Improved maturation medium

Abstract

Disclosed herein is a novel culture medium for the maturation of cardiomyocytes. More specifically, disclosed herein is an improved culture medium for cardiac maturation, and methods for using such media. Also disclosed are methods for screening candidate agents.

Description

TITLE OF THE INVENTION“IMPROVED MATURATION MEDIUM"RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2024902826 entitled “Improved Maturation Medium” filed 6 September 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present invention relates to culture medium. More specifically, the invention relates to improved culture medium for cardiac maturation, and methods for using such media.BACKGROUND

[0003] Maturation is the last phase of heart development that allows the heart to maintain its pumping function throughout a mammal’s lifespan. Maturation impacts almost every aspect of cardiac biology, structure and function (Guo, 2020; Mills, 2019). Understanding the maturation process is pivotal for many fields including the application of human pluripotent stem cell technologies for disease modelling and drug discovery. Although recent studies have begun to identify factors required for maturation (Mills, 2019; Karbassi, 2020), it has been difficult to pinpoint the upstream physiological stimuli that are sufficient to drive the maturation process.

[0004] hPSC-CMs are typically immature limiting some of their applications for disease modelling and drug screening. They therefore also provide an excellent model to understand the maturation process. Extended culture for up to a year does not result in equivalent maturation to that of time matched in vivo maturation (Bedada, 2014), indicating that either environmental conditions are inhibitory and / or additional stimuli are required. However, the challenge is that different stimuli sequentially drive distinct aspects of maturation, meaning that combinations of factors and precise timing of factors are essential. To this end, the five most successful methods for maturation thus far are multicellularity, mechanical loading / structural patterning, hormonal stimulation, metabolic switching, and pacing with electrical stimulation (Karbassi, 2020). Multicellular and organoid protocols have been established to promote cellular complexity and cell-cell interactions (Voges, 2023; Drakhlis, 2021 ; Lewis-Israeli, 2021 ; Hofbauer, 2021 ; Giacomelli, 2020). Tissue engineering approaches generally incorporate mechanical loading which drastically improves the morphology, alignment and maturity of the cardiomyocytes (Mills , 2017, Tiburcy, 2017; Schaaf, 2011 ; Zhang 2013). Metabolic maturation can also provide maturation of key features including metabolism, cell cycle and sarcomeric isoforms (Mills, 2017). Pacing has been also demonstrated to be one of the most potent inducers of functional maturation in terms of excitation- contraction coupling and drug responses (Zhao, 2019; Ronalsdon-Bouchard, 2018; Shen, 2022). Despite these advances, mechanistic understanding of the maturation stimuli are limited, and the induction of furthermaturation is challenging. This may be important for modelling complex genetic or environmental driven diseases.

[0005] HeartDyno® is a 96-well platform that facilitates the self-organisation of cardiac cell types into miniaturised, mechanically loaded hCOs (Mills, 2017; Voges, 2023). To create hCOs, hPSCs are carefully patterned into pre-cardiac mesoderm (Raad, 2021), which gives rise to cardiomyocytes and cardiac progenitor cells in a single differentiation protocol. Subsequently following hCO fabrication, this facilitates the differentiation and organisation of multiple cardiac cell types to form hCOs (Voges, 2023).SUMMARY OF THE INVENTION

[0006] Provided herein are compositions and methods related to development of mature cardiomyocytes and cardiac organoids

[0007] In one aspect, the present invention provides a cell culture medium for use in cardiomyocyte maturation, the medium comprising a 5' AMP-activated protein kinase (AMPK) activator, a carbohydrate source and a fatty acid substrate.

[0008] In some embodiments, the AMPK activator is a pan-AMPK activator. In some embodiments of this type, the AMPK activator is MK-8722.

[0009] In some preferred embodiments, the medium further comprises an ERRp / y agonist and / or interferon-M (IFN-M). In some embodiments of this type, the ERRp / y agonist is selected from DY131 , SLU-PP-332, ERRy inverse agonist 1 , and SR19881. Preferably, the ERRp / y agonist is DY131.

[0010] In some embodiments, the fatty acid substrate is selected from the group comprising palmitic acid, oleic acid, myristic acid, and linoleic acid.

[0011] In another aspect, the invention provides a method of maturing a cardiac organoid, the method comprising: exposing the cardiomyocytes to a culture condition that comprises an AMPK activator and one or both of an ERRp / y agonist and IFN-A1 , wherein the second culture conditions are sufficient to promote cardiac organoid maturation.

[0012] In some embodiments, the cardiomyocytes are exposed to the culture condition following the phase where the cardiac organoids are metabolically switched to fatty acid oxidation.

[0013] In some embodiments, the culture condition also comprises a fatty acid substrate. By way of an illustrative example, the fatty acid substrate can be selected from the group comprising palmitic acid, oleic acid, myristic acid, and linoleic acid.

[0014] In some embodiments, the cardiomyocytes have previously been exposed to a first culture condition that comprises a GSK3 inhibitor / Wnt activator.

[0015] In yet another aspect, the invention provides a cardiac organoid produced by the method described above and / or elsewhere herein.

[0016] In some embodiments, the cardiac organoid has high levels of cTnl expression. In some embodiments of this type, cTnl is the only isoform (i.e., cTnl / (cTnl + ssTnl) = 1).

[0017] In some embodiments, the cardiac organoid of the present invention has a TNNI3 proportion of TNNI1 and TNNI3 greater than about 0.1769 (the value observed for SF hCO). In some embodiments, the value is less than about 1 (the value observed for adult heart tissue). For example, the proportion of TNNI1 of the TNNI1 and TNNI3 is between about 0.2 and 0.6. In some more preferred embodiments, the proportion of TNNI1 of the TNNI1 and TNNI3 is between about 0.3 and 0.5. In some even more preferred embodiments, the TNNI3 fraction of TNNI1 and TNNI3 is about 0.43.

[0018] In still yet another aspect, the invention provides a method of screening for a candidate agent suitable for preventing cardiac dysfunction, the method comprising: contacting a cardiac organoid as described above and / or elsewhere herein with the candidate agent; determining the presence or level of one or more immunomodulatory or fibrotic biomarkers, wherein the immunomodulatory or fibrotic biomarkers are selected from the group comprising TGF-p1 , THBS1 , LOXL2, TNC and IL-10 as compared to a predetermined threshold; on the basis of the immunomodulatory or fibrotic biomarkers being above a predetermined threshold, determining that the candidate agent is suitable for preventing cardiac dysfunction.

[0019] A method of determining the effect on force of a candidate agent, the method comprising: applying a first pacing condition to a cardiac organoid produced by the method described above and / or elsewhere herein, or a cardiac organoid as described above and / or elsewhere herein; blocking spontaneous contractile activity of the cardiac organoid; contacting the cardiac organoid with the candidate agent; pausing the pacing of the cardia organoid; and applying a second pacing condition to the cardiac organoid; wherein the effect on force of the cardiac organoid during the second pacing condition determines the effect on force of the candidate agent. For example, an increase in force is indicative that the candidate agent increases force, whereas a decrease in force is indicative that the candidate agent has a negative effect on force.

[0020] In some embodiments, the spontaneous contractile activity is blocked by funny current ( / f) blockade.

[0021] In some embodiments the continued contraction of the cardiac organoid during the pacing pause step is indicative of whether the cardiac organise is disposed to a risk of arrhythmia. Therefore, the method can be used to determine whether the candidate agent can reduce or prevent the risk of arrhythmia.

[0022] In some embodiments, the first pacing condition and the second pacing condition are the same. In alternative embodiments, the first pacing condition and the second pacing condition are different.

[0023] In some embodiments, the contractile activity blocking is performed by exposing the cardiac organoid to cilobradine.

[0024] In some embodiments, the cardiac organoid comprises a gene mutation identified in tachyarrhythmias (e.g., polymorphic ventricular tachycardia). By way of an illustrative example, the gene mutation is in the CASQ2 gene or the RYR2 gene.

[0025] In some embodiments, the gene mutation is in the desmoplakin gene (DSP). By way of an illustrative example, the DSP gene may be a two base pair deletion at positions 4246 and 4247 (resulting in Leu1416Asn substitution, and a stop codon after 22 amino acids in the new reading frame).BREIF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows screening for combinatorial stimuli to promote hCO maturation. (A) Schematic workflow of the protocol. (B) Force of contraction, rate and time from peak to 50% relaxation (Tr50) at 30 days normalized to pre-treatment at 24 days, n = 3 experiments. (C) Cardiac troponin I (cTnl) intensity normalized to DNA and then to DMSO controls. (D)-(H) Addition of DY131 and MK8722 are optimal during the weaning media (WM) phase. (D) Schematic of the protocol. (E) Force of contraction at 30 days. (F) Rate at 30 days. (G) Time from peak to 50% relaxation (Tr50) at 30 days. (H) cTnl intensity normalized to DNA. s. Krusksal-Wallis test with Dunn’s multiple comparison to SF + CHIR. (I) Cardiac troponin (cTnl) staining of cardiomyocytes (a-actinin). (J) Comparison of SF and DM-hCO raw contraction curves. (K) Comparison of raw force of contraction, rate and Tr50 between SF and DM-hCO across three cell lines. (L) cTnl intensity normalized to DNA and then to DMSO controls. (M)-(Q) Type of fatty acid has limited impact on hCO function and cTnl expression. PLOM - 25 pM of each fatty acid. (M) Schematic of the protocol. (N) Force of contraction at 30 days. (O) Rate at 30 days. (P) Time from peak to 50% relaxation (Tr50) at 30 days. (Q) cTnl intensity normalized to DNA. Two experiments. Krusksal-Wallis test with Dunn’s multiple comparison to palmitic acid (N)-(Q). (R) Schematic of the protocol to show addition of DY131 and MK8722 improve maturation features in 2D. (S) Rate at 30 days. (T) Normalized (to DMSO) mean fluorescence intensity (MFI) of cTnl. Krusksal- Wallis test with Dunn’s multiple comparison to DMSO, bar = 20 pm. (S,T). (U) Phosphoproteomics comparing between electrically paced and DM treated hCO. Intensity of phosphopeptides across the different samples. Each sample is 15 hCO pooled. (V) Samples plotted on their principal components PC1 and PC2. (W) Volcanoplot of DM versus control. (X) Volcano plot of paced hCO versus control. DM - 10 pM MK8722 + 3 pM DY131 , ESTIM - 120 bpm paced hCO, both for 5 minutes. (Y) Regulated phosphosites in MK8722 + DY131 (DM) treated and 120 bpm electrically paced hCO (5 min stimulation each), highlighting shared sites, n = 3 biological replicates with 15 pooled hCO each. (Z) Heatmap of phosphosite Z-scores. (A’) Gene ontology analysis of phosphorylated proteins shared between DM and electrical pacing. (B’) The AMPK signalling network shared between DM and electrical pacing. Concentrations were: 10 pM progesterone, 3 pM DY131 , 10 pM MY8722 and 100 ng / mL IFN-A1 . Krusksal- Wallis test with Dunn’s multiple comparison to DMSO (B,C). For heatmaps (B,C) the p- values are given beneath the values if significant. Mann-Whitney tests (J,K).**** P < 0.0001 . Bar = 200 pm (except (S,T)).

[0027] Figure 2 provides a graphical representation of cellular composition in hCO. (A) Co-clustering of snRNA-seq from hCO and human heart data from GSE1567072, including proportions of cell populations, n = 2 snRNA-seq replicates for SF- and DM-hCO each from ~70 pooled hCO per sample. (B) Expression of pro-epicardial organ marker TCF21. (C) Generation of a human pluripotent stem cell TCF21 lineage tracing cell line. (D) Schematic of lineage tracing experiments. (E) Representative analysis (n = 3 experiments) of lineage Tracing 1 and 2. Bars = 200 pm.

[0028] Figure 3 shows DM induced sarcomeric and metabolic maturation in hCO. (A) UMAP projection of nuclei in SF- and DM-hCO. Nuclei labelled by cell type. (B) Expression of canonical cell markers in hCO. (C) Co-clustering of cardiomyocytes from hCO and human heart cardiomyocytes form GSE156707. (D) Number of regulated genes in different cellular populations from comparison DM vs SF using pseudo-bulk analysis (average log2FC > |0.25|, adjusted P < 0.05). (E) Top 10 gene ontology terms from the sub ontology ‘biological processes’ for upregulated genes in Cardiomyocyte 1 and 2 populations. (F) Expression of sarcomeric maturation genes TNNI3 and MYL2. (G) Top 10 gene ontology terms from the sub ontology ‘biological processes’ for upregulated genes in Cardiomyocytes 3. (H) Oxidation rate in hCO over the culture duration, n = 2 cell lines, HES3 and PB005.1. (I) Response to BAM15 in hCO. (J) Representative spatial expression of cardiomyocyte (MYH7) and nodal cardiomyocyte (MYH6) markers in a hCO section. Purple arrows point to MYH6 clusters. (K) Representative spatial expression of sarcomeric and metabolic genes in a hCO section, including a heatmap of average expression. Two-way ANOVA with Tukey’s multiple comparison test. **** P < 0.0001 . (L) Comparison of mRNA expression of sarcomeric protein ratios that correlate with maturation across multiple human pluripotent stem cell derived cardiomyocyte platforms. Fraction of MYH7. (M) Fraction of MYL2. (N) Fraction of TNNI3. Gene counts expressed as a ratio RNA-sequencing data collected from hPSC-CM cultures including GSE93841, GSE148025, GSE116464, GSE201437, GSE114976 and human hearts including ERP109940 and Hahn et al., 2021 (Pedram, 2013).

[0029] Figure 4 shows mechanisms of rate control and SR handling in hCO. (A)Dependence of force and rate on extracellular calcium concentration. (B) Contractile rate / burstbehaviour with blockade of It using 1 pM cilobradine. (C) Post-rest-potentiation assessment of SR loading and verification using blockade of SERCA using 5 pM thapsigargin. (D) SR and desmosomes are present in hCO. Overview of a hCO section. Bar = 2 pm. (E) Electron dense SR structures (indicated by arrows) and t-tubule (T). Bar = 500 nm. (F) Electron dense intercalated discs. Bar = 500 nm. (G) 3D transmission electron microscopy rendering of the SR (red). Scale bar = 500 nm. (H) Dose-response curve of DM-hCO treated with ryanodine and representative trace curves. (I) Influence of ryanodine on rate and time from 50% activation to peak (Ta50), including under 1 Hz pacing for DM-hCO. (J) Influence of CASQ knockout on rate and Ta50, including under 1 Hz pacing for DM-hCO. Three independent experiments for PB006.6 CASQ+ / +. (K) Quantification of ectopic contractions during the pause phase of post-rest-potentiation experiments. (L) Critical excitationcontraction genes regulated in DM versus SF-hCO cardiomyocyte populations. Mann-Whitney (B), Welch’s t test (G,I,J), Mixed-Effects testing with Dunnett’s post hoc analysis (E) and Kruskal-Wallis test with Dunn’s post-hoc analysis (I, J). **** P < 0.0001.

[0030] Figure 5 shows top 10 genes demarcating each cell population in SF and DM hCO.

[0031] Figure 6 (A) DM hCO predict CiPA compound low and high risk. Mixed -effects testing with Dunnett’s post-hoc analysis in comparison to baseline. Only the statistics for concentration closest to Cmax are shown. Cmax values were derived from the literature. hCO did not alter their force, rate or Ta50 more than 10% in response to the CiPA compounds at the closest concentrations to Cmax, except for high quinidine (force), ibutilide (force and rate), verapamil (force and rate) and terfenadine (Ta50). DM hCO responses to benign drugs (paracetamol and pravastatin) or drugs inhibiting the impact of systemic factors (atenolol and captopril). (C) DM hCO response to negative inotropes. Mixed -effects testing with Dunnett’s post-hoc analysis in comparison to baseline. **** P < 0.0001 . Only the statistics for lowest concentration with a decline in force are shown, as further force decline will impact the other parameters, n indicated number of hCO. DM hCO to predict positive inotropes. (D) DM hCO responses to drugs modulating L-type calcium channel (BAYK-8644), p-adrenergic signalling (isoprenaline), a1 -adrenergic signalling (phenylephrine) or Na+, K+- ATPase (ouabain). (E) DM hCO response to phosphodiesterase inhibition using milrinone (PDE3 / PDE4) and rolipram (PDE4). For isoprenaline curves 10 pM and 30 pM milrinone were used. (F) DM hCO response to sarcomeric acting inotropes targeting troponin (CK- 136) or myosin (omecamtiv mecarbil and danicamtiv). All experiments were performed at 0.6 mM Ca2+by mixing weaning medium made in RPMI and DMEM base. Mixed -effects testing with Dunnett’s post-hoc analysis in comparison to baseline (D-F), two-way ANOVA with Dunnett’s post hoc analysis. relative to DMSO (isoprenaline curves in E), one-way ANOVA with Dunnett’s post hoc analysis relative to DMSO (EC5o in E). **** P < 0.0001 . Statistics for highest concentration are shown, with the addition of intermediate concentrations where Tr50 is impacted, n indicates number of hCO unless stated otherwise.

