Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation

Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation Kaomei Guan, Wener Li, Xiaojing Luo, Anna Strano, Shakthi Arun, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3973784/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules, thus hindering their potential role in guiding personalised drug selection for patients. Here, we demonstrate an approach that combines lipid-enriched maturation medium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with an advanced electrophysiological, structural and metabolic phenotype. Through a systematic, stepwise parallel testing of the three stimuli, electrostimulation emerged as the pivotal factor to enhance mitochondrial development and to improve the electrophysiological properties of iPSC-CMs. The combined approach brought a substantial modification in their current composition by increasing I Na , I to , I K1 and I Kr but decreasing I Ca−L , resulting in a significant change in their sensitivity to cardioactive drugs. Transcriptome analysis revealed that activation of HMCES and TFAM targets played a role in mitochondrial development, whereas the downregulation of MAPK/PI3K signalling pathways and SRF targets were associated with polyploidy of iPSC-CMs. Taken together, our study provides mechanistic insights into the maturation of iPSC-CMs with a more adult-like drug response. Biological sciences/Stem cells/Stem-cell differentiation Health sciences/Cardiology/Cardiovascular biology/Heart development induced pluripotent stem cell-derived cardiomyocytes maturation nanopatterning electrostimulation drug testing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The discovery of human induced pluripotent stem cells (iPSCs) represents a breakthrough for medical research and clinical application. As an unlimited source of cardiomyocytes (CMs) with a patient-specific genetic background, iPSC-derived CMs (iPSC-CMs) can be employed to explore disease mechanisms and drug effects. Additionally, they hold the potential for regenerating lost myocardium in patients with heart failure 1 . Numerous studies have demonstrated the ability of iPSC-CMs to recapitulate clinical features of inherited cardiomyopathies 2 , arrhythmias 3 , 4 and cardiotoxic drug responses 5 , even with patient-specific sensitivities 6 , 7 , 8 . Despite these achievements, their immaturity remains a significant limitation. In comparison to adult CMs, iPSC-CMs exhibit considerable differences in their morphology, gene expression patterns, metabolism and functionality 9 , which may explain their low sensitivity to hypoxia(-reperfusion) injury 10 , 11 , or the lack of features expected from a clinical phenotype of an inherited disease 12 , 13 . The use of iPSC-CMs to predict the pro-arrhythmic activity of drugs in the Comprehensive in vitro Proarrhythmia Assay (CiPA) has shown good correlation with the clinical risk of torsade de pointes or QTc prolongation. However, discrepancies have been reported for some multichannel blockers. For example, verapamil has a good safety profile in the clinic, but abolishes the beating activity of iPSC-CMs at clinically relevant concentrations 14 , probably due to differences in the expression of genes encoding ion channels, such as SCN5A (Na V 1.5), CACNA1C (Ca V 1.2), KCNH2 (hERG) and KCNQ1 (K V 7.1) in iPSC-CMs compared to adult CMs 15 . Whether the establishment of more adult-like current patterns in iPSC-CMs affects their drug response remains elusive 16 . In recent years, several approaches have been developed to improve the maturation of iPSC-CMs. The integration of fibroblasts and/or endothelial cells into cardiac tissue models derived from iPSC-CMs significantly enhanced both structural and functional development, as demonstrated in various studies 17 , 18 . However, it is worth noting that 3D-tissue generation is challenging and the experimental throughput is lower compared to 2D-cultures 19 . Other approaches to enhance iPSC-CM maturation include supplementation