 70 / 30. In some embodiments, the ratio is, or is approximately, 50 / 50 to 80 / 20 (e.g., 50 / 50, 51 / 49, 52 / 48, 53 / 47, 54 / 46, 55 / 45, 56 / 44, 57 / 43, 58 / 42, 59 / 41, 60 / 40, 61 / 39, 62 / 38, 63 / 37, 64 / 36, 65 / 35, 66 / 34, 67 / 33, 68 / 32, 69 / 31, 70 / 30, 71 / 29, 72 / 28, 73 / 27, 74 / 26, 75 / 25, 76 / 24, 77 / 23, 78 / 22, 79 / 21, or 80 / 20).

[0061] The 70 / 30 ratio of hiPSC-aCMs to haCFs exhibited the most pronounced fibroblast-induced changes, including a significant upregulation of collagen III and TGFβ1, both of which are known to be involved in fibrosis and the proarrhythmic substrate in AF. TheUM-44787.601

[0062] electrophysiological remodeling observed in these co-cultures were reduced AP frequency and prolonged APD, which may be due to the altered ion channel expression and delayed repolarization kinetics due to fibrosis. Further, the slowed conduction velocity is consistent with the fibrotic remodeling seen in AF, where increased extracellular matrix deposition can impair electrical conduction and lead to reentrant circuits. Additionally, the changes in calcium handling, including prolonged CaTD80%, reduced calcium upstroke slope and amplitude of calcium release, indicate that fibroblast-induced alterations may contribute to the arrhythmogenic substrate in AF.

[0063] The co-culture experiments also revealed differences between the 70 / 30 and 90 / 10 hiPSC-aCM / haCF ratios. In the 90 / 10 ratio, a modest decrease in diastolic calcium levels was observed, indicating that a lower fibroblast content may have subtler effects on calcium handling. In contrast, the 70 / 30 ratio co-cultures exhibited more pronounced alterations in calcium homeostasis.

[0064] When examining the combined effects of ITPP and fibroblast co-culture, it was found that the 70 / 30 ratio of hiPSC-aCMs and haCFs exhibited a marked increase in the frequency of spontaneous activations and a shortening of APD. Also, a significant decrease in conduction velocity was observed, indicating that fibroblast presence exacerbates the conduction block and electrical instability induced by tachypacing. The lack of significant changes in AP frequency and APD between the F-ITPP 70 / 30 and F-ITPP 100 / 0 co-culture conditions supports that fibroblasts primarily contribute to the observed electrophysiological changes through modulation of conduction properties, rather than excitability or repolarization.

[0065] In some embodiments, one or more of the cells may be genetically modified. In some embodiments, cells are modified prior to maturation. In some embodiments, cells are modified during or after maturation. In some embodiments, modification involves incorporation of a heterologous sequence into a genome of the cell. In some embodiments, modification comprises episomal expression of a genetic element. Genetic modification can be carried out using any suitable methodology, including but not limited to, transfection with a viral vector (e.g., adenovirus, AAV, lentivirus, retrovirus, etc.) or plasmid and gene editing (e.g., CRISPR).

[0066] In some embodiments, hiPSC and / or aCMs are modified to comprise a genetically encoded calcium indicator (GECI) (e.g., GCaMP6 fast variant). In some embodiments,UM-44787.601

[0067] hiPSC and / or aCMs are modified to comprise a genetically encoded voltage indicator (GEVI) (e.g., ArcLight, ASAP1, ASAP3, Archons, SomArchon, Ace2N-mNeon, etc.). In some embodiments, genetic changes are made to determine the impact of the genetic change on AF or therapeutic response.

[0068] Genetic modification that further recapitulate aspects of AF may also be employed. For example, mutation in the ryanodine receptor 2 (RyR2), which is responsible for calcium release from the SR are associated with AF (49,50). In a mouse model subjected to rapid pacing, increased phosphorylation of RyR2 by calmodulin kinase II (CaMKII) was observed, mirroring findings in patients with AF (51). Moreover, inhibition of RyR2 in Ryr2S2814A mice reduced susceptibility to AF (52). Factors associated with calcium handling, such as SR Ca2+-ATPase (SERCA) pump and the sodium-calcium exchanger (NCX), both of which play pivotal roles in calcium regulation during AF (53,54), may be modified (e.g., mutated, overexpressed, under-expressed, etc.). Upon application of AIT, ITPP -treated cells exhibited a reduced calcium upstroke slope and impaired reconstitution of diastolic calcium. These findings further emphasize the compromised calcium handling observed in these cells, which could contribute to conditions that predispose cells to calcium overload.

[0069] In some embodiments, cells are treated to maximize cell homogeneity. In some embodiments, homogeneity is improved by selecting culturing conditions that reduce heterogeneity. For example, in some embodiments a retinoic acid (RA)-based atrial-specific monolayer differentiation protocol (33) is employed. In some embodiments, one or more purification steps are employed that enrich for desired cells. Purification can occur at one or more steps during the maturation process.

[0070] In addition to the characteristics identified above, cells may be evaluated (e.g., for maturity, response to stimuli, health, etc.) based on the measurement of one or more biochemical biomarkers, including, but not limited to gene expression, protein localization, indicators of metabolic activity, indicators of live / dead status, and secreted factors.