[0032] Figure 7 illustrates myosin activators differentially affect contraction duration in DM-hCO. (A-C) Testing of omecamtiv mecarbil and dancamtiv at Cmax values, 1 pM and 8 pM, respectively. Experiments were performed at 0.6 mM Ca2+by mixing weaning medium made in RPMI and DMEM base. (A) Representative force curves. (B) Force of contraction normalised to pre-drug baseline. Contraction duration between 50% activation and 50% relaxation. Mann-Whitney (B,C). (E) DM hCO predict positive inotropes at 1 .8 mM Ca2+. DM hCO responses to inotropes modulating L-type calcium channel (BAYK-8644), p-adrenergic signalling (isoprenaline, dobutamine), a-i.adrenergic signalling (phenylephrine) or Na+, K+- ATPase (ouabain), milrinone (PDE3 / PDE4) and myosin (omecamtiv mecarbil). Paracetamol and pravastatin were used as inert control drugs, n = 5-6 DM hCO. Brown-Forsythe and Welch’s ANOVA test with Dunnett T3 post-hoc analysis relative to paracetamol. **** P < 0.0001 .

[0033] Figure 8 shows modelling DSP cardiomyopathy in DM-hCO. (A) Family tree for the patient with dilated cardiomyopathy and arrhythmia, MCHTB11 . (B) Patient specific and CRISPR corrected human induced pluripotent stem cell lines. (C) MCHTB11 DSPmut / mutCRISPR corrected cell line and hCO function. Schematic of the CRISPR correction protocol. (D) DM-hCO staining for DSP and CX43. bars = 20 pm. (E) Schematic of functional assessment in hCO. (F) Contraction parameters of hCO in SF and DM. n = 3-4 experiments. (G) Representative force traces. (H) Time from peak to 50% relaxation (Tr50) with treatment of INCB054329 and danegaptide in DM hCO. n = 3-4 experiments. (I) Tr50 under acute 60 bpm pacing with treatment of INCB054329 and danegaptide in DM hCO. n = 3-4 experiments. (J) Contraction parameters with treatment of INCB054329 and danegaptide in DM hCO (as per E). n = 3-4 experiments. Kruskal-Wallis test with Dunn’s post-hoc analysis (J). Two-way ANOVA with Sidak’s post-hoc test between lines for DMSO or relative to DMSO for MCHTB11 (J). **** p < 0.0001 . (K) Principal component plot of proteomics data, n = 3-4 experiments with 3 hCO pooled in each. (L) Interaction map of regulated proteins in MTCHTB11 versus H.3 with highlighted circles also regulated by INCB054329 in MCHTB11. Two-way ANOVA with Sidak’s post-hoc test between lines for DMSO or relative to DMSO for MCHTB11 (H,l). **** p < 0.0001.

[0034] Figure 9 Characterization of DSP cardiomyopathy biopsy samples and generation of CRISPR-corrected hiPS cells. (A) Picrosirius red staining of cardiac biopsy samples from a healthy donor heart or at time of left ventricular assist device implantation for the patients. Scale bar, 500 pm. (B) Staining of cardiac biopsy samples in (A) for DSP and CX43. Scale bar, 20 pm. (C) Heat map of DSP signature protein expression in different human heart biopsy samples.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0035] For convenience, certain terms employed in the specification and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0036] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0037] The articles “a” and “an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0038] By “about’ is meant a quantity, level, value, frequency, percentage, dimension, size, or amount that varies by as much as 30%, preferably by as much as 20%, and more preferably by as much as 10% to a reference quantity, level, value, frequency, percentage, dimension, size, or amount

[0039] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein and / or a CTL provided herein.

[0040] The term “biological sample", “tissue sample", or simply “sample" each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.

[0041] As used herein “cardiomyocytes" are cardiac muscle cells (also known as myocard iocytes or cardiac myocytes) that make up cardiac muscle such as found in the atria and ventricles of the heart. Each myocardial cell contains myofibrils, which are the fundamental contractile units of cardiac muscle cells. Cardiomyocytes typically contain one or two nuclei, although they may have as many as four and a relatively high mitochondrial density, facilitating production of adenosine triphosphate (ATP) for muscle contraction. Myocardial infarction causes the death of cardiomyocytes. In adults, the heart’s limited capacity to regenerate these lost cardiomyocytes leads to compromised cardiac function and high morbidity and mortality.

[0042] As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising” , “comprises" and “comprised" , are not intended to exclude further additives, components, integers or steps.

[0043] The terms “differentiate" , “differentiating” , and “differentiated" , relate to progression or maturation of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway. It will be appreciated that in this context “differentiated” does not mean or imply that the cell is fully differentiated and has lost plu ropotentiality or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.

[0044] As will be well understood in the art, the stage or state of differentiation of a cell may be characterized by the expression and / or non-expression of one of a plurality of markers. In this context, by “markers” is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development. Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g., polymerase chain reaction) and nucleic acid hybridization (e.g., microarrays, Northern hybridization, in situ hybridization), although without limitation thereto. Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g., 2D gel electrophoresis), although without limitation thereto. Preferably, protein markers are detected by an antibody or antibody fragment (which may be polyclonal or monoclonal) that binds the protein marker. Suitably, the antibody is labeled, such as with a radioactive label, a fluorophore (e.g., Alexa dyes), digoxogenin or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), although without limitation thereto. Suitable markers, antibodies and fluorophores useful for marker detection according to the invention are known in the art, including as described in Table 1 and Table 2 of International Patent Publication No. WO 2018 / 035574 (the contents of which is incorporated by reference). Markers may alternatively be “metabolites" that are the product of metabolic processes accompanying cellular changes as a result of differentiation or development.

[0045] By “enriched" or “purified" is meant having a higher incidence, representation or frequency in a particular state (e.g., an enriched or purified state) compared to a previous state prior to enrichment or purification.

[0046] For the purposes of this invention, by “isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material (e.g., cells) may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

[0047] As used herein, a therapeutic that “prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0048] / ^“progenitor celf’ is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self-renewal.

[0049] The terms “human embryonic stem celf’, “hES celf’, and “hESC" refer to cells derived, obtainable or originating from human embryos or blastocysts, which are self-renewing andpluri- or totipotent, having the ability to yield all of the cell types present in a mature animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained from human in vivo preimplantation embryos, in vitro fertilized embryos, or one-cell human embryos expanded to the blastocyst stage.

[0050] The terms “induced pluripotent stem cell”, and “iPSC” refer to cells derivable, obtainable, or originating from human adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, KLF4, LIN28, Glisl, and c-Myc, although without limitation thereto.

[0051] As generally used herein, the term “serum" refers to a substantially cell-free proteinaceous blood fraction obtained or obtainable from an animal (e.g., fetal bovine serum) and does not include purified or recombinant synthetic serum components such as albumin. In this context “serum free”, as a serum free medium, means a complete absence of serum or may refer to no more than about 1 %, 0.5%, 0.2%, or 0.1 % (v / v) serum.

[0052] As used herein, “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a peptide to bind to its predetermined binding partner. Typically, an antibody or peptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 107M or less, and binds to the predetermined antigen / binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000-fold less than its affinity for binding to a non-specific and unrelated antigen / binding partner (e.g., BSA, casein).

[0053] As used herein, the term “subject’ means a human or non-human animal selected for treatment or therapy.

[0054] The phrases “therapeutically effective amount’ and “effective amount’ as used herein means the amount of an agent which is effective for producing the desired prophylactic and / or therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit / risk ratio applicable to any medical treatment.

[0055] As used herein, “treating” (or “treat’ or “treatment”) refers to a therapeutic intervention that ameliorates a sign or symptom of a HPV infection, inclusive of a HPV-associated disease, disorder or condition, after it has begun to develop. The term “ameliorating”, with reference to a HPV-associated disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.General

[0056] The present invention is predicated at least in part on the inventor’s realization of the effects of metabolism on cardiac maturation. One aspect of the technology is the determination of improved 3D culture conditions that promote structural, electrophysiological, metabolic andproliferative maturation of hPSC-derived cardiomyocytes and cardiac organoids. The present invention is therefore directed to a method and / or culture medium for facilitating structural, electrophysiological, metabolic and / or proliferative maturation of stem cell-derived cardiac tissue, such as in the form of cardiac organoids.

[0057] In certain aspects, the invention is broadly directed to a cell culture medium and / or method suitable for differentiating cardiac cells, such as card io myocytes and cardiac organoids, from progenitor cells such as human embryonic stem cells or induced pluripotent stem cells.

[0058] In one aspect of the invention provides a cardiac cell maturation medium comprising a base medium, an AMPK activator, a fatty acid substrate, and a carbohydrate source.

[0059] Another aspect of the invention provides a cardiac cell culture system comprising: the cardiac cell maturation medium as described above an / or elsewhere herein; and a cardiac cell culture vessel comprising a plurality of wells that each comprise opposed poles that extend substantially perpendicularly from a basal surface of the well.

[0060] Yet another aspect of the invention provides a method of culturing cardiac cells, said method including the step of contacting one or more progenitor cells with the cardiac cell maturation medium as described above and / or elsewhere herein, for sufficient time and under suitable conditions to induce or promote maturation of the one or more progenitor cells into cardiomyocytes.

[0061] A particular feature of the cardiac cell culture medium disclosed herein is the selection of components or constituents that optimise the maturation of cardiomyocytes and cardiac organoids.

[0062] Initial steps of the method differentiate progenitor cells, such as hESC or iPSC, into cardiac mesoderm and then into cardiomyocytes. Initially, progenitor cells are differentiated into cardiac mesoderm in a culture medium comprising a serum-free base medium (such as RPMI) together with a supplement such as B27 at a preferred concentration of about 2% (v / v), in the absence of insulin. In some embodiments, the first culture conditions further comprises one or more additional agent selected from a group comprising ascorbic acid 2 phosphate, BMP-4, Activin A, FGF-2, and a GSK-3 inhibitor (such as CHIR99021). Subsequently, cells are differentiated into cardiomyocytes in the presence of a Wnt inhibitor such as IWP-4 followed by addition of insulin and ascorbic acid 2 phosphate in supplemented medium (e.g., RPMI + 2% B27) until collagenase digestion at about 15 days.

[0063] The collagenase-digested cardiomyocytes may then be cultured in the cardiac cell maturation medium of the present invention. In some embodiments, the early-stage cardiac maturation medium comprises a base medium which may be any serum-free medium such as aMEM, DMEM, Iscove’s medium, or RPMI 1640. In some embodiments, the base medium is MEM alpha GlutaMAX.

[0064] Suitably, the initial conditions for early cardiomyocyte maturation is serum-free. The early-stage cardiac cell maturation medium may further comprise a supplement such as, but not limited to, B27 (GIBCO). In a preferred embodiment, the cardiac cell maturation medium comprises albumin, such as purified or recombinant bovine serum albumin or human serum albumin. The albumin may be present in supplements (such as B27, GIBCO) or may be included as a separate component of the medium. Suitably, the concentration of albumin is preferably no less than about 2 mg / mL, less than about 1 mg / mL, less than about 0.5 mg / mL, less than about 0.2 mg / mL or as low as about 0.1 or about 0.05 mg / mL. The cardiac cell maturation medium may further comprise L- ascorbic acid 2-phosphate.

[0065] In some particularly preferred embodiments, the initial conditions for early cardiomyocyte maturation comprise DMEM, no glucose, no glutamine, no phenol red (ThermoFisher Scientific) supplemented with 1 x GlutaMAX (ThermoFisher Scientific), 200 pM I-AA2P, 1% penicillinstreptomycin, 4% vol / vol B27 without insulin (ThermoFisher Scientific), 10 ng / mL FGF-2, 10 ng / mL PDGF-BB, 33 pg / mL aprotinin (Sigma or MedChemExpress), 100 pM palmitate (conjugated to bovine serum albumin in B27, Sigma), and 1 mM glucose.

[0066] The cardiac cell maturation medium may be suitable for producing cardiac cell suspensions, monolayers or two-dimensional “2D” cultures. In other embodiments where a three- dimensional (“3D”) cardiac tissue or organoid is to be produced, before culture in the cardiac cell maturation medium referred to above, cells are cultured in a gelling medium comprising serum- free base medium and supplement such as B27 (comprising albumin, but no insulin) as described above and extracellular matrix (ECM) or components thereof. As broadly used herein, ECM refers to a matrix or web of molecules located outside or external to cells that regulate cell-cell communication, cell signalling, cell adhesion, spacing, location and / or orientation, although without limitation thereto. The molecular components of ECM may include proteoglycans, heparan sulphate, chondroitin sulphate, keratin, collagens (e.g., types l-XIV), elastins, laminin and fibronectin, although without limitation thereto.

[0067] The earliest stage of cardiomyocyte maturation is preferentially glycolytic in the early stages of development, which switches to a metabolism that is almost exclusively oxidative at maturity. The optimized cardiomyocyte maturation medium replaces the fatty acid oxidative conditions of the intermediate maturation phase.

[0068] Cardiomyocytes are then matured in the optimized cardiac maturation medium, which comprises an AMPK activator, a fatty acid substrate, a carbohydrate source, and albumin. The fatty acid substrate may be any carboxylic acid with a saturated or mono- or poly-unsaturated aliphatic chain that is capable of acting as a substrate for oxidative fatty acid metabolism in a mammalian cell. Preferably, the fatty acid has an aliphatic chain that comprises 12 to 20 carbon atoms (i.e., C12- C20 fatty acids). Preferably, the fatty acid has an aliphatic chain that comprises 16 or 18 carbon atoms (i.e., a Cis or Cis fatty acids). The fatty acid substrate may be palmitic acid, linolenic acid, linoleic acid, or oleic acid, although without limitation thereto. In a particular embodiment, the fattyacid substrate is, or comprises, palmitic acid. The fatty acid such as palmitic acid may be present at a concentration in the range of about 1 to 1000 pM, 5 to 500 pM, or 5 to 100 pM. Preferably, the fatty acid (e.g., palmitate) is present at a concentration in the range of about 5 pM to about 15. A preferred concentration of a fatty acid substrate, such as palmitate, is about 10 pM.

[0069] In some embodiments, the carbohydrate source is, or comprises, glucose. It will also be understood that glucose includes D-glucose and any open chain, chiral and / or cyclic isomers of D- glucose that may act as substrates for glycolysis in a mammalian cell. A preferred concentration of glucose is about 1 to 50 mM, or advantageously about 5.5 mM.

[0070] The fatty acid substrate and carbohydrate source may be present at a concentration ratio of about 1 :1000 to about 1 :100, about 1 :700 to 1 :250. Preferably, the fatty acid substrate and carbohydrate source are present at a concentration ratio of about 1 :550.

[0071] In some embodiments, the cardiac cell maturation medium comprises insulin. Suitably, the concentration of insulin is a concentration of between about 0.5 nM to about 150 nM. Preferably, the concentration of insulin is about 1 nM. In some embodiments, the medium comprises insulin at a concentration of at least 0.5 nM.

[0072] In some preferred embodiments, the optimized maturation media comprises 5.5 mM glucose with 1 nM insulin and 10 pM palmitate.

[0073] The maturation medium preferably comprises one or both of an ERRp / y agonist and / or interferon-M (IFN-M). Some suitable examples of ERRp / y agonists include, but are not limited to DY131 , SLU-PP-332, ERRy inverse agonist 1 , and SR19881 . However, any alternative ERRp / y agonist known in the art would be suitable for use with the present invention. In some embodiments, the ERRp / y agonist is DY131 and present at a concentration of between about 1 pM and 10 pM. In even more preferred embodiments, DY131 is included in the maturation medium at a concentration of 3 pM.