of the culture medium with fatty acids (FA) 13 , 20 , 21 , hormones or small molecules 10 , 22 , micro- or nanopatterning (NP) of culture surfaces 23 , 24 , and electrostimulation (ES) 25 , 26 . As these stimuli have been investigated independently, the most effective factor for enhancing iPSC-CM maturation remains unclear. It is uncertain whether combined approaches could produce synergistic effects, and the underlying mechanisms driving the advanced maturation of iPSC-CMs remain to be elucidated. Here, we demonstrate that the combination of FA-enriched maturation medium (MM), NP and ES is an efficient approach to generate iPSC-CMs with an advanced maturation state and pharmacological response more closely resembling that of adult CMs. By systematically combining MM, NP and ES and using in-depth functional and molecular analyses, our data provide mechanistic insights into how these different stimuli influence the cellular structure, metabolism and electrophysiology of iPSC-CMs. Results We used the directed differentiation protocol to generate ventricular-like CMs from iPSCs derived from 3 healthy individuals 27 . On day 15, iPSC-CMs were digested and distributed to 4 experimental groups (Fig. 1 a). B27 medium, routinely used for iPSC-CM culture, served as a control. To unravel the synergistic effects of MM, NP, and ES on the maturation of iPSC-CMs, we systematically applied NP and ES to MM in a stepwise parallel manner (Fig. 1 a). MM was designed based on a published FA-supplemented medium that enhances the metabolic maturation of iPSC-CMs 13 , with some modifications (Suppl. Table 1). NP was used to induce cell alignment and ES was applied to induce a beating frequency of 2 Hz (Suppl. Video 1). Combined approach enhances structural maturation of iPSC-CMs We observed that NP application induced changes in cell shape and a significant increase in the alignment of iPSC-CMs in the MM + NP and MM + NP + ES groups compared to the B27 and MM groups (Fig. 1 b). In all three groups (MM, MM + NP, and MM + NP + ES) there was a noticeable increase in cell volume and granularity of iPSC-CMs compared to the B27 control. Compared to the MM group, NP did not induce an additional increase in cell volume and granularity, but the combination of MM + NP + ES induced further significant increases (Fig. 1 c,d), suggesting that ES plays an important role in the hypertrophic growth of iPSC-CMs. A comparable proportion of cardiac troponin T (cTNT)-positive cells was found in all conditions, with the highest cTNT mean fluorescence intensity in iPSC-CMs from the MM + NP + ES group (Fig. 1 e,f). Co-immunostaining for the sarcomeric protein α-actinin and cardiac ryanodine receptor (RYR2) revealed well-organised sarcomeric structures in iPSC-CMs under all conditions, but marked differences for the RYR2 localisation and the α-actinin/RYR2 colocalisation (Fig. 1 g,h). In B27-cultured CMs, robust RYR2 staining was observed in the nucleus and punctate staining in the cytosol, with a low degree of α-actinin/RYR2 colocalisation. In contrast, CMs from the other three groups revealed reduced nuclear RYR2 staining and an augmented presence of striated patterns. The α-actinin/RYR2 colocalisation was enhanced in both the MM and MM + NP groups, and this effect was further augmented by ES. In the B27 and MM groups, the gap junction protein connexin 43 (Cx43) was partially localised to perinuclear regions rather than the plasma membrane, whereas Cx43 membrane localisation was increased in CMs of the MM + NP group and further significantly improved by ES (Fig. 1 i). These results provide evidence for the additive effects of NP and ES to MM on the structural maturation of iPSC-CMs. Combined approach improves electrophysiological maturation of iPSC-CMs To evaluate the effect of MM, NP and ES on electrophysiological properties, we performed patch-clamp and multi-electrode array (MEA) studies to investigate the action potential (AP) and field potential (FP) parameters of single and monolayer iPSC-CMs, respectively. We