[0071] Intermittent Tachypacing-Induced Electrical Remodeling

[0072] In experiments conducted during the development of embodiments of the technology, ITPP treatment led to a significant increase in spontaneous AP frequency and a shortening of APD in hiPSC-aCM syncytia, which has been reported as a hallmark of electrical remodeling in AF (47). hiPSC-aCMs were cultured alone or co-cultured with haCFs. The ITPP was applied by field stimulation and the optical mapping was used to assess properties of actionUM-44787.601

[0073] potentials (APs) and calcium transients (CaTs). Immunostaining was performed to quantify the pro-fibrotic markers expression (Collagen III and, TGFβ1). In addition to the observed electrophysiological changes, significant alterations in calcium handling were identified in ITPP -treated cells. These included an increased variability in spontaneous calcium release events, reduced calcium release amplitude, and slower calcium wave propagation, all of which indicate disrupted intracellular calcium homeostasis. The slower calcium upstroke slope further demonstrates impaired calcium release from the SR. It is known that delayed afterdepolarizations (DADs) arise from spontaneous calcium release from SR that can lead to premature action potentials hence contributing as a trigger for the onset of AF (48), which is replicated in experiments of embodiments of the in vitro model.

[0074] ITPP led to a significant increase in the frequency of spontaneous APs (A = +31±6%, p<0.0001) and a reduction in AP duration 80% repolarization (APD80%) (A = -15±3%, p=0.0012). The upstroke slope (A = -41±7%, p=0.0012) and amplitude (dF / FO) (A = -51±4%, p=0.0005) of the intracellular CaT were significantly diminished under ITPP. Co-culture promoted structural remodeling at the 70 / 30 hiPSC-aCM / haCF ratio, including increased expression of Collagen III (>100-fold, p<0.0001). Electrophysiological changes associated with atrial fibrosis were observed, including diminished excitability (ΔHz=-61±1%, pO. OOOl; AAPD80%=+130%±l%, pO. OOOl), prolonged AP triangulation (AAPDTri=+143±9%, pO. OOOl), reduced upstroke slope (A =-66±2%, pO. OOOl), conduction block (A=-52±6%, p=0.0260), and diminished intracellular calcium handling (upstroke slope A =-50±3%, pO. OOOl; AdF / F0=-34±6%, p=0.0003). Finally, the application of ITPP to the 70 / 30 co-culture model recapitulated a tachycardic-like phenotype (AHz, p=0.0217; AAPD80%, p=0.0122 ) while introducing condition block (ACV100 / 0 vs 70 / 30= -47±9%; p=0.0005).

[0075] Electrical remodeling by ITPP altered the excitability and calcium handling properties of matured hiPSC-aCM monolayers. Structural and electrophysiological remodeling were observed in hiPSC-aCM-haCF co-cultures including conduction abnormalities. Thus, these models recapitulate key mechanisms of AF and their utility in, for example, the development of patient-specific therapies and drug discovery.

[0076] In some embodiments, cultured monolayers (e.g., comprising iACM and iACM-hACF co-cultures) are subjected to chronic electrical field pacing. In some embodiments, the intermittent tachypacing protocol applies an electrical field stimulation (e.g., at 20 V / cm withUM-44787.601

[0077] 0.5 ms pulse duration) at first frequency cycles (e.g., at 1.5 Hz) for a first time period (e.g., for 30-60 minutes; e.g., 45 minutes) followed by second frequency cycles (e.g., 3.5 to 5 Hz) for a second time period (e.g., 10-30 minutes; e.g., 15 minutes), repeated at a time frequency (e.g., 40 minutes to 1.5 hours; e.g., hourly) for a duration (e.g., 5-10 days; e.g., 7 days). Uses

[0078] Overall, ITPP, fibroblast co-culture in hiPSC-aCMs, and the combination of both, provides a robust in vitro model to study and assess the electrical and calcium-handling alterations that underlie AF. The insights gained from this technology find use to elucidate the AF mechanisms, offering potential targets for therapeutic intervention. This model can be further utilized in frameworks such as the Comprehensive In Vitro Proarrhythmia Assay (CiPA) and the Japan iPS Cardiac Safety Assessment (JiCSA) to enhance the specificity of proarrhythmic risk assessment during the screening of AF drugs (56,57). The in vitro models find use for target validation, drug repurposing efforts, safety testing of drugs and other interventions, and therapy selection. In some embodiments, the technology finds use in personal personalized medicine or precision medicine settings. For example, in some embodiments, cultured cells are derived from an individual and testing conducted on the cells provide information relevant to that specific individual, including but not limited to, therapy efficacy testing, therapy selection, therapy monitoring, and modulation of therapy. In some embodiments, two or more interventions (e.g., two or more drugs) are tested simultaneously in the model system to evaluating their coordinated action: e.g., additive affect, synergy, compatibility, negative effective, safety provide, etc.).

[0079] EXAMPLES

[0080] The following materials and methods were used in the Examples below:

[0081] Human iPSC Culture

[0082] This study utilized the DF19-9-11T human iPSC line (WiCell Research Institute, cell line name: iPS DF19-9-11T). Human iPSCs were cultured on 6-well plates coated with Matrigel (Coming) and maintained in either StemMACS iPS-Brew XF (Miltenyi Biotec) or mTESR Plus (STEMCELL Technologies) media. Cells were passaged every 6-7 days using Versene solution (Invitrogen). Prior to passaging, cells were washed with Hank's Balanced Salt Solution (HBSS) without calcium and magnesium (Gibco). The cells were then incubated with Versene solution at 37 °C for 5 minutes. After incubation, the VerseneUM-44787.601

[0083] solution was aspirated, and cells were dissociated by adding 2 mL of StemMACS iPS-Brew XF or mTESR Plus medium per well, followed by gentle pipetting to detach the cells. The dissociated cells were then replated onto fresh Matrigel-coated 6-well plates.