[0074] In some embodiments, the cardiac cell maturation medium comprises an AMPK activator. AMPK is an evolutionarily conserved metabolic sensorthat plays a vital role in regulation of energy balance. AMPK is expressed in all tissues as a heterotrimer composed of a catalytic a- (isoforms 1 or 2) subunit and p- (1 or 2) and y- (1 , 2, or 3) regulatory subunits. In some preferred embodiments, the AMPK activator is a pan-AMPK activator. By “pan-AMPK activator” means that the inhibitor selectively binds and inhibits at least two or more isoforms of AMPK (e.g., AMPKpl , AMPKp2). In some embodiments, the AMPK activator inhibits AMPKpl and AMPKp2. In some embodiments, the AMPK activator inhibits AMPKp2. In some embodiments, the AMPK activator is selected from the group comprising MK-8722, Activator 3 (2-(2-(4-(trifluoromethyl) phenylamino) thiazol-4-yl)acetic acid, 991 (Merk Sharpe & Dohme), C2 (Metabasis Therapeutics), and MT 63-78 (Mercury). In some particularly preferred embodiments, the AMPK activator is MK-8722.

[0075] In some embodiments, the AMPK activator is present in the maturation media at a concentration of between about 0.1 pM and 100 pM. More particularly, the AMPK activator is present at a concentration of around 0.5 pM to around 50 pM. In particular embodiments, the AMPK is present at a concentration of 10 pM. In some embodiments of this type, the AMPK activator is MK- 8722.

[0076] Although not wishing to be bound unnecessarily by theory, it is proposed that the heart shifts from a metabolism which is preferentially glycolytic in the early stages of development to a metabolism which is almost exclusively oxidative at maturity. Circulating levels of substrates thus play a major role in establishing the glycolytic metabolism encountered in the fetal heart since this stage of development is marked by a low level of fatty acids and a high level of lactate in the blood. Prenatal metabolism is characterized by a predominant use of carbohydrate and fatty acid oxidation contributes only about 15 % of total energy production. Adult cardiomyocyte metabolism is almost exclusively oxidative (about 90% of total energy production). The maturation media described above and / or elsewhere herein is effective following (but not during) the phase where hCO are metabolically switched to fatty acid oxidation. Data presented in the Examples suggest that this may be because during this phase the cardiomyocytes are already near maximum capacity as indicated by our data profiling the oxidation over the full hCO culture time-course, and further metabolic stress is detrimental during this phase. These conditions appear to favour cardiomyocyte maturation. However, a complete absence of fatty acid substrate is not preferable, in which case a particular fatty acid substrate:carbohydrate ratio is preferred, as hereinbefore described.

[0077] Typically, the AMPK activator and ERRp / y agonist are provided to the cell culture at intervals during the final stages of hCO maturation (see, for Example, Figure 1 A). This ensures that the AMPK activator and ERRp / y agonist remain available to the hCO at a sufficient amount to reach maximum maturation

[0078] Suitably, the cardiac cell maturation medium does not comprise TGF-p1 or comprises a minimal concentration or amount of TGF-p1 . Suitably, the minimal concentration of TGF is less than about 2 ng / mL or preferably no more than about 1 ng / mL. Suitably, the minimal concentration of TGF- p1 , if present, is for less than five days of culture, preferably the initial five days of culture. As will be described in more detail in the Examples, the prolonged presence of TGF-p1 within the maturation media caused significant cell death and compromised the contractile function of the cardiac organoids.

[0079] Suitably, the cardiac cell maturation medium does not comprise a fibroblast growth factor (FGF) such as FGF-2, or comprises a minimal concentration or amount of FGF-2. In some of the same embodiments, the cardiac cell maturation medium does not comprise PDGF-BB, or comprises a minimal amount of PDGF-BB.

[0080] In some preferred embodiments, the cell culture medium comprises DMEM, no glucose, no glutamine, no phenol red. The composition of DMEM, no glucose, no glutamine, no phenol red is as follows:TABLE 1DMEM, no glucose, no phenol red, no glutamine formulation

[0081] In some embodiments of this type, the optimised cardiac cell maturation medium also comprises L-alanyl-L-glutamine dipeptide (e.g., GlutaMAX™, ThermoFisher Scientific). Furthermore, the cell culture medium may also comprise penicillin-streptomycin (e.g., 1% penicillin-streptomycin). Furthermore, the cell culture medium may also comprise B27 without insulin (e.g., 4% v / v B27). In some embodiments, the cell culture medium further comprises aprotinin (e.g., 33 pg / mL aprotinin). Accordingly, in some particularly preferred embodiments of the invention, the optimised cardiac cell maturation medium comprises DMEM, no glucose, no glutamine, no phenol red (ThermoFisher Scientific) supplemented with 1 x GlutaMAX (ThermoFisher Scientific), 200 pM I-AA2P, 1% penicillin- streptomycin, 4% vol / vol B27 without insulin (ThermoFisher Scientific), 33 pg / mL aprotinin (Sigma or MedChemExpress), 10 pM palmitate (conjugated to bovine serum albumin in B27, Sigma), 5.5 mM glucose, and 1 nM recombinant human insulin (Gibco).

[0082] Notably, for directed maturation conditions, 2 pM CHIR99021 was added for the formation phase in the first two days for hCO formation, and the future media changes were instead done with weaning medium supplemented with 3 pM DY131 and 10 pM MK8722.

[0083] A related aspect of the invention provides a method of identifying one or more molecules that modulate cardiac cell maturation, said method including contacting one or more cardiomyocytes in the cardiac cell culture medium disclosed herein with one or more candidate molecules, whereby modification of the maturation of the one or more cardiomyocytes indicates that the candidate molecule is a modulator of cardiac cell maturation.

[0084] In some embodiments, the modulator at least partly enhances or promotes cardiac cell maturation. As will be appreciated from the foregoing, such modulators include AMPK activators,a carbohydrate source (such as glucose) and a fatty acid substrate (such as palmitate), particularly when present at a particular ratio. It will be appreciated that other modulators that at least partly enhance or promote cardiac cell maturation may be identified according to this method.

[0085] In another embodiment, the modulator at least partly inhibits or suppresses cardiac cell maturation. As will be appreciated from the foregoing, such modulators include relatively high concentrations of TGF-p1 for prolonged periods. It will be appreciated that other modulators that at least partly inhibit or suppress cardiac cell maturation may be identified according to this method. It will be understood that “at least partly inhibits or suppresses cardiac cell maturation” includes modulators that are toxic to cardiomyocytes and / or lead to cell death (e.g., inducers of cardiomyocyte apoptosis).

[0086] In some embodiments the cardiomyocytes have been differentiated from progenitor cells. The progenitor cells may be, or comprise, human embryonic stem cells or induced pluripotent stem cells.Methods of Screening

[0087] It will be appreciated that this aspect of the invention provides a method or system for identifying, assaying or screening candidate molecules that may modulate cardiomyocyte maturation. Candidate molecules may be present in combinatorial libraries, natural product libraries, synthetic chemical libraries, phage display libraries, lead compound libraries and any other libraries or collections of molecules suitable for screening. Alternatively, the candidate molecule may be a candidate molecule currently in clinical development in order to assess the capabilities and toxicity of the candidate molecule.

[0088] A further aspect of the invention provides one or more cardiomyocytes or cardiac tissues or organoids comprising same, produced by the method disclosed herein.

[0089] As described previously, the cardiac cell maturation medium, maturation system and method may be suitable for producing cardiac cell suspensions, monolayers or “2D cultures”.

[0090] In other particular embodiments, the cardiac cell maturation medium and method may be suitable for producing cardiac muscle tissue in three dimensional (3D) structures such as cardiac “organoids”. Organoids may be used for producing engineered or artificial cardiac tissue. For example, cardiac organoids may be incorporated within a scaffold, such as a de-cellularised human heart, polyester fleece or biodegradable polymer scaffold, to thereby produce a cardiac 3D structure. Also contemplated are “bioprinted” 3D cardiac structures.

[0091] By way of example only, an organ printing machine has been developed which uses a hydrogel scaffold to place human cells in a desired orientation to recreate human organs.

[0092] It will also be appreciated that the cardiomyocytes and / or cardiac organoids described herein may provide potential sources of purified, differentiated cardiomyocytes for cellulartherapy of the heart. In a particular embodiment, iPSC lines derived, obtained or originating from a patient with a genetic cardiac defect or disease may be used for repair of genetic mutation(s) in vitro. Such cardiac cells or organoids could be used according to the method of the invention and then administered to the patient for autologous cellular therapy. In some embodiments, cardiac cells, EHT or organoids produced according to the invention may be administered directly to the heart in the form of a tissue patch, mat, plug, bolus or other implantable form.

[0093] It will also be appreciated that the cardiomyocytes and / or cardiac organoids described herein may provide potential sources of purified, differentiated cardiomyocytes for cardiac disease modelling. By way of example, the effect of genetic defects upon heart function may be investigated, such as by determining the contractile properties of cardiac organoids comprising cardiomyocytes having the genetic defect. In a further embodiment, the efficacy of drugs or other molecules in treating or correcting the genetic defect may be assessed cardiomyocytes and / or cardiac organoids described herein.

[0094] In other embodiments, cardiomyocytes and / or cardiac organoids described herein may be used in applications such as patient specific cardiac disease modelling and cardiac biology, such as modelling, investigating or predicting the effects of modulating gene expression (e.g., gene “knock out”, “knock-down” or over-expression).

[0095] Accordingly, a particular aspect of the invention provides a method of determining, assessing or monitoring the effect of one or more molecules upon a cardiac cell, tissue or organoid, said method including the steps of contacting the cardiac cell, organoid or engineered heart tissue produced according to the method disclosed herein with the one or more molecules and determining assessing or monitoring the effect of the one or more molecules upon the cardiac cell, organoid or engineered heart tissue.

[0096] It will be appreciated that this aspect of the invention provides a method for determining, assessing or monitoring the effect of one or more molecules upon a cardiac cell, tissue or organoid. The effect may be, or relate to, therapeutic efficacy in treating diseases or disorders of the heart, drug dosage determination, toxicity and / or safety (e.g., assessing side effects of a drug) and contractile properties of the cardiac cell, tissue or organoid, although without limitation thereto.

[0097] The one or more molecules may be known or pre-existing drugs or may be present in combinatorial libraries, natural product libraries, synthetic chemical libraries, phage display libraries, lead compound libraries and any other libraries or collections of molecules suitable for the method.

[0098] In particular embodiments of the method, cardiomyocytes and / or cardiac organoids may be useful for toxicity screening or for in vitro drug safety testing. There are several drugs and other molecules that are cardiotoxic, particularly causing cardiac arrhythmias, cardiomyopathy and / or acute coronary syndrome. These include cisapride, Ca2+, K+and Na+channel blockers, p-blockers and chemotherapeutic agents such as anthracyclines. Drugs may be screened against cardiomyocytes and / or cardiac organoids to determine general cardiotoxocity or to determine ifcardiomyocytes or organoids obtained from progenitor cells of a particular individual display sensitivity, or not, to potentially cardiotoxic drugs or other molecules or compounds.

[0099] As previously described, in some embodiments the cardiac cell, tissue or organoid may be obtained from progenitor cells of an individual having one or more particular genetic defects. By way of example, the invention contemplates a “genetic background test” where a candidate drug or other molecule could be tested against cardiomyocytes and / or cardiac organoids disclosed herein having different genetic backgrounds to determine whether there are differential drug efficacies and / or side effects that correlate with a particular genetic background. This may enable selection of appropriate drug therapies for patients with a particular genetic background.

[0100] In some embodiments, methods of assessing the ability of a candidate agent to impact on the force of a cardiomyocyte are performed as described above and / or elsewhere herein. Such methods are also described in detail in Pieske, B. et al. (1996), which is herein incorporated by reference in its entirety.

[0101] By way of an illustrative example, such methods include those for determining the effect on force of a candidate agent, the method comprising: applying a first pacing condition to a cardiac organoid produced by the method or cardiac organoid described above or elsewhere herein; blocking spontaneous contractile activity of the cardiac organoid; contacting the cardiac organoid with the candidate agent; pausing the pacing of the cardiac organoid; and applying a second pacing condition to the cardiac organoid; wherein the effect on force of the cardiac organoid during the second pacing condition determines the effect on force of the candidate agent. For example, an increase in force is indicative that the candidate agent has a positive effect (increases) force, whereas a decrease in force is indicative that the candidate agent has a negative effect on force.

[0102] In some embodiments, the spontaneous contractile activity is blocked by funny current ( / f) blockade. In some embodiments of this type, the spontaneous contractile activity blocking is performed by exposing the cardiac organoid to cilobradine. Cilobradine causes contraction to stop, but still leaves the tissues capable of pacing (just like adult heart). After such treatment the cardiac organoids much be electrically paced for contraction.

[0103] On stopping the electrical pacing, the cardiac organoids begin accumulating calcium in the SR. This will either leak, causing ectopic beats (indicative of arrhythmia) or build up in the SR.

[0104] Accordingly, in some embodiments the continued contraction of the cardiac organoid during the pacing pause step is indicative of whether the cardiac organise is disposed to a risk ofarrhythmia. Therefore, the method can be used to determine whether the candidate agent can reduce or prevent the risk of arrhythmia.

[0105] In some embodiments, the first pacing condition and the second pacing condition are the same. In alternative embodiments, the first pacing condition and the second pacing condition are different. Typically, the pacing is performed by an electrical current, which may be between around 0.5 Hz and 3 Hz. By way of an example, the current maybe at 0.5 Hertz.

[0106] Upon initiating the second pacing condition, the first beat is typically large because of the build-up calcium in the SR for non-arrhythmic tissues. Thus, a decreased beat can be used to determine that a drug is not an effective treatment or prevention for arrythmia.

[0107] In some embodiments, the cardiac organoid comprises a gene mutation identified in tachyarrhythmias (e.g., polymorphic ventricular tachycardia). By way of an illustrative example, the gene mutation is in the CASQ2 gene or the RYR2 gene.

[0108] In some embodiments, the gene mutation is in the desmoplakin gene (DSP). By way of an illustrative example, the DSP gene may be a two base pair deletion at positions 4246 and 4247 (resulting in Leu1416Asn substitution, and a stop codon after 22 amino acids in the new reading frame)

[0109] So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting Examples.EXPERIMENTAL

[0110] The present inventors recently developed a serum-free (SF) protocol for hCO maturation (SF-hCO), featuring switching of metabolic substrates and vascularisation promoting growth factors (Voges, 2023). The inventors compared SF-hCOs to various other hPSC-CM 3D cultures using mRNA expression ratios of sarcomeric proteins that are indicative of maturation (Mills, 2017; Hofbauer, 2021 ; Giacomelli, 2020; Zhao, 2019; Shen, 2022). MYH7 as a fraction of MYH7 and MYH6, did not correlate with maturation in any system, and is perhaps rate dependent (Figure 1 A). MYL2 as a fraction of MYL2 and MYL7 (Figure 1 B) and TNNI3 as a fraction of TNNI3 and TNNI1 (Figure 1C) were stronger indicators, with TNNI3 in particular correlating strongly with maturation and being elevated by pacing protocols that are proven methods of maturation (Zhao, 2019; Shen, 2022). It is noted that the SF-hCO displayed advanced levels of maturation as a strong baseline to screen for further maturation (Figure 1C).