observed the ‘notch-and-dome’ AP morphology only in iPSC-CMs (43%) of the MM + NP + ES group (Fig. 2 a). The resting membrane potential (RMP) was found to be progressively more negative in CMs from the MM (-49.7 ± 1.7 mV), MM + NP (-58.2 ± 1.6 mV) and MM + NP + ES (-65.6 ± 2.1 mV) groups compared to the B27 group (-44.1 ± 2.1 mV), while the maximum AP upstroke velocity (Vmax) was gradually increased in iPSC-CMs from the MM (5.0 ± 0.2 V/s), MM + NP (6.6 ± 0.6 V/s) and MM + NP + ES (11.0 ± 2.0 V/s) groups compared to the B27 control (4.2 ± 0.3 V/s). Similarly, a gradual increase in AP amplitude (APA) was observed in the four groups (Fig. 2 b). The AP duration at 90% repolarisation (APD 90 ) was significantly shorter in iPSC-CMs paced at 0.5 Hz in the MM + NP + ES group than in the B27 control (Fig. 2 c). As the transient outward K + current ( I to ) underlies the prominent phase 1 repolarisation of cardiac APs and the ‘notch-and-dome’ AP morphology, we measured I to and found a significantly higher I to density in iPSC-CMs from the MM + NP + ES group, but only a slight increase in MM and MM + NP conditions compared to the B27 group; and NP itself has less effect on I to when comparing the MM + NP group with the MM group (Fig. 2 d,e). Intercellular electrotonic coupling and conduction velocity (CV) across CMs are largely dependent on Cx43 expression at the gap junction 28 . Consistent with the Cx43 localisation data (Fig. 1 i), heatmaps of electrical signal propagation analysed by MEA illustrate the stepwise increase in CV in iPSC-CM monolayers from the MM (22.3 ± 0.8 cm/s), MM + NP (25.6 ± 0.9 cm/s) and MM + NP + ES (27.8 ± 1.5 cm/s) groups, compared to the B27 condition (12.5 ± 1.2 cm/s). Similar stepwise changes in spike amplitude and slope were observed in the four groups (Fig. 2 f,g). To analyse which changes in specific ion currents underlie the improved electrophysiological functionality, we recorded I Na , I K1 , and I Kr using the patch-clamp technique. We found that I Na density was significantly higher in MM-cultured iPSC-CMs with a mean peak current density of -87.9 ± 8.9 pA/pF at -20 mV compared to the B27 group (-40.2 ± 7.3 pA/pF). Notably, NP induced only a small increase in I Na density with a mean peak of -100.1 ± 9.6 pA/pF at -25 mV when compared to the MM group, but I Na density was further significantly induced by ES in the MM + NP + ES group with a mean peak of -136.9 ± 13.5 pA/pF (Fig. 3 a,b). These data are consistent with the AP- (Vmax, APA) and FP-metrics (CV, spike amplitude and slope) shown in Fig. 2 . The electrophysiological immaturity of iPSC-CMs compared to adult CMs is partly attributed to the low density of the hyperpolarising K + current I K1 , which is important for stabilising the RMP 29 . We found a very low I K1 density in B27-cultured iPSC-CMs (-2.8 ± 0.4 pA/pF at -130 mV), and only a slight increase in I K1 in the MM group (-6.1 ± 1.2 pA/pF). Interestingly, NP induced a significant increase in I K1 in the MM + NP group (-13.1 ± 2.6 pA/pF), which was further induced by ES (-15.5 ± 2.6 pA/pF in the MM + NP + ES group) (Fig. 3 c,d). HERG channels conducting the rapid delayed rectifier K + current I Kr are involved in phase 3 repolarisation of cardiac APs. Similar to I K1 , both E-4031-sensitive I Kr step and tail current densities were only slightly induced by MM, but significantly induced by NP and further enhanced by ES (Fig. 3 e-g). These data are consistent with the most negative RMP and the shortest APD 90 in iPSC-CMs from the MM + NP + ES group (Fig. 2 b,c). Taken together, these findings highlight the distinct effects of the three stimuli on specific ion currents. This is evidenced by the strong influence of NP on I K1 and I Kr , with less or no effect on I Na and I to , and the robust effect of MM on I Na , but less on I Kr . Importantly, the data underline that the combined approach significantly enhanced electrophysiological maturation of iPSC-CMs. Combined approach improves calcium handling and contractility of iPSC-CMs Since