[0084] Differentiation of hiPSCs to iACMs

[0085] Human iPSCs were differentiated an into an atrial-specific lineage using a modified GiWi protocol (34). After seeding the iPSCs onto Matrigel-coated 6-well plates in either StemMACS iPS-Brew XF or mTESR Plus medium, cells were cultured for 1-2 days to reach 90-100% confluency. At the initiation of differentiation (day 0), the cells were washed once with 2 mL / well HBSS containing calcium and magnesium (Gibco). The medium was then replaced with 2 mL Basal Medium (RPMI 1640 supplemented with L-Glutamine and 25 mM HEPES (Gibco)), supplemented with 0.5 mg / mL BSA (Sigma), 0.2 mg / mL L- Ascorbic Acid (Sigma), and 4 pM CHIR99021 (Selleck). 48 hours following CHIR99021 treatment (day 2), medium was changed to 2 mL Basal Medium containing 5 pM IWP4 (Selleck). On day 3, medium was switched to 2 mL Basal Medium containing 5 pM IWP4 and 1 pM Retinoic Acid (Selleck). On day 4, the medium was changed with 2 mL Basal Medium containing 1 pM Retinoic Acid. On day 6, the medium was changed to 2 mL of Basal Medium without any small molecules. On day 8, the medium was switched to 2 mL of RPMI 1640 with L-Glutamine (Gibco) supplemented with B-27 (Gibco). After day 8, the medium was refreshed every 48-72 hours with 2 mL of RPMI 1640 with L-Glutamine supplemented with B-27 until cell purification.

[0086] Purification and Maturation of iACMs

[0087] Human iPSC-derived atrial differentiations were purified by negative-selection using biotin-conjugated magnetic bead sorting with an autoMACS Pro Separator (Miltenyi). Atrial differentiations were washed with HBSS without calcium and magnesium (Gibco), and then dissociated with 0.25% Trypsin / EDTA (Fisher). Following trypsinization, 2 mL of EB20 Medium (80% DMEM / F12, 0.1 mmol / L nonessential amino acids, 1 mmol / L L-glutamine, 0.1 mmol / L P -mercaptoethanol, 20% FBS and 10 pmol / L blebbistatin) with 1 pM ROCK inhibitor (ROCKi; Y-27632, Tocris) was added to each well. The cells were dissociated and then strained through a 70 pm cell strainer. Total cell count was estimated using a hemocytometer. The cell suspension was centrifuged at 1000 RPM for 5 minutes and the supernatant was aspirated. The cell pellet was resuspended in cold autoMACS Running Buffer (Miltenyi) and centrifuged again at 1000 RPM for 5 minutes. The cells were purifiedUM-44787.601

[0088] by depleting iPSC-derived non-cardiomyocytes by magnetic separation using Miltenyi isolation kit according to the manufacturer’s instruction.

[0089] Maturation of i ACMs on MatrixPlus was facilitated as previously described (29). A 96-well black microclear plate was coated with MatrixPlus, then rehydrated with HBSS with calcium and magnesium at 37 °C for 30 mins. MatrixPlus-coated 96-well plates were washed twice with HBSS with calcium and magnesium. The purified hiPSC-derived atrial cardiomyocytes were replated at a density of 95,000 cells / well. The iACMs were initially cultured in EB20 Medium with ROCKi for 48 hours, after which the medium was replaced with RPMI 1640 with L-Glutamine, supplemented with B-27 for 5 days, with media changes every 48 hours. All experiments were initiated 7 days post-purification, at which point iACMs had formed mature syncytia.

[0090] Culture of hACFs

[0091] Cryopreserved human donor atrial cardiac fibroblasts (hACFs) (NHCF-A Human Cardiac Atrial Fibroblasts; Lonza, #CC-2903) were obtained from Lonza Biosciences (Walkersville, MD) and thawed into FBMTM Basal Medium (Lonza, #CC-3131) supplemented with FGMTM-3 SingleQuot Supplements (Lonza, #CC-4525). The cells were cultured in T-25 cell culture flasks coated with 0.1% gelatin (Type A, Sigma) in IX PBS. The hACFs were maintained for 2-4 days until they reached 70-80% confluency, at which point they were passaged according to the manufacturer’s protocol. Cells were washed with 4 mL / flask HEPES Buffered Saline Solution (Lonza, #CC-5022), then incubated with 2 mL / flask 0.025% Trypsin / EDTA (Lonza, #CC-5012) at 37 °C for 3-5 minutes. Following incubation, 4 mL of Trypsin Neutralizing Solution (Lonza, #CC-5002) was added to the flask and hACFs were collected and centrifuged at 1000 RPM for 5 minutes. The supernatant was aspirated, and the cells were resuspended in FBMTM Basal Medium supplemented with FGMTM-3 SingleQuot Supplements and replated onto T25 flasks for subculturing. The hACFs were not cultured for more than 5 passages, in accordance with the manufacturer’s guidelines.

[0092] Co-Culture of iACMs and hACFs

[0093] Human iACMs were co-cultured with varying percentages of hACFs: 100%, 90%, 70%, or 50%. For pacing experiments, the co-cultures were plated at a seeding density of 500,000 cells per 400 pL drop of EB20 Medium in 8-well plates coated with MatrixPlus (StemBioSys) and fitted with a 3.5 x 2.5 cm polydimethylsiloxane silicone sheetingUM-44787.601

[0094] (Specialty Manufacturing, Inc, Saginaw, MI) imprinted with a 3.14 cm2circle of free space to restrict the seeding area of the cells. For experiments conducted in a 96-well format, cocultures were seeded at 90,000 cells per well coated with MatrixPlus (StemBioSys).

[0095] Genetically Encoded Calcium Indicator Transfection of iACMs and iACM-hACF CoCultures

[0096] To permit long-term, successive optical mapping, cells were transfected with the genetically encoded calcium indicator, GCaMP6 fast variant, as previously described (30). Recombinant adenoviruses (AdGCaMP6f, Vector Biolabs, Malvern, PA, #1910) were prepared and aliquoted for single-use in DMEM with 2% bovine serum albumin (BSA) and 2.5% glycerol. The viral stock was stored at -80°C and had a titer of 1x1010 PFU / mL.