[0111] The present inventors previously performed transcriptional profiling of human heart maturation identified a key oxidative metabolism and interferon signature that strongly underpins cardiomyocyte maturation (Sim, 2021). In the current study, the inventors screened for conditions that promote these features and determined their impact on cardiac maturation. Cardiac maturation is characterised by many different parameters, with different stimuli influencing distinct maturational properties (Mills, 2019; Karbassi, 2020). The inventors focused on key functional changes that occurwith maturation - increased force, decreased automaticity and unaltered or reduced time to 50% relaxation (Tr50) and mature sarcomeric markers - cTnl (TNNI3) (Bedada, 2014).AMPK and ERR agonists drive maturation

[0112] The present inventors firstly determined critical factors regulating metabolism or interferon signalling for a factorial maturation screen. 10 pM progesterone, 3 pM DY131 (ERRp / y agonist), 10 pM MK8722 (AMPK activator), and 100 ng / mL IFN-A1 were identified (Figure 1A). Conditions with MK8722 consistently reduced rate without impacting Tr50 (Figure 1 B). MK8722 combined with DY131 or IFN-A1 increased cTnl (Figure 1C). When MK8722 and DY131 were further combined with either progesterone or IFN-A1 , there was no additional increase in cardiac-specific troponin I (cTnl) (Figure 1C). The timing of DY131 and MK8722 addition was also assessed (Figure 1 D-H). If added during the maturation medium period (days 17-22) or immediately after this period at the start of the weaning medium period (days 22-27), the rate was still reduced but cTnl increase was limited. The inventors next assessed the impact of the most abundant fatty acids in human breast milk and / or blood (Sarkadi, 2022; Zeleniuch-Jacquotte, 2000) on maturation (Figure 1 M-Q). Fatty acids were used during maturation (100 pM) and weaning media (10 pM) phases including palmitic acid, linoleic acid, oleic acid, myristic acid, and an equimolar combination of all four. It was found that all fatty acids performed equally well, except for a combination of all four causing functional decline. The inventors therefore continued to use palmitic acid for the rest of the experiments.

[0113] The inventors next defined the new maturation conditions and designated it directed maturation (DM-hCO). In summary, the key additions to our SF-hCO conditions (Voges, 2023) are the addition of 2 pM CHIR99021 during the first 2 days of hCO formation and transient four-day addition of 3 pM DY131 combined with 10 pM MK8722 (days 24-28). The ability of these conditions to reduce rate and increase cTnl was confirmed in multiple additional hPSC cell lines (Figure 11-L). To determine whether these conditions are broadly applicable to the field, the inventors assessed the impact of our protocol in a 2D culture. The addition of MK8722 during a four-day period (days 24-28) was also sufficient to reduce rate and increase cTnl in 2D cardiomyocytes (Figure R-T).

[0114] As AMPK and ERR agonism may drive metabolic programs similar to pacing, the inventors compared DM-hCOs to paced hCOs using phosphoproteomics (Figure 1 U-X). The inventors utilized a custom platform, in which HeartDyno inserts into a C-pace system in 24-well plates enabling 120 bpm pacing for 5 minutes, without causing toxicity. Phosphoproteomics revealed that there was substantial overlap of 1 ,024 (48.2% of regulated) phosphosites shared between DM and electrical pacing (Figure 1Y). Key phosphosites were increased in both conditions including HDAC2 S394 (Pedram, 2013), GJA1 (CX43) S365 (Solan, 2007), MLC2v S15 (Sheikh, 2023), and PLN S16 / T17 (Traaseth, 2008) (Figure 1Z). In particular PLN S16 / T17 and MLC2v S15, which have been shown to drive increased SR calcium cycling and increased contractile force (Sheikh, 2012; Traaseth, 2008) respectively. Similar KEGG pathways were activated in both DM and electricallypaced conditions (Figure 1 ), including an AMPK network (Figure 1 B). This results in inhibition of the mevalonate / cholesterol biosynthesis program. hCO Display Cellular Complexity Consistent with Human Hearts

[0115] Using snRNA-seq and integrated clustering with previous human cardiac maturation datasets, the inventors found that multiple cardiac cell types including cardiomyocytes, endothelial cells, smooth muscle cells, fibroblasts and epicardial cells all co-cluster (Figure 2A). Thus, the single optimised differentiation-hCO protocol (Voges, 2023) yields a complex mixture of cells similar to native human heart tissue, but with a higher percentage of cardiomyocytes, lower percentage of endothelial cells and lack of immune cell populations (Figure 2A) (and neural population at higher clustering resolution).

[0116] Further exploring fibroblast identity, the inventors found some expression of the epicardial gene TCF21 in the fibroblastic population indicative of epicardial origins (Figure 2B). As TCF21 is reduced during differentiation and maturation (Fernandes, 2023), the inventors created a TCF21 lineage tracing tool (Figure 2C). The inventors found that early, but not late lineage tracing marked the epicardial cells surrounding the hCOs along with some infiltrating cells (Figure 2D,F). Thus, hCO contain TCF21 derived epicardium and stromal cells. hCO display complex cell-cell interactions

[0117] The DM-hCO protocol does not alter the cellular composition, and immunostaining confirmed the presence of the major cardiac cell types in the hCOs including cardiomyocytes, epicardial cells, endothelial cells, pericytes and fibroblasts (Figure 2A). When clustering hCOs alone, the nuclei also segregate into distinct cellular populations (Figure 3A), which are demarcated by canonical marker genes (Figure 3B). The hCO MYH6+ cardiomyocytes express critical lfchannel genes HCN1 and HCN4 and a critical pacemaker transcription factor SHOX2 (Espinoza-Lewis, 2009). This population likely represents the pacemaker population within the hCOs, which the inventors had previously confirmed using patch-clamping on isolated cells (Mills 2017).

[0118] During the hCO protocol the inventors only added exogenous growth factors FGF2 and PDGF-BB during the first week of hCO culture. This suggests that maintenance of the fibroblasts and endothelial cells occur via endogenous paracrine factor production. Consistent with paracrine support, cardiomyocytes express FGF10 for the fibroblast receptor FGFR2. While PDGFRB was strongly expressed by the fibroblasts, the ligand PDGFB was expressed at very low levels. This is potentially why over-stimulation of PDGF-BB can lead to fibroblast overgrowth and irregular contraction patterns (Voges, 2023).

[0119] The endothelial cells have strong expression of key endocardial markers including NRG1, NFATC1 and GATA4, whilst no expression of coronary markers APLN and FABP4. The endothelial cells and some fibroblasts express FLT1, and cardiomyocytes expressing its ligandVEGFA, which is potentially why additional VEGF-A does not further support additional endothelial cells in hCOs (Voges, 2023).DM Induces Sarcomeric and Metabolic Maturation

[0120] When comparing the most populous cardiomyocyte cluster in SF-hCOs and adult hearts are already similar, and there are only 185 and 98 transcripts two-fold lower or higher, respectively. Therefore, cardiomyocyte populations tightly clustered with human heart isolated cardiomyocytes (Figure 3C), so the inventors instead compared transcriptional changes. The DM- hCO protocol increases 20 and decreases 37 of these transcripts >log2|0.2| towards adult heart expression, respectively. Thus, indicating maturation, including a repression of immature cardiac genes highlighted by increased TNNI3 as a fraction of TNNI3 and TNNI1 of 0.43, surpassing all other models (Figure 3L-M).

[0121] DM induced transcriptional changes predominantly in the cardiomyocyte and fibroblast populations in hCO (Figure 3D). Gene ontology analysis on Cardiomyocytes 1 and 2 revealed numerous terms related to heart contraction and development were upregulated in DM- hCOs (Figure 3E). This includes key sarcomeric maturation markers TNNI3 and MYL2 (Figure 3F) and key transcriptional regulators FOXP1, SOX6, and CSRP3 controlling heart development (Wang, 2004; Saleem, 2020; Vafiadaki, 2015). Genes upregulated in DM-hCO compared to SF-hCO in Cardiomyocytes 3 were enriched for gene ontology terms mostly associated with oxidative phosphorylation (Figure 3G). This includes direct ERRa / y target genes particularly those involved with mitochondrial electron transport (COX5A, COX5B, COX6A1, COX7C, COX8A and CYCS) and ATP synthesis (ATP5F1D, ATP5F1B, ATP5PO, ATP5MC1, ATP5PD, ATP5MC3 and ATP5PF). Realtime monitoring of intact-cell respiration revealed similar changes in respiration upon maturation of SF and DM hCO, which peaks during the maturation medium phase (Figure 3H). However, spare respiratory capacity was higher in DM hCO, assessed by treatment with the mitochondrial uncoupler BAM15 at the end of the maturation protocol (Figure 3I). Together, DM enhanced metabolic capacity consistent with cardiac maturation in vivo.

[0122] To assess whether these changes occurred homogenously across the tissue spatial transcriptome analysis using the STOMICS platform identified 953 genes, which were mostly cardiomyocyte transcripts. The cardiomyocytes were spread throughout the hCO in both SF and DM conditions (MYH7), whereas the MYH6+ pacemaker cardiomyocytes were present sporadically throughout the hCO with some clustered regions which may help facilitate endogenous contractile activity (Figure 3J). DM treated hCO had uniform induction of the sarcomeric maturation markers MYL2 and TNNI3 and metabolic genes MT-ND1 and COX7C (Figure 3K). This highlights that the DM conditions penetrate the hCO effectively, there are limited diffusion barriers in this miniature system and that Cardiomyocytes 3 is not confined to a particular hCO region.

[0123] While the DM stimulation primarily activates transcription in the cardiomyocytes, there are also some regulated genes in the stromal cells. Some of these are also fibroblast sub-typespecific such as C0L15A1, C0L19A1 and TGFBR3 which may play a key role in extracellular matrix biology and paracrine interactions.DM decreases automaticity in hCO

[0124] Immature hPSC-CMs typically display robust automaticity by depolarization largely driven by membrane currents with some sarcoplasmic reticulum (SR) contribution (Kim, 2015; Marchiano, 2023). Engineered heart tissues display increased maturity, and the automaticity has been shown to be regulated by both It and SR calcium cycling clocks (consistent with experiments in rabbit sinoatrial nodal cells) (Mannhardt, 2016; Bogdanov, 2001). In this series of experiments, the inventors investigated whether hCO rate is controlled by similar processes to understand why DM- hCO have reduced rates. Both SF and DM-hCO have similar calcium sensitivities for force and no calcium-rate relationship, thus ruling out changes in calcium sensitivity (Figure 4A). The inventors blocked cardiac pacemaker current (A) using 1 pM cilobradine because <10 pM ivabradine only partially blocks lfand prolonged Tr50 at higher concentrations (data not shown). Following blockade of h with 1 pM cilobradine, hCOs became dormant with ‘bursts’ of activity which are far less common in DM-hCOs in comparison to SF-hCOs (Figure 4B). This indicates that DM-hCO are more stable under 1 pM cilobradine blockade.

[0125] To determine the role of SR cycling in pacing, the inventors first demonstrated that hCO have a functional SR. The ability to block contractile activity in DM-hCOs allowed us to perform post-rest-potentiation experiments, which enable measurement of functional SR in cardiac muscle (Pieske, 1996). After a 10 second pause the increase in force of the first re-paced contraction is indicative of increased SR filling. In DM-hCOs this was 36% (Figure 3C), which is similar to human hearts (43%) at the same time interval (Pieske, 1996). This increase no longer occurs when sarcoendoplasmic reticulum calcium ATPase (SERCA) is inhibited using 5 pM thapsigargin, thus confirming a functional SR in DM-hCOs (Figure 4C). Three-dimensional transmission electron microscopy of a random section in a DM-hCO also revealed the presence of an extensive SR network (Figure 4G) and was confirmed in both SF- and DM-hCOs (Figure D-F).

[0126] The present inventors next sought to determine the contribution of SR calcium release on rate control by blocking type-2 ryanodine receptor (RR2) with 25 pM ryanodine, a concentration that sufficiently blocks the channel. This decreased the rate with a larger magnitude in DM-hCOs (15%) compared to SF-hCOs (6%), indicating greater basal RR2 leakiness in SF-hCOs (Figure 4H). Force remained constant and Ta50 increased, which remained elevated when paced at 60 bpm to correct for frequency-dependent acceleration of relaxation (Figure 4I). To further confirm the role of SR handling in dictating hCO rate, the inventors knocked out the SR calcium buffering protein calsequestrin 2 (CASQ2) using CRISPR gene editing. In comparison to an isogenic control, rate declined in both SF- and DM-hCOs (Figure 4J). There were no changes in force, and Ta50 was elevated in DM-hCOs when paced at 60 bpm (Figure 4J). These consistent forces combined with increased Ta50, is consistent with SR blockade in larger mammals (that are less reliant on SR calcium) and experiments performed with higher extracellular calcium concentrations (Mannhardt,2016; Sutko, 1980; Chiesi, 1994). This mechanism of increased Ta50 is potentially due to L-type calcium channel compensation (Xia, 2024; Chung 2018). Together, this confirms that hCOs have a functional SR that regulates endogenous pacing rate, and this is potentially less leaky in DM-hCOs and why the SF-hCOs undergo rapid bursts with 1 pM cilobradine.

[0127] In the DM-hCOs where spontaneous contractions are less frequent, the inventors were able to determine whether SR leakiness contributes to contractile instability. For this, the inventors knocked out the SR calcium buffering protein CASQ2 and also generated a pathogenic variant / ?Y / ?2+ / N4104Kthat creates RR2 leak and arrhythmia in human patients using CRISPR approaches. Using the post-rest potentiation protocol, ectopy in CASQ2-'- and RY / ?2+ / N4104KDM-hCOs was observed (Figure 4K).

[0128] These changes between SF- and DM-hCOs are underpinned by multifactorial expression changes. In cardiomyocytes PLN was increased (Figure 4L), which may be in part responsible for the reduced SR impact on rate and reduction in SR leak in DM-hCOs. However, there were also additional changes that could also play a role including decreased expression of CACNA1D (with a respective increase in CACNA1C), and increased GJA1 (CX43) expression (Figure 5 and Figure 4L). A reduction in CACNA1D activity has been shown to reduce rate in nodal cells (Baig, 20111), and CX43 enhances cell-cell coupling, which also alters rate in hPSC-CM. Together all of these may decrease the rate.

[0129] Together these experiments reveal a functional role for the lfchannel and SR in hCO automaticity. The lower rate and resistance to bursts under It blockade and a greater reduction in rate with ryanodine are the main functional differences in DM-hCOs and are underpinned by gene expression changes.DM-hCO Accurately Predicts Responses to Cardioactive Drugs

[0130] Productivity of drug responses is a core property required for widespread use of hCOs in drug discovery applications. DM-hCOs were benchmarked using 12 Comprehensive In Vitro Proarrhythmia Assay (CiPA) compounds that bind the human ether-or-go-go (hERG) channel with different risk stratifications for arrhythmia (Gintant, 2020) and a set of 17 compounds that are inert, negative or positive inotropes covering a diverse array of calcium transient and sarcomere regulators.

[0131] The time to relaxation (Tr50) is a major predictor of compound activity in the hERG used in the CiPA screen and contraction duration is also a key arrhythmia predictor clinically (Abdelsayed, 2020). The inventors therefore used Tr50 to screen the CiPA compounds (Figure 6A). Low risk compounds including ranozaline, mexiletine, diltiazem and verapamil did not increase Tr50, whereas high risk compounds including quinidine, DL-sotalol, ibutilide and dofetilide increased Tr50 (32-64%) at the concentration closest to Cmax. In addition, further increases in concentrations of high-risk compounds stopped contraction (quinidine) or induced arrhythmias (ibutilide and dofetilide). None of the intermediate risk compounds elevated Tr50 at the concentration closest to Cmax. If doses were further increased, then the response was variable, with cisapride and terfenadine leadingto dose-dependent reductions in force and odansetron leading to a substantial increase in Tr50 (50%). Together this indicates that the hCO are reliable in segregating low- and high-risk compounds.

[0132] In the boutique set of cardioactive compounds, the inert compounds paracetamol and pravastatin did not alter any of the contractile parameters (>10%) at the highest concentrations (Figure 6B). Drugs inhibiting systemic cardiovascular factors including atenolol (p-adrenoreceptor inhibitor) and captopril (angiotensin converting enzyme inhibitor) did not directly alter any of the contractile parameters (>10%) at the highest concentrations (Figure 6B), confirming that there is limited basal adrenergic drive or a renin-angiotensin system in the hCO. Likewise, clonidine (ci2- adrenorecptor agonist) which primarily acts on neurons to alter heart function had no impact on the hCO, consistent with the lack of neurons in our culture (Figure 2).

[0133] Negative inotropes sunitinib (RTK inhibitor), verapamil ( / Ca,L inhibitor), flecainide (lNainhibitor), mavacamten (myosin conformation) and aficamten (myosin conformation) all doses dependently decrease force (Figure 6C). Flecainide also initially increases Tr50 at low doses as expected. These drugs also impact other parameters once forces start to substantially decline.

[0134] Inotropic compounds were screened at 0.6 mM Ca2+given the higher calcium sensitivity in hCO (Figure 3A) compared to the human heart ~2.6 mM (Toischer, 2010). BAYK-8644 ( / ca,L activator) also increased force, but with a large liability on diastolic function with 250% Tr50 increase. Isoprenaline (p-adrenoreceptor agonist) dose dependently increased the rate, then force at higher concentrations together with decreasing Ta50 and Tr50 (Figure 6D). Phenylephrine (a-i- adrenoreceptor agonist) increased force at the highest doses (Figure 6D). The present inventors also found that ouabain ( / NQ / K inhibitor) increased force, prior to contraction cessation at higher doses (Figure 6D). Milrinone (PDE3 inhibitors) was able to increase the force of contraction in the presence of 10 nM isoprenaline, with limited changes in other parameters (<10%) (Figure 6E). The confirmed that PDE inhibition, particularly PDE4 with rolipram, resulted in increased force production and isoprenaline sensitivity in hCO from multiple cell lines (Figure 6E).

[0135] For sarcomeric acting inotropes, CK-136 (nelutroctiv, troponin activator), which is under clinical assessment (Romero, 2024), increases force prior to increasing Tr50 (102%) at the highest dose tested (Figure 6F). Omecamtiv mecarbil (myosin activator) dose dependently increases force until >1 pM where it starts to substantially increase Tr50 and force declines (Figure 6F) similar to other formats (Rhoden, 2022). This is consistent with data in human ventricular muscle preparations, and as an inotropic effect was not observed in human atrial preparations, this also further confirms the predominantly ventricular phenotype of the inventor’s DM-hCO (Dashwood, 2021). More recently, danicamtiv (myosin activator) has been developed to overcome this liability and the inventors found it substantially increases force with limited increases in Tr50 (20%) (Figure 6E). When comparing omecamtiv mecarbil and dancamtiv at their upper serum concentrations in the clinic (1 pM and 8 pM, respectively) it was found that danicamtiv consistently increased force with reduced contraction duration liability in hCO made from multiple cell lines (Figure 7A-C).

[0136] Taken together this suggests that DM-hCO accurately predict many drug responses including similar IC50 / EC50 to that reported (Table 2). However, there are still attributes consistent with an immature phenotype. In comparison to adult hearts DM-hCO have a higher calcium sensitivity, PDE4>PDE3 activity (although similar EC50 for milrinone as the human heart), and less sensitive ai-adrenoreceptor stimulation (Table 2). These features are all consistent with the expression of the key genes involved in our RNA-sequencing data: PDE3 (lower than human heart), PDE4 (higher than human heart) and ADRA1A (lower than human heart). The inventors also found that using our weaning medium culture conditions, positive inotropes can still be detected (Figure 7D), which is important for statistical power and comparisons in screening applications as otherwise small changes in calcium EC50 can have large impacts on the magnitude of force increases.TABLE 2Comparison of hCO drug sensitivity with in vivo derived muscle or card io myocytesDM-hCO enable modelling of complex DSP cardiomyopathy

[0137] DSP cardiomyopathies are driven by complex cell-cell interactions and changes in excitation-contraction coupling (Yuan, 2021). As DM-hCO more faithfully recapitulates the multicellular composition of heart tissue and mature excitation-contraction coupling, the inventors chose to model a DSP mutation to demonstrate the utility of this platform for disease modelling. The inventors identified a patient presenting with dilated cardiomyopathy and arrhythmia (MCHTB11), diagnosed with a homozygous 2 bp deletion in the DSP gene DSP c.4246_4247del; p.Leu1416AsnfsTer23. This results in leucine being replaced by asparagine at amino acid position 1416, followed by a termination codon after 22 amino acids in the new reading frame. The variant affects the longer isoform of DSP (NM_004415.3; 2871 amino acids), but not the shorter isoform (NM_001319034.2; 2428 amino acids). The family was screened, and the parents found to be heterozygous, and two deceased siblings were homozygous for the mutation (Figure 8A). The cardiac pathology was associated with substantial cardiac fibrosis (Figure 9A) and loss of DSP at thejunctions with CX43 dysregulation (Figure 9B). Proteomic profiling revealed that a diverse array of factors including extracellular matrix, metabolism regulators, ion channel regulators, sarcomeric regulators and growth factors were dysregulated in diseased hearts (Figure 9C, and Table 1).

[0138] The inventors created an induced pluripotent cell line (MCHTB11) and corrected line (H.3) using a simultaneous CRISPR correction protocol (Figure 8B,C). DM-hCO created from these cell lines displayed normal junctional expression of DSP and CX43 in the corrected H.3 line, but largely absent DSP expression and aberrant CX43 localisation together with disordered sarcomeres in the patient line (Figure 8D). Next, the inventors sought to determine whether there were functional differences between MCHTB11 and H.3 (Figure 6D). There were large differences in rate in SF-hCO between MCHTB11 and H.3, which may mask other key phenotypes (Figure 8F). In contrast, in DM- hCO rate was relatively stable, which unmasked a phenotype whereby Tr50 was increased by 30% (Figure 8G, F). MCHTB11 had an enhanced activation velocity and reduced relaxation velocity (Figure 8G-I).

[0139] Two drugs were trialled in an attempt to improve the relaxation phenotype. INCB054329, a bromodomain extra-terminal inhibitor that the inventors recently discovered can correct inflammation induced diastolic dysfunction (Mills, 2021). Danegaptide, gap junction enhancer was also assessed due to the CX43 remodelling the inventors observed (Figure 6C) and putative interest in gene therapies for DSP cardiomyopathy. INCB054329 and danegaptide had no substantial effects in the H.3 corrected line other than a slightly reduced rate for INCB054329 (Figure 8J). In the MCHTB11 hCOs, danegaptide slightly improved Tr50 under acute pacing. In the same line, INCB054329 increased force, slightly decreased rate and fully corrected the Tr50 phenotype (Figure 8J) and greatly enhanced the relaxation kinetics (Figure 6G).

[0140] The present inventors next compared MCHTB11 to H.3 DM-hCO with and without treatment of INCB054329 using proteomics. There were no major changes in cellular population markers, and MCHTB11 clustered separately to H.3 regardless of INCB054329 treatment (Figure 8K). This was due to regulation of more proteins by the DSP mutation in MCHTB11 hCOs in comparison to INCB054329. There was a major upregulation of fibrotic proteins in the MCHTB11 line including many markers of cardiac fibrosis, strongly underpinned by TGF-p1 activation (Figure 8L). Only a subset of these proteins were reverted by INCB054329 treatment which appeared to regulate a sub-set of proteins in the MCHTB11 hCOs compared to the H.3 hCOs (Figure 8L). This was in the absence of rescuing the expression of the desmosome proteins that decrease in MCHTB11. Furthermore, there was a strong upregulation of immunomodulatory proteins including CD47, THBS1 , IL-10, TGF-p1. Taken together there is a strong immunomodulatory response caused by DSP mutation which drives fibrosis and alters contraction kinetics. INCB054329 can improve function by only reverting the expression of a minor subset of these proteins rather than controlling the overall fibrotic response (Alexanian, 2021).Discussion

[0141] These data seek to investigate whether the key in vivo signatures identified in human hearts play a role in in vitro cardiac maturation.

[0142] Together, MK8722 and DY131 (DM) were the most effective drivers of increased maturation. In contrast to previous studies, activation of ERR (using DY131) had a more subtle effect whereas activation of AMPK using MK8722 dramatically enhanced maturation. The timing was very important, and these factors were most effective following (but not during) the phase where hCO are metabolically switched to fatty acid oxidation. This may be because during this phase the cardiomyocytes are already near maximum capacity as indicated by our data profiling the oxidation over the full hCO culture time-course, and further metabolic stress is detrimental during this phase. Additionally, the impact of DM was not dependent on the particular fatty acid substrate. Together, these data further support our previous findings that oxidative metabolism is not just a feature, but a driver of cardiac maturation.

[0143] The DM conditions induced a similar signature to pacing with over 48% of phosphopeptides being similar. Given DM directly activates these, the critical AMPK signalling network the identified by the inventors may play a key role in maturation under both AMPK and DM protocols. The inventors found that high rates alone do not promote maturation, as PB010.5 hCOs had endogenous beating rates >150 bpm but low levels of cTnl expression, which were then greatly enhanced (the most of any cell line tested) with DM treatment. Therefore, forced increases in metabolic capacity is essential for maturation. Even after DM stimuli is removed the increase in maturation indicators such as cTnl and reduced endogenous beating rate are sustained, indicating that DM results in a stable maturation cellular state in the hCOs. This may be a key adaptation during cardiac maturation to enable large increases in demand from basal function to increased function during exercise or stressed conditions.

[0144] One key feature of DM induced maturation was the lower and much more consistent rate across hCO from different hPSC lines. This made it possible to (1) assess SR function under reduced If in >90% of hCO, (2) assess ectopy as a surrogate readout of arrhythmia for SR protein mutations, and (3) reveal a stiffening phenotype in DSP mutant hCO. The mechanisms underlying automaticity in hPSC-CM have been recently shown to involve relatively high expression of HCN4, CACNAH1 and SLC8A1, with low expression of KCNJ2 (Marchiano, 2023). These data show that DM induced reduction in rate via alternative mechanisms as expression of these ion channels were not altered. Instead, the inventors found a more stable SR, a switch in hCO from CACNA1D to CANACA1C, and increased CX43 expression as key changes and are also consistent with human heart maturation. The DM therefore also has some applications in cellular therapies to help facilitate a reduction in arrhythmic risk conferred by automaticity, and it is clearly shown that the DM protocol can also reduce rate in 2D hPSC-CM culture.

[0145] One of the key findings of our protocol was also the cellular complexity of our hCO model which was important for modelling a DSP mutation. The inventors found a striking pro-fibrotic signature underpinned by TGF-p1 and kinetic dysfunction in DSP mutant DM-hCO. TGF-p1 and IL-10were increased and are heavily involved in anti-inflammatory signalling (Li, 2009) and may be a response to protect the heart against the inflammation caused by desmosome breakdown. In DSP cardiomyopathy in the clinic there are multiple inflammatory “myocarditis” episodes that cause cardiac remodelling and eventually lead to decreased heart function (Smith, 2020). This indicates that the DSP mutation may be an initiating event that drives immune and fibroblast driven pathological remodelling leading to decreased function and arrhythmia overtime. Indeed, myocardial injury is one of the major risk factors for arrhythmia and heart failure for DSP probands. Such a structural remodelling basis for clinical disease has recently been proposed to be a key determinant of arrhythmia in Brugarda syndrome (Nademanee, 2015). Therefore, multicellular hPSC models with cellular complexity may be pivotal in the study of these diseases at these early stages, and screening for preventative therapeutics with the most benefit.Materials & Methods hPSC Cell Lines and Culture hPSC Lines

[0146] hPSC lines were maintained in mTeSR PLUS (Stem Cell Technologies) in Matrigel coated flasks Matrigel (Millipore) and passaged using ReLeSR (Stem Cell Technologies). Routine quality control was performed for Karyotyping using G-banding (Sullivan Nicolaides) and more recently Molecular Karyotyping Analysis (Victorian Clinical Genetics Service and Ramaciotti Centre for Genomics) and mycoplasma testing. Some HES3 clones had gained a copy number at q11 .21 , which is likely mosaic in the parental line.Generation of hPSC transgenic lines

[0147] For the LED pacing line, neomycin resistance followed by CAG-C1 V1 (E122T / E162T) channel rhopdopsin-P2A-super ecliptic pHurlion was cloned into an AAVS1 targeting construct with ~800 bp homology arms. For the TCF21 reporter line a homologous directed recombination template was synthesized as depicted in the relevant figure. Transgenic cell lines were generated, cloned and quality controlled. A detailed protocol for the generation of the TCF21 line is given below.

[0148] Reprogramming and gene-editing factors (Cas9-Gem mRNA, TCF21 :CreERT2 template and plasmid encoding TCF21 -specific sgRNA) were introduced into peripheral blood mononuclear cells using the Neon transfection system (1150 V, 30 ms, 2 pulses). Transfected cells were plated over 3 wells of a Matrigel / MEF coated 6-well dish in StemSpan Media. E8 medium was added to each well two days post-transfection and half medium changes were performed every other day until adherent iPSC colonies became visible (~1 week post-transfection) when full E8 media changes were performed. Individual iPSC colonies were isolated and expanded in E8 medium.

[0149] Successfully edited cells were identified by PCR using primers flanking the recombination junctions. Homozygous knock-in of the CreERT2 was confirmed by PCR analysis using primers which flank the target site. A clone harbouring homozygous insertion of CreERT2 was selected for incorporation of the fate-mapper cassette into the EEF2 locus. Gene-editing factors(Cas9-Gem mRNA, EEF2 fate-mapper HDR template and plasmid encoding EEF2-specific sgRNA) were introduced into iPSCs using the Neon transfection system (1100 V, 30 ms, 1 pulse). Transfected cells were plated over 4 wells of a Matrigel coated 6-well dish in mTesR medium supplemented with 10 pM Y-27632 which was removed from medium after 24 h. Successfully edited iPSC colonies (expressing EYFP) were identified by fluorescence microscopy. EYFP+ colonies were isolated and expanded in E8 medium. A clone with a high proportion of EYFP+ cells was selected for subcloning to attain a pure population. Subcloning was performed by dissociating cells with TryPLE and plating at low density in mTesR supplemented with Y- 27632 (removed from medium after 24 h). Individual colonies were picked and expanded in E8 medium. One of these clones was confirmed by flow cytometry to consist entirely of EYFP+ cells and showed homozygous knock-in of the EEF2 fatemapper transgene as evidenced by PCR analysis using primers flanking the EEF2 target site. sgRNAs are details are provided in the relevant figures.Generation of CASQ2-'- and / ?y / ?2+ / N4104KhPSC lines

[0150] Two hours prior to electroporation, media on the PSCs was changed to fresh mTeSR Plus supplemented with 10 pM ROCK inhibitor Y-27632 (Stem Cell Technologies). For the CASQ21- mutation, sgRNAs (Synthego) was conjugated to TrueCut Cas9 Protein v2 (Thermo Fisher) in a 9:1 molar ratio of sgRNA:Cas9 in a 10 pL reaction. For the Ry / ?2+ / N4104Kcell line, 1 .0 pL of 60 pM Alt-R CRISPR-Cas9 sgRNA (Integrated DNA Technologies) was conjugated to 1.0 pL of 3 mg / mL TrueCut Cas9 Protein v2, in a 10 pL reaction. Cells were passaged with ReLeSR, 1.6 x 105cells resuspended in Buffer R (12 pL) with the sgRNA-Cas9 RNP prepared above. For RyR2

[0151] + / N4104Kcell line, 0.6 pL of each 100 pM ssODN was added. sgRNAs and ssODN details are provided in the relevant figures.

[0152] The Neon Transfection System 10 pL Kit (mCell Technologies) was used according to the manufacturer’s instructions. Electroporation settings were 1200V, with 1 pulse, and 30 ms pulse width. Cells were immediately plated onto 24-well tissue culture plates coated with Matrigel, containing mTeSR Plus with 10 pM ROCK inhibitor Y-27632, or 1X CloneR2 (Stem Cell Technologies). Post- electroporation survival was assessed the next day and media changed to mTeSR Plus without further supplements. Media was changed every two to three days with mTeSR Plus until PSCs reached 70% confluency and subsequently cryopreserved with mFreSR (Stem Cell Technologies) upon reaching 70% confluency. Concurrently, hPSCs were also harvested for DNA with the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions.

[0153] Amplicons of target sites were generated from DNA of electroporated hPSCs with Hifi Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific) according to manufacturer’s instructions. PCR purification was performed with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. Sanger sequencing was performed by the QIMR Berghofer Analytical Facility. The sequence traces from the wild-type parental cell lines and theelectroporated hPSC populations were analysed with Synthego’s ICE Analysis tool (available at https: / / ice.synthego.com / ) to determine editing efficiency and incorporation rate of ssODNs.