[0097] Human iACM and iACM-hAF co-cultures were transfected with a load of 5 multiplicity of infection (MOI) GCaMP6f in 100 pL of RPMI with L-Glutamine and without phenol red (Gibco), supplemented with B-27 (Gibco) for 48 hours. The media was replenished for an additional 24 hours prior to optical mapping.

[0098] High-Throughput Optical Mapping of iACMs and iACM-hACF Co-Cultures Optical mapping was performed using a CARTOXTM device (StemBioSys). All iACM and iACM-hACF monolayers demonstrated spontaneous pacemaker activity. All experiments were performed at 37 °C. For calcium transient (CaT) and propagation experiments, monolayers were pre-loaded with the GCaMP6f. Optical action potentials (APs) were recorded using the FluoVolt membrane potential probe (F10488; Life Technologies). Following a 30-minute incubation, monolayers were washed with HBSS with Ca2+and Mg2+, then incubated at 37 °C for 1 hour prior to optical mapping. Data analysis was conducted using StemBioSys software.

[0099] Intermittent Tachypacing of iACMs and iACM-hACF Co-Cultures

[0100] Human iACM and iACM-hACF monolayers cultured in 8-well plates were subjected to chronic electrical field pacing using a C-PACE EP Cell Culture Stimulator Bank (lONOptix) coupled with C-Dish™ 8-well carbon electrodes (lONOptix). An intermittent tachypacing protocol was established by applying electrical field stimulation at 20 V / cm with 0.5 ms pulse duration at frequency cycles at 1.5 Hz for 45 minutes followed by 5 Hz for 15 minutes, repeated hourly for a duration of 7 days. For all pacing experiments, cells wereUM-44787.601

[0101] maintained in RPMI with L-Glutamine without Phenol Red, and the media was refreshed every 24 hours.

[0102] Arrhythmia Inducibility Test of iACMs and iACM-hACF Co-Cultures

[0103] Following 7 days of intermittent-tachypacing, iACM and iACM-hACF monolayers were subjected to a point stimulation test with simultaneous optical mapping. Optical mapping of calcium transients (CaTs) was conducted by placing the 8-well plate beneath a high-speed CCD camera (200 fps, 80 x 80 pixels; Red-Shirt Little Joe, Scimeasure, Decatur, GA). A heating block was used to maintain physiological temperature (37 °C) during recordings. An emission filter (515 nm; Chroma) appropriate to the loaded GCaMP6f, was used in combination with blue LED illumination to capture spontaneous or electrically induced CaTs. Point stimulation of the monolayers was facilitated using a dual -pronged electrode coupled to a MyoPacer TM power bank (lONOptix). The electrode was placed in direct contact with the monolayers under the guidance from the CCD camera and the monolayers were tachypaced at 7 Hz (25 V) for 30s. Optical mapping of CaTs began immediately upon termination of point stimulation, and resultant spontaneous CaTs were recorded.

[0104] Immunocytochemistry

[0105] Cells were washed with HBSS with Ca2+and Mg2+and then fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized in 0.1% Triton-X100 (Fisher) for 10 minutes at room temperature. After permeabilization, cells were washed once with 1x PBS and incubated for 30 minutes at room temperature in a blocking solution of 1% normal donkey serum in 1x PBS containing 0.1% Triton-X100. Primary antibodies were diluted in the blocking solution and incubated with the cells for 1 hour at room temperature. Cells were then washed with lx PBS twice. Following primary antibody incubation, cells were washed twice with lx PBS. Secondary antibodies (1:1000 dilution) were then applied in the blocking solution and incubated for 30 minutes at room temperature in the dark. Cells were then washed once with lx PBS and stained with 1:1000 DAPI in 1x PBS for 5 minutes. Cells were then washed with 1x PBS prior to imaging. Imaging was performed using either a Cytation 5 Imaging Reader (Biotek), Incucyte SX5 (Sartorius), or a Nikon AIR confocal microscope (Nikon).

[0106] Real-Time Quantitative PCR (qPCR)UM-44787.601 After intermittent tachypacing, hiPSC-aCM and haCF were processed for RNA extraction using the Nucleospin RNA II kit (Macherey-Nagel, 740955.250) following the manufacturer's instructions. RNA concentration was quantified using a Nanodrop spectrophotometer. Complementary DNA (cDNA) synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368814, USA). qPCR was performed using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, 4444557) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Relative mRNA expression levels were determined using the comparative Ct (2^-ΔΔCt) method, normalized to the geometric mean of the housekeeping genes 18S rRNA, HPRTL and GAPDH. The following TaqMan probes (ThermoFisher Scientific) were used for this study: RYR2 (Hs00181461_m1); ATP2A2 (Hs00544877_m1); PLN (Hs00160179_m1); NPPA (Hs00383230_g1); NPPB (Hs00173590_m1); TGFB1 (Hs00998133_m1); 18S rRNA (Hs99999901_s1); HPRT1 (Hs99999909_m1) and GAPDH (Hs99999905_m1).

[0107] Western Blot Analysis

[0108] hiPSC-aCM and hiPSC / haCF co-cultures were lysed in cold RIPA buffer (Thermo) treated with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo). Lysates were combined with Laemmeli sample loading buffer (Thermo) with 10% P-Mercapoethanol and heated at 95°C for 5 minutes. Samples were then resolved using SDS-PAGE on a 4-20% MiDi gradient gel at 120 V for 90 minutes and transferred to nitrocellulose membranes using a Power Blotter semi-dry system (Invitrogen). Membranes were stained with No-Stain™ Protein Labeling Reagent (Invitrogen) and total protein quantification was imaged using a ChemiDoc Imaging System (Bio-Rad). Membranes were then washed with 0.1% Tween-20 in PBS and blocked with a solution of 0.1% Tween-20 and 5% non-fat dry milk in PBS for 60 minutes. Overnight incubation with primary antibodies was carried out at 4°C with constant agitation. Membranes were triple-washed with a solution of 0.1% Tween-20 in PBS andUM-44787.601 incubated with secondary antibodies at room temperature for 1 hour with constant agitation. Detection of protein bands was performed using Western Lightning Plus ECL reagent (PerkinElmer, Waltham, MA, USA) and visualized using the ChemiDoc Imaging System (ImageLab 6.1; Bio-Rad). Protein levels were quantified using Bio-Rad image analysis software and normalized to total protein levels from the same membranes. Blot images were not subjected to any adjustments in contrast or brightness. The pseudocolor display was set to an inverted monochrome in selected blots for presentation purposes. Details of the antibodies used are listed in Table 1. Images provided within supplement were obtained by direct screenshot and exported within ImageLab 6.1 software without further manipulation or use of outside software.

[0109] Table 1: Antibodies utilized

[0110] Antibody Company Catalog # Species Dilution Primary antibodies:

[0111] Anti-TE-7 (ER-TR7) Millipore CBL271 Mouse 1:100

[0112] Anti-a-Actinin Sigma A7811 Mouse 1:500

[0113] Anti-cTnT Abcam 45932 Rabbit 1 pg / mL Anti-TGFβ1 Santa Cruz sc-146 Rabbit 1:100

[0114] Anti-Collagen III Invitrogen PA5- Rabbit 1:500

[0115] 34787

[0116] Anti-SERCA2A Invitrogen MA3-919 Mouse 1:500

[0117] Anti-PLN Abcam ab219626 Rabbit 1:2000 Anti-pPLN (S16 / T17) Invitrogen 702369 Rabbit 1:500 Secondary antibodies:UM-44787.601

[0118] Donkey anti-Rabbit Thermo Fisher A21207 Anti- 1:500

[0119] (594) Rabbit

[0120] Donkey anti-mouse (488) Jackson 715-545- Anti- 1:500

[0121] ImmunoResearch 150 Mouse

[0122] Statistical analysis

[0123] All data is presented normalized to control. Outlier analysis was performed using ROUT with 1% sensitivity. Outliers between interrelated measurements (dF / FO, F0, Upstroke Slope, Conduction Velocity; or Frequency, CaTD80 / APD80, CaTDTri / APDTri) were removed. For comparison of 2 groups, Kolmogorov- Smirnov test of normality was performed with subsequent unpaired parametric T-Test or Mann-Whitney U Test. For comparison of >2 groups, 1-Way-ANOVA was performed with Bonferroni comparison of individual groups.

[0124] Example 1: ITPP alters action potential and conduction velocity in hiPSC-aCM syncytia Experiments were conducted that determined that hiPSC-aCM subjected to ITPP would induce electrophysiological characteristics observed in chronic human AF patients. Optical mapping experiments were performed using voltage-sensitive dye and calcium indicator in cells subjected to ITPP and control hiPSC-aCM syncytia.

[0125] Optical mapping of APs revealed that ITPP -treated hiPSC-aCM syncytia exhibited a significantly higher frequency of spontaneous APs relative to control (CTRL: 1.00±0.03, n=13, N=2; ITPP: 1.31±0.06, n=13, N=2; p<0.0001; Figure IB). Fridericia-corrected APD80% (CTRL: 1.00±0.02, n=13, N=2; ITPP: 0.85±0.03, n=13, N=2; p=0.0012; Figure 1C) and AP triangulation (APDTri = APD90%-APD30%) (CTRL: 1.00±0.03, n=13, N=2; ITPP: 0.87±0.03, n=13, N=2; p=0.0104; Figure ID) were both shortened in ITPP -treated cells, indicating a reduction in phase 3 repolarization. The upstroke slope of the AP, a measurement of phases 0 / 1, was not affected by ITPP (CTRL: 1.00±0.03, n=l 1, N=2; ITPP: 1.02±0.05, n=13, N=2; p=0.7753; Figure IE). The conduction velocity of the AP was significantly faster following ITPP (CTRL: 1.00±0.07, n=l 1, N=2; ITPP: 1.40±0.06, n=13, N=2; p=0.0002; Figure IF).UM-44787.601

[0126] EXAMPLE 2: Calcium handling changes in ITPP-treated hiPSC-aCM

[0127] To assess the effects of ITPP on intracellular calcium handling, hiPSC-aCM syncytia were further evaluated by optical mapping to study CaTs. Foremost, the variability of spontaneous calcium release events was measured using the standard deviation of frequency of spontaneous CaTs. While the baseline frequency of spontaneous CaTs was not significantly different between ITPP-treated hiPSC-aCM syncytia relative to controls (CTRL: 1.00±0.06, n=15, N=2; ITPP: 0.90±0.03, n=16, N=2; p=0.1603; Figure 2B), there was a significant increase (A = +332±88%) to the variability of calcium release events following ITPP (CTRL: 1.00±0.16, n=15, N=2; ITPP: 4.32±0.88, n=16, N=2; p=0.0006; Figure 2C).