[0154] hPSC pools that had a rate of successful edits greater than 1 % were thawed into T25 flasks for subsequent single-cell cloning. Cloning Medium was prepared by supplementing mTeSR Plus with 10% of CloneR or CloneR2. Cells were passaged using ReLeSR and sorted using a BD FACSARIA I Hu Cell Sorter (BD Biosciences) into 96-well plates at a density of 1 cell per well. Culture continued in CloneR2 Cloning Medium, and once all colonies were over 25% confluency they were each passaged with ReLeSR into one well of a 96-well plate and 24-well plate and then maintained in mTeSR Plus. The Extract- N-Amp for Blood Kit (Sigma-Aldrich) was used according to the manufacturer’s instructions to generate DNA lysates from the 96-well plate, and to produce PCR amplicons of the edited sites. PCR amplicons were purified with the QIAquick PCR Purification Kit and submitted for Sanger Sequencing as above. For each clone the Sanger chromatograms were manually interrogated to determine the presence and mono- or bi- allelic editing. hPSC clones were maintained in 24-well plates until genotyping was complete. Positive clones were expanded and banked, negative clones were discarded.

[0155] For each gRNA sequence, the top five non-overlapping sites predicted by COSMID (available at https: / / crispr.bme.gatech.edu / ) and CRISPOR (available at http: / / crispor.tefor.net / ) were amplified with PCR and Sanger sequenced. Traces for each off-target site from clones were manually compared to traces from the relevant parental cell line. Clonal edited hPSC cell lines were also karyotyped as described above. hCO formation and culture

[0156] Heart-Dyno culture inserts were manufactured with polydimethylsiloxane (PDMS), SYLGARD 184 Silicone Elastomer (CBC Australia) at a 10:1 ratio of base to curing agent. The base and curing agent were thoroughly mixed and degassed in a vacuum chamber. Heart-Dyno plates were cast in a machined aluminum mold (ANFF-SA). After pouring into a mold, the PDMS was degassed again in a vacuum chamber then cured for 1 h at 70°C.

[0157] Heart-Dyno culture plates were fabricated using two different methods. For one method, individual well inserts were manually “glued” into 96-well flat-bottom tissue culture plates (Greiner and Corning) with uncured PDMS (10:1 ratio of base to curing agent) and were left for at least 24 h at room temperature to cure. For the new method, the whole 96-well Heart-Dyno layer was bonded to 96-well flat-bottom tissue culture plates (Greiner and Corning) using chemical bonding and uncured PDMS (10:1 ratio of base to curing agent)82.

[0158] Tissue culture plates with Heart-Dyno inserts were sterilized in a biosafety cabinet by immersion in 80% vol / vol ethanol for 2 h followed by 1 h of ultraviolet exposure in the biosafety cabinet. Before seeding of hCOs, the HeartDynos were coated with 1-3% wt / vol bovine serum albumin (Sigma; in PBS without Ca2+and Mg2+) for 2 h to limit cell adhesion to the PDMS. This was aspirated immediately before seeding.

[0159] Before seeding of hCOs, hPS cell-CCs (differentiation described above) were centrifuged at 300 x g for 3 min and resuspended in serum-free medium: MEMa GlutaMAX supplemented with 200 pM I-AA2P, 1% penicillin-streptomycin, 4% vol / vol B27 with insulin, 10 ng / mL FGF-2 and 10 ng / mL PDGF-BB (RnD Systems). Each Heart-Dyno plate was seeded with a 3.5 pL mix of 5 x 1 o4cardiac cells in serum-free medium, 2.6 mg / mL acid-solubilized collagen I (Devro) that was salt balanced with 10x DMEM (Thermo Fisher Scientific) and pH neutralized with 0.1 M NaOH and 9% vol / vol Matrigel. All components were mixed over ice, as was the manual pipetting of the mix into the Heart-Dyno plates, to prevent premature gelling. After seeding, Heart-Dyno plates were incubated at 37°C in a 5% CO2 humidified incubator for 30-45 min to allow the mix to gel. After gelling, 150 pL of serum-free medium warmed to 37°C was added to each well. hPS cell-CCs were allowed to condense and self-organize into hCOs for 2 days in the serum-free medium. On day 17, the medium was changed to maturation medium: DMEM, no glucose, no glutamine, no phenol red (Thermo Fisher Scientific) supplemented with 1 x GlutaMAX (Thermo Fisher Scientific), 200 pM I- AA2P, 1% penicillin-streptomycin, 4% vol / vol B27 without insulin (Thermo Fisher Scientific), 10 ng / mL FGF-2, 10 ng / mL PDGF-BB, 33 pg / mL aprotinin (Sigma or MedChemExpress), 100 pM palmitate (conjugated to bovine serum albumin in B27, Sigma) and 1 mM glucose (Sigma). Maturation medium was refreshed on day 20. On day 22, the medium was changed to weaning medium. The composition of weaning medium has the following differences to maturation medium: no FGF-2, no PDGF-BB, 10 pM palmitate instead of 100 pM, 5.5 mM glucose instead of 1 mM, and 1 nM recombinant human insulin (Gibco). Weaning medium was refreshed on days 24 and 27. For directed maturation conditions, 2 pM CHIR99021 (GSK-3a / p inhibitor) was added for the formation phase in the first 2 days for hCO formation and the media changes on days 24 and 27 were instead done with weaning medium supplemented with 3 pM DY131 and 10 pM MK8722 (both MedChemExpress). All further media changes were performed with weaning medium every 2 to 3 days with no further supplements. For some experiments, VIAFLO 96 (Integra) semiautomated pipetting electronic pipetting systems were used for seeding the hCOs and media changes.Cardiac differentiation and hCO culture

[0160] The single step cardiomyocyte and stromal cell differentiation protocol, dissociation and hCO formation was performed as recently described (Voges, 2023, Voges, 2023b).

[0161] For pacemaker differentiation, hPSC were differentiated using RPMI base media (Thermo Fisher Scientific) supplemented with 1% penicillin / streptomycin (Thermo Fisher Scientific), 200 mM L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), and B27 (insulin minus) supplement (Thermo Fisher Scientific). For mesodermal induction, 5 ng / mL BMP4, 9 ng / mL Activin A, 5 ng / mL bFGF (all RnD Systems), 1 pM CHIR99021 (Stem Cell Technologies) was added on each day for the first 3 days of differentiation. On day 4 of differentiation 5 pM IWP4 (Stem Cell Technologies), 2.5 ng / mL BMP4, 5.4 mM SB431542 (Sigma), 0.24 mM all-trans retinoic acid (Stem Cell Technologies), 0.5 mM PD173074 (Tocris) was added to RPMI base medium to pattern the cardiac cells. B27 (plus insulin) RPMI basal medium supplemented with 5 pM IWP4 wasadded for the next 7 days with media changes every 2 / 3 days. On day 15 of differentiation B27 (insulin minus) DMEM no glucose, no glutamine, no phenol red (Thermo Fisher Scientific) supplemented with 5 mM lactic acid (Sigma) was added for the next 7 days with media changes every 2 / 3 days. On day 22 of differentiation, cells were dissociated and hCO formed as recently described (Voges, 2023, Voges, 2023b).Pacing conditions

[0162] hCOs were paced using different methods. Isoprenaline (Sigma). They were exposed to pulses of green light using a 96-well green LED array (Lumidox) connected to a Panlab / Harvard Apparatus Digital Stimulator as recently described (Mills, 2019c). The Heart-Dyno inserts were also fabricated in a custom plate format to fit into a 24 well plate for acute pacing using a C-Pace Cell Culture EP Stimulator (lonoptix) using 10 V with 1 ms pulses at 120 bpm (Figure 1).Maturation factors

[0163] Various factors were added at the times and concentrations outlined in the different figures. Small molecules GSK4716, DY131 , MK-8722, 0304 were purchased from MedChem Express. Interferons IFN-y, IFN-A1 , IFN-A2, IFN-p and IFN-w were purchased from Peprotech. Fatty acids palmitic acid, oleic acid, myristic acid and linoleic acid were purchased from Sigma.Force analysis

[0164] hCOs were imaged under environmentally controlled conditions at 37°C with 5% CO2 on a Leica Thunder microscope. Image series were taken of each hCO at 50 frames per second generally for 10 seconds, but up to 40 seconds for experiments using cilobradine. Contraction analysis was performed using custom Matlab scripts or Tempo. ai analysis software developed by Dynomics (Voges, 2023b).

[0165] For calcium sensitivity experiments Tyrode’s solution (120 mM NaCI, 5 mM KCI, 22.6 mM NaHCOs, 2 mM MgCh and pH adjusted to 7.4) was used and calcium concentration adjusted using a 0.2 M CaCh stock solution.

[0166] To assess post-rest potentiation, hCOs were pre-treated with 1 pM cilobradine (and in some cases 5 pM thapsigargin) for 2 hours before the experiment. For experiments with acutely paced hCO using custom designed gold-plated electrode lids. Pacing was performed at 30-40 mA with 5 ms square pulses for at least 30 seconds prior to recordings. qPCR

[0167] hCO were manually homogenised in 500 pL Trizol (Thermo Fisher Scientific) with a stainless-steel ball bearing and 10 sec vortex pulses until solubilised. Samples were stored at -80°C before RNA was extracted using Trizol as per manufacturer’s instructions. RNA was DNase treated as per the manufacturer’s instruction (Roche) before cDNA synthesis (Thermo Fisher Scientific).Powerup SYBR Green Master Mix (Thermo Fisher Scientific) was used for RT-qPCR using StepOne software v2.3 to determine gene expression.Immunostaininq

[0168] hCOs were fixed with 1% paraformaldehyde (Sigma) for 60 min. Cells were stained with primary antibodies (see Methods Table 1) in 5% FBS (Thermo Fisher Scientific) and 0.2% Triton X-100 (Sigma) in PBS (Blocking Buffer) at 4°C overnight on a rocker. Cells were washed 2X for 1 h with Blocking Buffer and labelled with secondary antibodies (see Methods, Table 1) and Hoechst33342 at 4°C overnight on a rocker. Cells were again washed with Blocking Buffer 2X for 1 h and imaged in the Heart- Dyno or mounted on microscope slides in Prolong Glass (ThermoFisher Scientific). Low magnification images were taken on a Leica Thunder microscope. High magnification images were taken using Zeiss 780-NLO Point Scanning Confocal or a Leica Stellaris 5.Drug screening

[0169] Individual compounds were purchased from Sigma to form our boutique drug libraries. These were added to hCO for at least 15 minutes for equilibration prior to video recordings. For dose- response curves, cumulative drug additions were applied to the same hCO.Transmission Electron Microscopy

[0170] hCOs were fixed in 2.5% glutaraldehyde in PBS for 1 hour at room temperature and then processed for embedding in situ as described previously (Takasato, 2015). For electron tomography 200-300 nm sections were prepared parallel to the base of the plate on a Leica Ultracut 6 ultramicrotome. The grid was then coated with a thin carbon layer. Tomography was performed as described previously (Ariotti, 2015) on a Tecnai F30 transmission electron microscope (FEI) at 300 kV. A dual-axis tilt series spanning ± 60° with 1 ° increments was acquired with a Gatan OneView camera under the control of Serial EM. Tilt series were reconstructed using IMOD (https: / / bio3d.colorado.edu / imod / ) with segmentation performed by density thresholding using the Isosurface Render program in IMOD.DSP proteomics

[0171] Each sample contained three hCOs and stored (-20°C) prior to preparation. Samples were thawed on ice, and residual supernatant removed prior to addition of 150 pL chilled lysis buffer (4% sodium deoxycholate in TRIS-buffered saline). Samples were immediately boiled, 95°C, for 5 minutes to inactivate endogenous enzymatic activity and assist lysis. Four volumes of chilled acetone was added to each sample and sonicated in 1 min bouts alternating with incubation on ice for a total of five cycles. Protein was pelleted by centrifugation at 20,000 x g for 30 min at 4°C. Supernatant was removed, and protein pellet washed twice in acetone prior to resuspending in 150 pL 50 mM TEAB (Triethylammonium bicarbonate buffer, Sigma). Reduction alkylation buffer was added to a final concentration of 10 mM TCEP (Sigma) and 40 mM 2-CAA (Sigma, equilibrated with KOH to pH 8)and incubated for 10 min 45°C with 1500 rpm agitation. 3 pg Trypsin enzyme mix (Thermo Fisher Scientific) was added to sample and incubated overnight (17 hours) at 37°C, with 1500 rpm agitation. 50 pL of 10% Trifluoroacetic acid solution was added to each sample to halt digest. Peptides were desalted using SDB-RPS tips, according to standard protocol (Tappsilber, 2007). Samples were resuspended in 2% ACN / 0.3% TFA, with sonication to assist solubility.

[0172] For each sample, a uniform volume (3 pL) was resolved across a 65 min gradient in data-independent acquisition mode using a Thermo PepMap100 analytical column equipped on a Thermo Ultimate 3000 LC interfaced with a Thermo Exactive HF-X mass spectrometer. Single injection DIA method utilised parameters established previously (Pino, 2020). Briefly, MS1 included survey scans of 1e6 ions at resolution of 60,000, with a max isolation time of 60 ms within the scan range of 390-101 m / z and collision energy of 27. MS2 was performed with isolation windows of 12 m / z spanning 400-1000 m / z range, AGC target of 1e6 and resolution of 15,000.CASQ2-KO proteomics

[0173] PB006.6 wild-type and CASQ2-KO hCOs were snap frozen on dry ice and stored at --80°C until extraction. 8-16 hCOs were lysed in 300 pL of 1% SDS in Milli-Q water with complete ULTRA Protease Inhibitor Cocktail (Roche). Samples were vortexed, transferred to 2 mL Precellys Lysing Kit tubes and then pulsed three times for 30 seconds at 5000 rpm with a Minilys homogenizer (Bertin Technologies). Samples were centrifuged at 8000 x g for 10 minutes to remove bubbles. Transferred to Eppendorf tubes and stored at -20°C. Four volumes of chilled acetone was added to each sample, and sonicated in 1 min bouts alternating with incubation on ice for a total of 5 cycles. Protein was pelleted by centrifugation at 20,000 x g for 30 min at 4°C. Supernatant was removed, and protein pellet was washed twice in acetone prior to resuspending in 1 x TBS Buffer (ThermoFisher Scientific).

[0174] Sodium deoxycholate was added to protein extracts for improved protein solubility, to a final SDC concentration of 1% (v / v). Reduction alkylation buffer was added to a final concentration of 10 mM TCEP (Sigma) and 40 mM 2-CAA (Sigma, equilibrated with KOH to pH 8) and incubated for 10 min 45°C with 1500 rpm agitation. 1 pg Trypsin (ThermoFisher Scientific) was added to sample and incubate overnight (17 hours) at 37°C, with 1500 rpm agitation. 50 pL of 10% Trifluoroacetic acid solution was added to each sample to halt digest. Peptides were desalted using SDB-RPS tips, according to standard protocol (Pino, 2020). Samples were resuspended in 2% ACN / 0.3% TFA, with sonication to assist solubility.

[0175] For each sample, a uniform volume (3 pL) was resolved across a 55 min gradient in data-independent acquisition mode using a Thermo PepMap100 analytical column equipped on a Thermo Vanquish Neo UHPLC interfaced with a Thermo Exactive HF-X mass spectrometer. Single injection DIA method utilised parameters established previously (Humphrey, 2018). Briefly, MS1 included survey scans of 1 e6 ions at resolution of 60,000, with a max isolation time of 60 ms within the scan range of 390-1010 m / z and collision energy of 27. MS2 was performed with isolationwindows of 16 m / z spanning 400-1000 m / z range, AGC target of 1 e6 and resolution of 15, 000. DSP and CASQ2-KO proteomics bioinformatics

[0176] Raw spectra were analysed using DIA-NN, version 1 .8.1 against the human proteome (20,399 sequences, downloaded April 19, 2021 from UniProt) using the library-free method. Carbamidomethylation of cysteines and N-term N excision was set as a fixed modification, matching between runs enabled, cross-run normalisation used RT-dependent method, and small-profiling method was used to generate in silico library from the human proteome.

[0177] For the DSP samples the mean values for each protein was calculated for each condition, and Iog2 fold change values generated, including p value (two-sided parametric t test) and false discovery rate corrected p value (Benjamini Hochberg method). PCA plots were generated using R (v4.2.2) and ggfortify package (vO.4.16). Within Cytoscape (v3.10.2) a protein-protein interaction network was generated by querying the STRING database (stringApp v2.1 .1) with the 4473 proteins detected in the DSP hCO dataset. A confidence score of 0.9 was used, and the maximum number of additional interactors was set to 40. Default edge filters were used. The largest network of 3516 proteins was isolated from all other smaller networks and nodes. An Edge-weighted Spring Embedded layout was used. The Markov clustering algorithm was used to cluster the network and functional enrichment was performed on the 10 largest sub- clusters. Clusters were coloured according to the consensus description from ‘GO Biological Process’ and ‘I Reactome Pathways’. Node size and transparency was mapped to Iog2 fold change between DSP and WT groups. Proteins that were regulated by INCB in the DSP hCOs (p value < 0.05) were given a black border.