[0128] Although there was no change observed in the duration at 80% decay of the CaT between the groups (CaTD80%; CTRL: 1.00±0.04, n=15, N=2; ITPP: 1.04±0.03, n=16, N=2; p=0.4704; Figure 2D), there was an increase in the standard deviation of CaTD80% (CTRL: 1.00±0.06, n=13, N=2; ITPP: 1.83±0.08, n=15, N=2; pO. OOOl; Figure 2E). The upstroke slope of the CaT measures the speed of calcium release from the sarcoplasmic reticulum. Compared to controls, ITPP-treated hiPSC-aCM syncytia exhibited a significantly slower upstroke slope of calcium release (CTRL: 1.00±0.08, n=13, N=2; ITPP: 0.59±0.07, n=13, N=2; p=0.0012; Figure 2F), indicating impaired release from the SR. Calcium release amplitude was also significantly diminished in ITPP-treated cells (CTRL: 1.00±0.12, n=13, N=2; ITPP: 0.49±0.04, n=13, N=2; p=0.0005; Figure 2G), and the propagation speed of the intracellular calcium wave across the monolayer was significantly slower (CTRL: 1.00±0.10, n=13, N=2; ITPP: 0.19±0.01, n=13, N=2; p<0.000; Figure 2H). The duration of intracellular calcium reuptake into the sarcoplasmic reticulum was assessed using a CaT triangulation measure (CaTDTri = CaTD90% - CaTD30%); nevertheless, no significant, relative change to the value of the CaTDTri (CTRL: 1.00±0.07, n=15, N=2; ITPP: 1.00±0.04, n=16, N=2; p=0.9768; Figure 21) was observed. However, there was a significant, relative increase to the beat-to-beat variability in CaTDTri (CTRL: 1.00±0.08, n=13, N=2; ITPP: 2.03±0.19, n=15, N=2; p<0.0001; Figure 2J) and in ITPP-treated hiPSC-ACMs compared to controls, consistent with the observation of increased variability in the frequency of spontaneous CaT events. There were no significant changes in diastolic calcium levels (F0) between groups (CTRL: 1.00±0.07, n=13, N=2; ITPP: 1.15±0.07, n=13, N=2; p=0.1607; Figure 2K).

[0129] Optical mapping was performed before and after administration of Arrythmia Inducibility Test (AIT; 3.5 Hz) in control and ITPP-treated hiPSC-aCM syncytia.UM-44787.601

[0130] Comparison of the difference in average baseline fluorescence during and after AIT (AFO = F0, Pacing - F0, Rest) serves to proxy the change in diastolic calcium levels following burst tachypacing. In post-rest potentiation (PRP) of CaTs, ITPP -treated hiPSC-ACMs exhibited a reduced upstroke slope (CTRL: 1.00±0.18, n=15, N=2; ITPP: 0.35±0.05, n=14, N=2; p=0.0004; Figure 3C) and decreased amplitude (CTRL: 1.00±0.20, n=15, N=2; ITPP:

[0131] 0.33±0.05, n=14, N=2; p=0.0008; Figure 3D) relative to controls. ITPP -treated hiPSC-aCM syncytia also exhibited a significantly diminished reconstitution of diastolic calcium post-AIT, (CTRL: 1.00±0.18, n=15, N=2; ITPP: 0.35±0.05, n=14, N=2; p=0.0004; Figure 3B) consistent with observed alterations to PRP CaT dynamics.

[0132] EXAMPLE 3: hiPSC-ACM-hACF co-culture subjected to ITPP shows AF characteristics in-a-dish

[0133] Co-culture of purified and matured hiPSC-aCM with haCFs was conducted at different seeding density ratios of 100 / 0, 90 / 10, 70 / 30 to explore fibroblast-induced electrophysiological and calcium-handling changes. Cocultures maintained the seeding ratios following 7 days of culture (100 / 0 cTnT: TE7: 0.96±0.03:0.01±0.001; 90 / 10 cTnT: TE7: 0.84±0.03:0.15±0.01; 70 / 30 cTnT: TE7: 0.72±0.03: 0.34±0.03; Figure 4A-B). To verify pro-fibrotic effects of haCF culture, Collagen III expression was measured using immunocytochemistry. The 70 / 30 ratio (hiPSC-aCM / haCF) exhibited the highest expression of collagen III, which was fibroblast-dependent (100 / 0: 1.00±0.06, n=3; 90 / 10: 36.11±1.59, n=4; 70 / 30: 103.95±7.25; pO. OOOl; Figure 4C-D). Expression of the upstream cytokine TGFβ1 was also significantly increased in ratios consistent with increased collagen III expression (100 / 0: 1.000.16, n=4; 90 / 10: 1.360.04, n=4; 70 / 30: 1.610.05; p=0.0026; Figure 4E-F).

[0134] A previous report (36) has demonstrated that the effect of fibroblast co-culture on the electrophysiology of hiPSC-aCMs was ratio-dependent. Therefore, hiPSC-aCM-haCF coculture preparations were subjected to voltage and calcium optical mapping. Voltage mapping revealed fibroblast-dependent decrease to spontaneous AP frequency (100 / 0:

[0135] 1.000.06, n=ll; 90 / 10: 0.84±0.06, n=8; 70 / 30: 0.390.01, n=24; pO. OOOl; Figure 5B) with concomitant prolongation of both the Fridericia-corrected AP duration at 80% repolarization (100 / 0: 1.000.04, n=ll; 90 / 10: 1.070.03, n=8; 70 / 30: 2.30±0.09, n=24; pO. OOOl; Figure 5C) and Fridericia-corrected APD triangulation (100 / 0: 1.00±0.09, n=ll; 90 / 10: 0.91±0.03, n=8; 70 / 30: 2.43±0.09, n=24; pO. OOOl; Figure 5D) at 70 / 30 ratio only. Substantially, theUM-44787.601

[0136] upstroke slope of the AP (100 / 0: 1.00±0.06, n=ll; 90 / 10: 1.01±0.08, n=10; 70 / 30: 0.34±0.02; p<0.0001; Figure 5E) and conduction velocity of the AP (100 / 0: 1.00±0.17, n=ll; 90 / 10: 1.94±0.29, n=10; 70 / 30: 0.48±0.06; p<0.0001; Figure 5F) were also decreased at 70 / 30 ratio.

[0137] In terms of calcium handling, co-culture did not affect the frequency of intracellular calcium release events (100 / 0: 1.00±0.07, n=10; 90 / 10: 0.94±0.09, n=10; 70 / 30: 0.90±0.08, n=10; p=0.6530; Figure 6B), however it did prolong both duration of 80% decay of the CaT (CaTD80%; 100 / 0: 1.00±0.03, n=10; 90 / 10: 1.34±0.10, n=10; 70 / 30: 1.53±0.09, n=10; p=0.0002; Figure 6D) and the duration of SR calcium reuptake (CaTDTri; 100 / 0: 1.00±0.03, n=10; 90 / 10: 1.34±0.10, n=10; 70 / 30: 1.39±0.09, n=10; p=0.0022; Figure 6E) in a ratiodependent manner. Calcium release events became markedly more variable at 70 / 30 ratio (A = +169±44%) (HzSD; 100 / 0: 1.00±0.16, n=7; 90 / 10: 1.53±0.24, n=10; 70 / 30: 2.69±0.44, n=9; p=0.0039; Figure 6C), with diminished intracellular calcium upstroke slope (100 / 0: 1.00±0.08, n=10; 90 / 10: 1.14±0.08, n=9; 70 / 30: 0.50±0.03, n=9; pO. OOOl; Figure 6F) and intracellular calcium release amplitude (100 / 0: 1.00±0.07, n=10; 90 / 10: 1.04±0.05, n=9; 70 / 30: 0.66±0.06, n=9; p=0.0003; Figure 6G). The propagation of the intracellular wave was slowed significantly in 70 / 30 preparations (100 / 0: 1.00±0.11, n=10; 90 / 10: 0.74±0.04, n=9; 70 / 30: 0.34±0.05, n=9; p<0.0001; Figure 61). The 90 / 10 coculture presented a modest (90-10 A = l±0.1%) but significant decrease to diastolic calcium; however this was not recapitulated in 70 / 30 preparations (100 / 0: 1.000±0.002, n=10; 90 / 10: 0.990±0.001, n=9; 70 / 30:

[0138] 0.998±0.002, n=9; p=0.0028; Figure 6H).

[0139] Given the similarities in the functional electrophysiology and calcium handling of 70 / 30 coculture to atrial fibrosis - a critical structural component of AF propagation, it was contemplated that coculture preparations subjected to electrical remodeling by ITPP would present conduction block analogous to this known proarrhythmic substrate. Voltage mapping of ITPP-treated 100 / 0 (Control; F-ITPP 100 / 0) and 70 / 30 (ITPP; F-ITPP 70 / 30) verified that 70 / 30 preparations recapitulated tachycardic-like electrical remodeling observed in 100 / 0 iPSC-aCM syncytia, including increased frequency of spontaneous activations (100 / 0:

[0140] 1.00±0.05, n=8; F-ITPP 100 / 0: 1.36±0.07, n=8; F-ITPP 70 / 30: 01.25±0.07, n=8; p=0.0018; Figure 7D) and shortening of the Fridericia-corrected APD80% (100 / 0: 1.00±0.05, n=8; F-ITPP 100 / 0: 0.87±0.03, n=8; F-ITPP 70 / 30: 0.84±0.03, n=8; p=0.0135; Figure 7F). Critically, F-ITPP 70 / 30 preparations did not demonstrate facilitated increase in the conduction velocity of the AP (100 / 0: 1.00±0.10, n=8; F-ITPP 100 / 0: 1.45±0.09, n=8; F-ITPP 70 / 30: 0.74±0.11, n=8; p=0.0002; Figure 7H). Rather, conduction velocity was significantly decreased (A =UM-44787.601

[0141] 50±9%) by inclusion of fibroblasts in F-ITPP 70 / 30 preparations in relation to F-ITPP 100 / 0 (F-ITPP 100 / 0: 1.00±0.04, n=8; F-ITPP 70 / 30: 0.53±0.09, n=8; p=0.0005; Figure 71). The magnitude of change in frequency (F-ITPP 100 / 0: 1.00±0.04, n=8; F-ITPP 70 / 30: 0.93±0.07, n=8; p=0.3604; Figure 7E) or APD80% (F-ITPP 100 / 0: 1.00±0.02, n=8; F-ITPP 70 / 30:

[0142] 0.98±0.05, n=8; p=0.8785; Figure 7G) was preserved between F-ITPP 70 / 30 or F-ITPP 100 / 0 conditions.

[0143] EXAMPLE 4: Molecular remodeling of calcium-handling and fibrotic signaling under intermittent tachypacing and fibroblast co-culture

[0144] To determine whether the electrophysiological and calcium-handling alterations observed under ITPP and fibrotic co-culture were associated with changes in calcium regulatory proteins, the expression of phospholamban (PLN), phospho-phospholamban, pPLN(S16 / T17), and sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a) was analyzed by Western blot (Figure 8 A). Total PLN expression was significantly reduced in both ITPP 100 / 0 (P=0.0008) and ITPP 70 / 30 (P=0.0006) (Figure 8B) compared with control conditions, whereas pPLN and SERCA2a protein levels remained unchanged (Figure 8C-D).

[0145] Consequently, with no difference in pPLN / PLN ratio (Figure 8E). Nevertheless, the (pPLN / PLN) / SERCA2a ratio was markedly elevated in the 70 / 30 ITPP group (P=0.0022; Figure 8F), suggesting a relative disinhibition of SERCA2a activity. This alteration in the PLN-SERCA2a regulatory balance is indicative of disturbed SR calcium reuptake dynamics and may underlie the spontaneous calcium release variability observed in the functional assays.