[0178] For each CASQ2 sample and the WT PB006.6 sample, GAPDH was used as the normalization factor for CASQ2, HRC, RYR2, ATP2A2, PLN, and CALR. Normalized spectral intensities for each protein in the CASQ2 samples were then expressed as fold difference to the respective protein from the WT PB006.6 sample.Phosphoproteomics

[0179] Control hCO or hCO treated with 3 pM DY131 and 10 pM MK8722, or pacing at 120 bpm for 5 mins were immediately quenched in ice cold tris-buffered saline. 16 hCO were pooled per condition for phosphoproteomics preparation, based on the EasyPhos protocol (Humphrey, 2018). Briefly, samples were suspended in sodium deoxycholate containing buffer (4% SDC in tris-buffered saline, pH 7.5) and heated for 5 min at 95°C, with 1500 rpm agitation, to assist lysis and inhibit endogenous enzymatic activity. This was followed by sonication, using five 30 sec on / off intervals in a chilled water bath sonicator. Reduction alkylation buffer was added to a final concentration of 10 mM TCEP (Sigma) and 40 mM 2-CAA (Sigma, equilibrated with KOH to pH 8) and incubated for 10 min 45°C with 1500 rpm agitation. 1 pg Trypsin / Lys-C enzyme mix (ThermoFisher Scientific) was added to sample and incubated overnight at 37°C, with 1500 rpm agitation. The following day, 75 pL of isopropanol was added to each sample and thoroughly mixed, before adding 25 pL enrichment buffer (48% v / v TFA and 8 mM KH2PO4). 45 mg TiO2 titansphere beads were washed three times with 6%TFA / 80% ACN, resuspended and 5 mg added to each sample. All samples were incubated for 10 min at 40°C, with 2000 rpm agitation. All samples were moved to new tubes, and centrifuged (2,000 x g for 1 min) to remove non-phosphorylated peptides (supernatant). Beads were washed five times with 1 mL 5% TFA / 60% isopropanol wash buffer using centrifugation. A final wash used 0.1% TFA / 60% isopropanol. Lastly, 50 pL elution buffer (200 pL of ammonia solution to 800 pL of 40% ACN) was added to beads twice, to collect two subsequent eluents. Phosphopeptides were dried, using a Genevac sample concentrator, approximately 25 min on aqueous setting. Peptides were desalted using SDB-RPS tips, according to standard protocol (Rapppsilber, 2007). Samples were resuspended in 2% ACN / 0.3% TFA, with sonication to assist solubility.

[0180] For each sample, a uniform volume (5 pL) was resolved across a 65 min gradient in data-dependent acquisition mode using a Thermo PepMap100 analytical column equipped on a Thermo Ultimate 3000 LC interfaced with a Thermo Exactive HF-X mass spectrometer. The mass spectrometer performed survey scans of 3e6 ions at a resolution of 60,000 from 300-1600 m / z. The 10 most abundant precursors from the survey scan with charge state >1 and <5 were selected for fragmentation. Precursors were isolated with a window of 1 .6 m / z and fragmented in the HCD cell with NCE of 27. Maximum ion fill times for the MS / MS scans were 50 ms, with a target of 2e4 ions. Fragment ions were analysed with high resolution (15,000) in the Orbitrap mass analyser. Dynamic exclusion was enabled with duration 30 sec.Phosphoproteomics bioinformatics

[0181] Raw LC-MS data was searched against the reviewed Uniprot human database (20399 sequences, downloaded April 19, 2021) using Sequest HT on the Thermo Proteome Discoverer software (Version 2.3), with matching between runs enabled. Precursor and fragment mass tolerance were set to 20 ppm and 0.05 Da respectively. A maximum of two missed cleavages were allowed. A strict false discovery rate (FDR) of 1% was used to filter peptide spectrum matches (PSMs) and was calculated using a decoy search. Carbamidomethylation of cysteines was set as a fixed modification, while oxidation of methionine, N-terminal acetylation and S / T / Y phosphorylation were set as dynamic modifications, up to three phosphosites per peptide.

[0182] Differential expression analysis was performed in Proteome Discoverer software, to return Iog2 fold change values and an adjusted p value. Fold changes were made between electrical pacing or directed maturation conditions to control conditions. Differentially altered phosphoproteins (adj.p < 0.05) were analysed in EnrichR (Kuleshov, 2016) using the KEGG 2021 Human gene set. All phosphoproteomics graphs were generated in GraphPad Prism, and diagrams made in Inkscape (v1.2.2).

[0183] Live-cell respiration was measured in real-time using Resipher (Lucid Scientific). hCO were cultured in 96-well microplates (ThermoFisher Scientific) using hCO culture inserts (height approximately 1 .5 mm) that were fabricated by PDMS molding (Voges, 2023b) The Resipher sensor lid (9.4 mm probe length, Lucid Scientific, NS32-94A) was pre-equilibrated with naive media in acell-free 96-well microplate at 37°C and 5% CO2 overnight prior to seeding. After hCO formation, the Resipher sensor lid was transferred to the hCO culture plate and media 02 was measured with an operating height between 1600 and 2100 pm. During subsequent media changes, the sensor lid was stored in pre-equilibrated naive media in a cell-free microplate. On Day 30 of culture, hCO were treated with DMSO control or 10 pM BAM-15 (MedChemExpress) for 6 h. Respiration rates of hCO were normalised to cell-free control wells on the same plate, which contained naive media for each condition (SF or DM culture conditions).Measurement of hCO respiration

[0184] Live-cell respiration was measured in real-time using Resipher (Lucid Scientific). hCOs were cultured in 96-well plates (ThermoFisher Scientific) using Heart-Dyno inserts (height approximately 1 .5 mm) that were fabricated by PDMS molding.; . The Resipher sensor lid (9.4 mm probe length, Lucid Scientific, NS32-94A) was pre-equilibrated with naive media in a cell-free 96-well microplate at 37°C and 5% CO2 overnight before seeding. After hCO formation, the Resipher sensor lid was transferred to the hCO culture plate and media C>2was measured with an operating height between 1 ,600 and 2,100 pm. During subsequent media changes, the sensor lid was stored in preequilibrated naive media in a cell-free microplate. On day 30 of culture, hCOs were treated with DMSO control or 10 pM BAM15 (MedChemExpress) for 6 h. Respiration rates of hCOs were normalized to cell-free control wells on the same plate, which contained naive media for each condition (SF or DM culture conditions).Bulk RNA-sequencinq dataset

[0185] For maturation marker analysis bulk RNA-sequencing data from published datasets were collected. To ensure cell compositions did not impact analysis sarcomeric isoform fractions were used for the analysis including MYH7-MYH6, MYL2-MYL7 and TNNI3-TNNI1. Data were collated from hPSC-CM cultures including GSE93841 , GSE148025, GSE116464, GSE201437, GSE114976 and human hearts including ERP109940 and Hahn et al., 2021. hCO nuclei isolation for snRNA-sequencinq hCO were matured using the SF or DM protocol. At the conclusion of the experiment, media was aspirated and hCO were washed with ice cold PBS’. Intact hCO were pooled with up to 70 hCO per sample (n = 2 per maturation protocol). All PBS’ was removed and hCO were snap frozen in liquid nitrogen and stored at -80°C. Nuclei isolation was completed using the 10x Chromium Nuclei Isolation Kit (PN-1000494, 10x Genomics) and 10x Genomics Chromium Nuclei Isolation Kit Protocol for Single Cell 3' Gene Expression (RevA) with modifications. All subsequent steps in the nuclei isolation steps were performed on ice. The samples were homogenised in 200 pL Lysis Reagent using the pestle supplied in the 10x kit. 300 pL Lysis Reagent was added and samples were pipette mixed and left to incubate on ice for 5 minutes. The sample was then transferred into a prechilled Nuclei Isolation Column and centrifuged at 16 000 x g for 20 sec at 4°C. Samples were vortexed for 5 seconds at max speed to resuspend nuclei and centrifuged again at 500 x g for 5 minat 4°C. 300 pL of supernatant was removed and nuclei re- suspended in 500 pl of Debris Removal Buffer. Samples were centrifuged at 700 x g for 10 min at 4°C. 500 pL of supernatant was removed and nuclei were gently re-suspended in 1 mL of Wash and Resuspension Buffer. Samples were centrifuged at 500 x g for 5 min at 4°C. Nuclei pellets were then re- suspended in 250 pL Wash and Resuspension Buffer containing 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Samples were sorted on the FACSAria III Cell Sorter (BD Biosciences) using a 70 pM nozzle. Nuclei were captured in 1.5 mL Eppendorf tubes containing 1 mL Wash and Resuspension Buffer. Recovered nuclei were centrifuged again at 500 x g for 5 min at 4°C. The supernatant was removed to leave ~100 pL and nuclei were resuspended by pipette mixing. Nuclei density counting was completed on the Countess III FL (Thermo Fisher Scientific).

[0186] Nuclei were processed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (PN-1000128, 10X Genomics). Nuclei were loaded into a Chip G (PN-1000127, 10X Genomics) and run on the Chromium Controller (10X Genomics) for gel bead emulsion (GEM) formation. Reverse transcription, barcoding, complementary DNA amplification and purification for library preparation were performed according to the Chromium Single Cell 3' Reagent Kits User Guide (CG000204_ChromiumNextGEMSingleCell3'v3.1_Rev). GEM formation and library preparation was completed by the Sequencing Facility at the Institute of Molecular Biosciences. All libraries were pooled and sequenced across two NovaSeq 6000 S1 flow cells (Illumina) to a depth of ~50,000 reads per nuclei (~10,000 per sample). snRNA-sequencinq bioinformatics

[0187] For human fetal, young, and adult hearts snRNAseq Fastq files were obtained from GEO accession GSE1567072. Fastq files for human and hCO samples were aligned using 10x Cell Ranger (version 7.0). The cellranger count command with default parameters was used to align the sequencing reads to the GRCh38 build of the human transcriptome (refdata-gex-GRCh38-2020-A) and generate a gene expression count matrix.

[0188] Sample filtering and quality control analysis: All subsequent analysis was performed using the R statistical programming language, using the Seurat package (version 4.3.0.1). There was an initial filtering step to keep genes that were expressed in three or more nuclei and nuclei with at least 200 detected genes. The quality of the cells was assessed for each sample independently by examining the total number of nuclei, the distributions of total unique molecular identifier (UMI) counts, the number of unique genes detected, and the proportions of ribosomal and mitochondrial content per nuclei. Nuclei displaying high expression of mitochondrial genes (using a cut-off of < 5%) were removed. For the nine human samples, nuclei with a UMI count depth of under 1 ,000 and higher than 40,000 were filtered out to remove debris and potential doublets. For the four hCO samples, nuclei with a UMI count depth of under 1 ,000 and higher than 30,000 were filtered out. No filtering was applied based on nFeature_RNA. SoupX (version 1.6.2) was used to estimate ambient RNA from empty droplets and correct the expression metrics by removing counts related to ambient RNA molecules.

[0189] Normalisation, integration and clustering: For each Seurat object, transformation and normalisation was performed using SCTransform to fit a negative binomial distribution and regress out mitochondrial read percentage. Integration of the four samples was performed using the following functions with default parameters: SelectlntegrationFeatures, FindlntegrationAnchors, Integrate Data. Principle components (PCs) were then calculated using the RunPCA function, and an elbow plot generated to select the cut-off for significant PCs to use for downstream analysis. UMAP dimensional reduction was then computed using the top 20 PCs using the RunUMAP command. Unsupervised clustering was then performed using the FindNeighbors and FindClusters functions. For exploratory purposes, Leiden clustering with a resolution range of 0 - 1 increasing at increments of 0.1 was performed to identify clusters within the data.

[0190] Differential gene expression testing: Differential gene expression analysis was performed using the FindMarkers command using the default Wilcoxon rank-sum. The logFC_cutoff was set to 0.25, assay set to ‘SCT’, and slot set to ‘data’. Mitochondrial genes were excluded from differential expression analysis. P-value adjustment was performed using bonferroni correction based on the total number of genes in the dataset, clusterprofiler (version 4.2.2) was used for enrichment analysis of gene ontology (GO) terms, using the enrichGO function. Adjusted p-values were calculated by the BH method and results were visualised using the dotplot function from enrichplot (version 1 .14.2).Spatial RNA-sequencinq

[0191] hCOs were washed in PBS and evenly coated in pre-chilled Tissue-Tek O.C.T (Sakura), then placed on a 1 x 1 cm plastic mold containing OCT. hCOs were arrayed in different orientations to contain 6 SF-hCO and 6 DM-hCO embedded in Tissue-Tek O.C.T and frozen in an ethanol slurry. Tissue sections were sectioned at 10 pm thickness using a CM3050S cryostat (Leica), mounted onto a Stereo-seq T-Chip slide (Stereo-seq, 210CT114) pre-coated with 0.01% poly-L-lysine (Sigma) and allowed to incubate for 5 min at 37°C. Tissue sections were then fixed in methanol for 30 min at -20°C then stained with ssDNA fluorescent staining solution to identify cell boundaries and imaged using Z2 Axio Imager (Ziess) using the FITC channel. The sections were permeabilised using 1 x Permeabilsation Reagent Solution (Stereo-seq Transcriptomics T Kit, 111 KT114) for 18 minutes at 37°C, then rinsed in 1 x PR Rinse Buffer.

[0192] RNA was released and reverse transcribed using Reverse Transcription Mix (Stereo- seq Transcriptomics T Kit, 111 KT114) at 42°C for 3 hours. Following reverse transcription, hCOs were digested with TR Buffer (Stereo-seq Transcriptomics T Kit, 111 KT114) at 55°C for 10 minutes. To release cDNA from the chip, samples were incubated with cDNA Release Mix for 16 hours at 55°C. cDNA was collected, purified using 0.8x AMPure XP Beads (Beckman Coulter) then amplified using cDNA Amplification Mix (Stereo-seq Transcriptomics T Kit, 111 KT114). Samples were incubated at 95°C for 5 min, then 15 cycles of 98°C for 20 secs, 58°C for 20 sec, 72°C for 3 min and a final elongation step at 72°C for 5 min.

[0193] DNA concentration was quantified using Qubit™ dsDNA Assay Kit (Thermo Fisher Scientific) and cDNA fragmentation was carried out using 20 ng of cDNA and 1 x Fragmentation Reaction Mix (Stereo-seq Library Preparation Kit, 111 KL114) at 55°C for 10 min. Fragmented product was amplified using PCR Barcode Primer and Amplification Mix (Stereo-seq Library Preparation Kit, 111 KL114) using the following thermal cycler settings: 95°C for 5 min, 13 cycles of 98°C for 20 sec, 58°C for 20 sec, 72°C for 3 min and a final step at 72°C for 5 min. The PCR product was purified using 0.55X AMPure XP Beads and sequenced using MGI DNBSEQ™-T7 sequencer.Spatial RNA-sequencinq bioinformatics

[0194] Unless otherwise indicated each n is designated as an individual hCO, cultured independently for the entire period following hCO formation. An experiment is designated as an independent cardiac differentiation performed on an entirely different week from a different passage number of hPSCs.

[0195] For functional data, in most cases pre-treatment baseline functional recordings of hCO were performed and post-treatment recordings normalized to these as the baseline. An additional normalization to the control conditions was also performed in many cases to account for any time- dependent changes in function.