[0146] At the transcriptional level, qPCR analysis showed that RYR2 and ATP2A2 expression remained unaltered across all conditions (Figure 8G-H), whereas PLN mRNA was significantly upregulated in the 70 / 30 ITPP group (P=0.01; Figure 81), suggesting a compensatory transcriptional response to altered SR calcium regulation. Atrial stress and remodeling markers NPPA, NPPB, and TGFB1 were markedly increased following ITPP, with the most pronounced upregulation observed in the 70 / 30 co-culture condition, P=0.004, P=0.02, and P=0.04 respectively (Figure 8J-L). The concurrent elevation of TGFB1 and natriuretic peptides indicates activation of a pro-fibrotic and stress-responsive transcriptional regulation consistent with advanced atrial remodeling. Together, these molecular data demonstrate that intermittent tachypacing in a fibrotic environment induces coordinatedUM-44787.601

[0147] alterations in the PLN-SERCA2a axis and TGFβ1 -driven signaling, recapitulating key aspects of calcium dysregulation and structural remodeling characteristic of atrial fibrillation.

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[0220] All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

UM-44787.601CLAIMS1. An in vitro atrial fibrillation (AF) cell culture system comprising: a co-culture of human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) and human atrial cardiac fibroblast (haCFs) having one or more characteristics of AF cardiomyocytes relative to non-AF cardiomyocytes selected from the group consisting of reduced action potential (AP) frequency, prolonged action potential duration (APD), changes in calcium handling (e.g., prolonged calcium transient duration 80% (CaTD80%), calcium upstroke slope, amplitude of calcium release), upregulation of collagen III and TGFβ1, and increase in frequency of spontaneous activations.

2. The system of claim 1, wherein said co-culture comprises a hiPSC-aCM / haCF ratio of 50 / 50 to 80 / 20.

3. The system of claim 2, wherein the ratio is approximately 70 / 30.

4. The system of claim 1, wherein the haCFs are derived from stem cells.

5. The system of claim 1, wherein the haCFs are obtained from a human donor.

6. The system of claim 1, wherein the hiPSC-aCMs and haCFs are obtained from or derived from a single individual.

7. The system of claim 1, wherein the hiPSC-aCMs and / or haCFs are modified to express a heterologous factor.

8. The system of claim 7, wherein the heterologous factor is a genetically encoded indicator.

9. The system of claim 8, wherein the genetically encoded indicator is a genetically encoded calcium indicator.

10. The system of claim 1, comprising a culture medium.

11. The system of claim 10, wherein the culture medium comprises a test compound.

12. The system of claim 1, comprising a culture surface upon which a monolayer of the co-culture is attached.

13. The system of claim 12, further comprising an electrode in contact with the monolayer.UM-44787.60114. The system of claim 1, further comprising an instrument that measures one or more electrical, chemical, or optical properties of the co-culture.

15. A method comprising: analyzing a system of any of claims 1-14.

16. The method of claim 15, wherein said analyzing comprises determining a change in the co-culture in response to a stimulus (e.g., a change in an AF-related characteristic in response to the stimulus).

17. The method of claim 16, wherein the stimulus comprises administration of a test compound.

18. The method of claim 17, wherein the test compound comprises a drug.

19. A method for generating an in vitro atrial fibrillation (AF) cell culture system comprising:a) co-culturing human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) and human atrial cardiac fibroblast (haCFs); andb) remodeling co-cultured cells using an intermittent tachypacing protocol (ITPP).

20. The method of claim 19, wherein the remodeled cells have one or more characteristics of AF cardiomyocytes relative to non-AF cardiomyocytes selected from the group consisting of: reduced action potential (AP) frequency, prolonged action potential duration (APD), changes in calcium handling (e.g., prolonged calcium transient duration 80% (CaTD80%), calcium upstroke slope, amplitude of calcium release), upregulation of collagen III and TGFβ1, and increase in frequency of spontaneous activations.

21. The method of claim 19, wherein the co-culturing comprises generating a hiPSC-aCM / haCF ratio of 50 / 50 to 80 / 20.

22. The method of claim 21, wherein the ratio is approximately 70 / 30.

23. The method of claim 19, wherein the haCFs are derived from stem cells.

24. The method of claim 19, wherein the haCFs are obtained from a human donor.

25. The method of claim 19, wherein the hiPSC-aCMs and haCFs are obtained from or derived from a single individual.UM-44787.60126. The method of claim 19, wherein the hiPSC-aCMs and / or haCFs are modified to express a heterologous factor.

27. The method of claim 26, wherein the heterologous factor is a genetically encoded indicator.

28. The method of claim 27, wherein the genetically encoded indicator is a genetically encoded calcium indicator.

29. The method of claim 19, wherein the ITPP comprises subjecting a cultured monolayer of the co-cultured cells to chronic electrical field pacing.

30. The method of claim 29, wherein the chronic electrical field pacing comprises applying an electrical field stimulation at first frequency cycles for a first time period followed by second frequency cycles for a second time period, repeated at a time frequency for a duration.

31. The method of claim 30, wherein the electrical field stimulation comprises 20 V / cm with 0.5 ms pulse duration.

32. The method of claim 30, wherein the first frequency cycles are at 1.5 Hz.

33. The method of claim 30, wherein the first time period is 30-60 minutes (e.g., 45 minutes).

34. The method of claim 30, wherein the second frequency cycles are at 3.5-5 Hz (e g., 4 Hz, 5 Hz).

35. The method of claim 30, wherein the second time period is 10-30 minutes (e.g., 15 minutes).

36. The method of claim 30, wherein the time frequency is hourly.

37. The method of claim 30, wherein the duration is 5-10 days (e.g., 7 days).

38. The method of claim 19, wherein prior to the co-culturing, the cardiomyocytes (hiPSC-aCMs) and / or human atrial cardiac fibroblast (haCFs) are purified to reduce cellular heterogeneity.UM-44787.601

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