[0196] Most data were analysed using Prism v8.2 (GraphPad) with the appropriate statistical tests depending on normality, equal variances, experimental setup and required comparisons. The tests applied and the p-values are given in each figure and legend. For omics data, data were analysVed as described in the bioinformatics sections.REFERENCES1. Guo, Y. & Pu, W. T. Cardiomyocyte Maturation: New Phase in Development. Circ Res 126, 1086-1106, (2020).2 Sim, C. B. et al. Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor. Circulation 143, 1614- 1628, (2021).3 Mills, R. J. et al. Functional screening in human cardiac organoids reveals a metabolic mechanism for card io myocyte cell cycle arrest. Proc Natl Acad Sci U S A 114, E8372-e8381 , (2017).4 Voges, H. K. et al. Vascular cells improve functionality of human cardiac organoids. Cell Rep 42, 112322, (2023).5 Mills, R. J. & Hudson, J. E. Bioengineering adult human heart tissue: How close are we? APL Bioeng 3, 010901 , (2019).6 Karbassi, E. et al. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol 17, 341-359, (2020).7 Bedada, F. B. et al. Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes. Stem Cell Reports 3, 594-605, (2014).8 Drakhlis, L. et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat Biotechnol 39, 737- 746, (2021).9 Lewis-Israeli, Y. R. et al. Self-assembling human heart organoids for the modeling of cardiacdevelopment and congenital heart disease. Nat Commun 12, 5142, doi:10.1038 / s41467-021-25329-5 (2021).10 Hofbauer, P. et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell 184, 3299-3317. e3222, (2021).11 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.e811 , (2020).12 Tiburcy, M. et al. Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation 135, 1832-1847, (2017).13 Schaaf, S. et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One 6, e26397, (2011).14 Zhang, D. et al. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials 34, 5813-5820, (2013).15 Zhao, Y. et al. A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling. Ce / / 176, 913- 927. e918, (2019).16 Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239-243, (2018).17 Shen, S. et al. Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue. Stem Cell Reports 17, 2037-2049, (2022).18 Raad, F. S. et al. Chaicone-Supported Cardiac Mesoderm Induction in Human Pluripotent Stem Cells for Heart Muscle Engineering. ChemMedChem 16, 3300-3305, (2021).19 Simon Sarkadi, L. et al. Fatty Acid Composition of Milk from Mothers with Normal Weight, Obesity, or Gestational Diabetes. Life (Basel) 12, (2022).20 Zeleniuch-Jacquotte, A., Chajes, V., Van Kappel, A. L., Riboli, E. & Toniolo, P. Reliability of fatty acid composition in human serum phospholipids. EurJ Clin Nutr 54, 367-372, (2000).21 Pedram, A. et al. Estrogen regulates histone deacetylases to prevent cardiac hypertrophy. Mol Biol Cell 24, 3805-3818, (2013).22 Solan, J. L. et al.. Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down- regulation by PKC. J Cell Biol 179, 1301-1309, (2007).23 Sheikh, F. et al. Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease. J Clin Invest 122, 1209-1221 , doi: 10.1172 / jci61134 (2012).24 Traaseth, N. J. et al. Structural and dynamic basis of phospholamban and sarcolipin inhibition of Ca(2+)-ATPase. Biochemistry 47 , 3-13, (2008).25 Mills, R. J. et al. Drug Screening in Human PSC-Cardiac Organoids Identifies Pro-proliferative Compounds Acting via the Mevalonate Pathway. Cell Stem Cell 24, 895-907. e896, (2019b).26 Espinoza-Lewis, R. A. etal. Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5. Dev Biol 327, 376-385, (2009).27 Fernandes, I., Funakoshi, S., Hamidzada, H., Epelman, S. & Keller, G. Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells. Nat Commun 14, 8183, (2023).28 Wang, B. et al. Foxpl regulates cardiac outflow tract, endocardial cushion morphogenesis and myocyte proliferation and maturation. Development 131 , 4477-4487, (2004).29 Saleem, M., Barturen-Larrea, P. & Gomez, J. A. Emerging roles of Sox6 in the renal and cardiovascular system. Physiol Rep 8, e14604, (2020).30 Vafiadaki, E., Arvanitis, D. A. & Sanoudou, D. Muscle LIM Protein: Master regulator of cardiac and skeletal muscle functions. Gene 566, 1-7, (2015).31 Sakamoto, T. et al. A Critical Role for Estrogen-Related Receptor Signaling in Cardiac Maturation. Circ Res 126, 1685- 1702, (2020).32 Kim, J. J. et al. Mechanism of automaticity in card io myocytes derived from human induced pluripotent stem cells. J Mol Cell Cardiol 81, 81-93, (2015).33 Marchiano, S. et al. Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy. Cell Stem Cell 30, 396-414. e399, (2023).34 Mannhardt, I. et al. Human Engineered Heart Tissue: Analysis of Contractile Force. Stem Cell Reports 7, 29-42, (2016).35 Bogdanov, K. Y, Vinogradova, T. M. & Lakatta, E. G. Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation. Circ Res 88, 1254-1258, (2001).36 Peters, C. H. et al. Bidirectional flow of the funny current (1(f)) during the pacemaking cycle in murine sinoatrial node myocytes. Proc Natl Acad Sci U S A 118, (2021).37 Pieske, B. et al. Diminished post-rest potentiation of contractile force in human dilated cardiomyopathy. Functional evidence for alterations in intracellular Ca2+ handling. J Clin Invest 98, 764-776, (1996).38 Miotto, M. C. et al. Structural analyses of human ryanodine receptor type 2 channels reveal the mechanisms for sudden cardiac death and treatment. Sci Adv 8, eabo1272, (2022).39 Sutko, J. L. & Willerson, J. T. Ryanodine alteration of the contractile state of rat ventricular myocardium. Comparison with dog, cat, and rabbit ventricular tissues. Circ Res 46, 332-343, (1980).40 Chiesi, M., Wrzosek, A. & Grueninger, S. The role of the sarcoplasmic reticulum in various types of card io myocytes. Mol Cell Biochem 130, 159-171 , (1994).41 Xia, Y, Zhang, X. H., Yamaguchi, N. & Morad, M. Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling. Cardiovasc Res 120, 44-55, (2024).42 Chung, J. H., Canan, B. D., Whitson, B. A., Kilic, A. & Janssen, P. M. L. Force-frequency relationship and early relaxation kinetics are preserved upon sarcoplasmic blockade in human myocardium. Physiol Rep 6, e13898, (2018).43 Priori, S. G. et al. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation 106, 69-74, (2002).44 Baig, S. M. et al. Loss of Ca(v)1.3 (CACNA1 D) function in a human channelopathy with bradycardia and congenital deafness. Nat Neurosci 14, 77-84, (2011).45 Al-Attar, R. et al. Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes. Biomolecules 14, (2024).46 Gintant, G. et al. Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations. Regul Toxicol Pharmacol 117, 104756, (2020).47 Abdelsayed, M., Bytygi, I., Rydberg, A. & Henein, M. Y. Left Ventricular Contraction Duration Is the Most Powerful Predictor of Cardiac Events in LOTS: A Systematic Review and Meta-Analysis. J Clin Med 9, (2020).48 Toischer, K. et al. K201 improves aspects of the contractile performance of human failing myocardium via reduction in Ca2+ leak from the sarcoplasmic reticulum. Basic Res Cardiol 105, 279-287, (2010).49 Romero, A. et al. Discovery of Nelutroctiv (CK-136), a Selective Cardiac Troponin Activator for the Treatment of Cardiovascular Diseases Associated with Reduced Cardiac Contractility. J Med Chem 67, 7825- 7835, (2024).50 Rhoden, A. et al. Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat andhuman cardiac preparations. Am J Physiol Heart Circ Physiol 322, H373-h385, (2022).51 Dashwood, A. et al. Effects of omecamtiv mecarbil on failing human ventricular trabeculae and interaction with (-)- noradrenaline. Pharmacol Res Perspect Q, e00760, (2021).52 Teerlink, J. R. et al. Dose-dependent augmentation of cardiac systolic function with the selective cardiac myosin activator, omecamtiv mecarbil: a first-in-man study. Lancet 378, 667-675, (2011).53 Voors, A. A. etal. Effects of danicamtiv, a novel cardiac myosin activator, in heart failure with reduced ejection fraction: experimental data and clinical results from a phase 2a trial. EurJ Heart Fail 22, 1649-1658, (2020).54 Yuan, R et al. Single-Cell RNA Sequencing Uncovers Paracrine Functions of the Epicardial-Derived Cells in Arrhythmogenic Cardiomyopathy. Circulation 143, 2169-2187, (2021).55 Howden, S. E., Thomson, J. A. & Little, M. H. Simultaneous reprogramming and gene editing of human fibroblasts. Nat Protoc 13, 875-898, (2018).56 Mills, R. J. et al. BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection. Cell 184, 2167- 2182. e2122, (2021).57 Laurent, G. et al. Effects of chronic gap junction conduction-enhancing antiarrhythmic peptide GAP- 134 administration on experimental atrial fibrillation in dogs. Circ Arrhythm Electrophysiol 2, 171-178, (2009).58 Alexanian, M. et al. A transcriptional switch governs fibroblast activation in heart disease. Nature 595, 438-443, (2021).59 Ashour, D. et al. An interferon gamma response signature links myocardial aging and immunosenescence. Cardio vase Res 119, 2458-2468, (2023).60 Kirchhof, C. J., Bonke, F. I., Allessie, M. A. & Lammers, W. J. The influence of the atrial myocardium on impulse formation in the rabbit sinus node. Pflugers Arch 410, 198-203, (1987).61 Arad, M., Seidman, C. E. & Seidman, J. G. AMP-activated protein kinase in the heart: role during health and disease. Circ Res 100, 474-488, (2007).62 Fujiwara, Y. et al. ERRy agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation. Stem Cell Reports 18, 2108- 2122, (2023).63 Sakamoto, T. & Kelly, D. P. Cardiac maturation. J Mol Cell Cardiol 187, 38-50, (2024).64 Li, M. O. & Flavell, R. A. Contextual regulation of inflammation: a duet by transforming growth factor-beta and interleukin-10. Immunity 28, 468-476, (2008).65 Smith, E. D. etal. Desmoplakin Cardiomyopathy, a Fibrotic and Inflammatory Form of Cardiomyopathy Distinct From Typical Dilated or Arrhythmogenic Right Ventricular Cardiomyopathy. Circulation 141 , 1872- 1884, (2020).66 Wang, W. et al. Clinical characteristics and risk stratification of desmoplakin cardiomyopathy. Europace 24, 268-277, (2022).67 Nademanee, K. et al. Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome. J Am Coll Cardiol 66, 1976-1986, (2015).68 Voges, H. K., Mills, R. J., Porrello, E. R. & Hudson, J. E. Generation ofvascularized human cardiac organoids for 3D in vitro modeling. STAR Protoc 4, 102371 , (2023b).69 Mills, R. J. et al. Development of a human skeletal micro muscle platform with pacing capabilities. Biomaterials 198, 217- 227, doi:10.1016 / j.biomaterials.2018.11.030 (2019c).70 Takasato, M. et al. Kidney organoids from human IPS cells contain multiple lineages and model human nephrogenesis.Nature 526, 564-568, doi:10.1038 / nature15695 (2015).71 Ariotti, N. et al. Molecular Characterization of Caveolin-induced Membrane Curvature. J Biol Chem 290, 24875-24890, (2015).72 Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2, 1896-1906, (2007).73 Pino, L. K., Just, S. C., MacCoss, M. J. & Searle, B. C. Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries. Mol Cell Proteomics 19, 1088-1103, (2020).74 Humphrey, S. J., Karayel, O., James, D. E. & Mann, M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat Protoc 13, 1897-1916, (2018).75 Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44, W90-97, (2016).76 Pervolaraki, E., Dachtler, J., Anderson, R. A. & Holden, A. V. The developmental transcriptome of the human heart. Sci Rep 8, 15362, (2018).77 Hahn, V. S. et al. Myocardial Gene Expression Signatures in Human Heart Failure With Preserved Ejection Fraction. Circulation 143, 120-134, (2021).78 Vizcaino, J. A. etal. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res 41 , D1063-1069, (2013).79 Kao, T. et al. GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives. Stem Cell Reports ! , 518-526, (2016).

Claims

AMENDED CLAIMS received by the International Bureau on 12 February 2026 (12.06.2026)1. A cell culture medium for use in cardiomyocyte maturation, the medium comprising a 5' AMP- activated protein kinase (AMPK) activator, a carbon source and a fatty acid substrate.

2. The cell culture medium of claim 1 , further comprising an ERRp / y agonist and / or interferon-A1 (IFN-A1).

3. The cell culture medium of claim 1 or claim 2, wherein the AMPK activator is a pan-AMPK activator.

4. The cell culture medium of any one of claims 1 to 3, wherein the AMPK activator is MK-8722.

5. The cell culture medium of any one of claims 2 to 4, wherein the ERRp / y agonist is DY131.

6. The cell culture medium of any one of claims 1 to 5, wherein the fatty acid substrate is selected from the group comprising palmitic acid, oleic acid, myristic acid, and linoleic acid.

7. The cell culture medium of any one of claims 1 to 6, wherein the fatty acid substrate is present at a concentration of less than 100 pM.

8. The cell culture medium of any one of claims 1 to 7, wherein the fatty acid substrate is present at a concentration of about 10 pM.

9. A method of maturing a population of cardiomyocytes and / or a cardiac organoid, the method comprising exposing the population of cardiomyocytes or the cardiac organoid to a culture condition that comprises an AMPK activator and one or both of an ERRp / y agonist and IFN-A1 , wherein the culture condition is sufficient to promote maturation of the population of cardiomyocytes and / or the cardiac organoid.

10. The method of claim 9, further including the step of metabolically switching the population of cardiomyocytes and / or the cardiac organoid to fatty acid oxidation.1 1. The method of claim 9 or claim 10, wherein the culture condition also comprises a fatty acid substrate.

12. The method of claim 11 , wherein the fatty acid substrate is selected from the group comprising palmitic acid, oleic acid, myristic acid, and linoleic acid.

13. The method of any one of claims 9 to 12, wherein the method further comprises the earlier or initial step of exposing the population of card io myocytes and / or the cardiac organoid to a further culture condition that comprises a Wnt activator.

14. The method of claim 13, wherein the WNT activator is a GSK3 inhibitor.

15. A cardiac organoid or a population of cardiomyocytes produced by the method of any one of claims 9 to 14.

16. The cardiac organoid or the population of cardiomyocytes of claim 15, wherein the cardiac organoid or the population of cardiomyocytes has high levels of cTnl expression.

17. The cardiac organoid or the population of cardiomyocytes of claim 15 or claim 16, wherein the cardiac organoid or cardiomyocyte mRNA expression of sarcomeric TNNI3 as a fraction of TNNI3 and TNNI1 is about 0.43.

18. A method of screening for a candidate agent suitable for preventing cardiac dysfunction, the method comprising: contacting the cardiac organoid of any one of claims 15 to 17 with the candidate agent; determining the presence or level of one or more immunomodulatory or fibrotic biomarkers as compared to a predetermined threshold, wherein the immunomodulatory or fibrotic biomarkers are selected from the group comprising TGF-01 , THBS1 , LOXL2, TNC and IL-10; and on the basis of the immunomodulatory or fibrotic biomarkers being above a predetermined threshold, determining that the candidate agent is suitable for preventing cardiac inflammation.

19. A method of determining the effect on force of a candidate agent, the method comprising: applying a first pacing condition to the cardiac organoid produced by the method of any one of claims 9 to 14, orthe cardiac organoid of any one of claims 15 to 17; blocking spontaneous contractile activity of the cardiac organoid; contacting the cardiac organoid with the candidate agent; pausing the pacing of the cardiac organoid; and applying a second pacing condition to the cardiac organoid; wherein the increase in force of the cardiac organoid during the second pacing condition determines the effect on force of the candidate agent.

20. The method of claim 19, wherein the spontaneous contractile activity is blocked by funny current blockade.

21. The method of claim 19 or claim 20, wherein the first pacing condition and the second pacing condition are the same.

22. The method of claim 19 or claim 20, wherein the first pacing condition and the second pacing condition are different.

23. The method of any one of claims 19 to 22, wherein the spontaneous contractile activity blocking isperformed by exposing the cardiac organoid to cilobradine.

24. The method of any one of claims 19 to 23, wherein the cardiac organoid comprises a gene mutation identified in tachyarrhythmias (e.g., polymorphic ventricular tachycardia).

25. The method of claim 24, wherein the gene mutation is in the CASQ2 gene or the RYR2 gene.

26. The method of claim 24, wherein the gene mutation is in the desmoplakin (DSP) gene.

27. The method of claim 26, wherein the gene mutation in the DSP gene is a two base pair deletion at positions 4246 and 4247 (resulting in a Leu1416Asn substitution, and a stop codon after 22 amino acids in the new reading frame).

28. A cardiac organoid with mRNA expression of sarcomeric TNNI3 as a fraction of TNNIS and TNNI1 of0.43.

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