excitation-contraction coupling in CMs involves calcium cycling to convert electrical signals into mechanical output (contraction), we next examined L-type calcium channel (LTCC) current I Ca−L and calcium transients in iPSC-CMs. I Ca−L densities exhibited similar reductions in both the MM and MM + NP groups when compared to the B27 group, which were further reduced by ES (Fig. 4 a,b). To quantify intracellular Ca 2+ dynamics, we performed Fura-2-based calcium imaging in iPSC-CMs paced at 0.5 Hz (Fig. 4 c-e). Significantly reduced diastolic and systolic Ca 2+ levels were observed in the MM, MM + NP and MM + NP + ES groups compared to the B27 group, but Ca 2+ transient amplitudes were comparable between all groups despite the reduced I Ca−L density in the MM, MM + NP and MM + NP + ES groups. The Ca 2+ transient decay time constant (tau) is also significantly shortened in the MM group compared to the control, whereas no further shortening was observed in the MM + NP and MM + NP + ES groups. Application of 10 mM caffeine resulted in significantly increased Ca 2+ release from the sarcoplasmic reticulum (SR) in the MM, MM + NP and MM + NP + ES groups compared to the B27 group (Fig. 4 e). These data suggest a more efficient coupling between I Ca−L and SR Ca 2+ release, enhanced Ca 2+ decay kinetics and a higher SR calcium content in these three groups. Video-based analysis of iPSC-CM beating properties 5 showed that the changes in calcium handling were associated with improved contractile function. Stopping ES in the MM + NP + ES group resulted in cessation of beating, followed by regaining of spontaneous beating activity within 15–30 minutes at a rate comparable to the other three groups (Fig. 4 f). Significantly shorter beating duration, contraction and relaxation times were observed in the MM + NP + ES group compared to the other groups (Fig. 4 g; Extended Data Fig. 2 a,b). We found a similar trend in the MM and MM + NP groups compared to the B27 control, but no significant difference between the two groups. Consistent with this observation, all cultures in the MM + NP + ES group successfully captured high-frequency (2 Hz) field stimulation, whereas none of the B27 cultures demonstrated this capability (Fig. 4 h; Extended Data Fig. 2 c). This improved contractile function was accompanied by an increased gene expression ratio of TNNI3 / TNNI1 and MYL2/MYL7 , whereas the expression of MYH6 , encoding the fast-twitch MHC isoform, was upregulated in response to sustained ES, leading to a reduced MYH7 / MYH6 ratio (Fig. 4 i). These results demonstrate that MM, NP and ES individually and synergistically induce electrophysiological and functional maturation of iPSC-CMs. Combined approach improves drug response of iPSC-CMs To investigate whether the maturation state of iPSC-CMs influences their drug response, we chose verapamil (calcium-channel blocker), E-4031 (hERG-channel blocker) and isoprenaline (β-adrenergic stimulus) as model substances to detect pro-arrhythmic activity based on changes in FP parameters (Fig. 5 ; Extended Data Fig. 3 a,b). We observed beating arrest in cultures from the B27 (all cultures), MM (9/17) and MM + NP (7/18) groups at 1 µM verapamil. Concentration-dependent reductions in spike amplitude were observed in the B27 group and, to a lesser extent, in the MM and MM + NP groups. In contrast, no effect of verapamil on beating activity and spike amplitude was observed in the MM + NP + ES group (Fig. 5 b; Extended Data Fig. 3 c). Verapamil-induced shortening of FP duration (FPDc), which corresponds to QT-shortening in the clinic, was comparable in iPSC-CMs from the MM, MM + NP and MM + NP + ES groups. However, this effect was more pronounced compared to iPSC-CMs cultured in B27 (Fig. 5 c,d). Previous studies showed that immature iPSC-CMs failed to produce APD prolongation after E-4031 treatment, even at high concentrations 30 . Similarly, we found that E-4031 induced only minor changes in FPDc in B27-cultured iPSC-CMs, whereas significant concentration-dependent FPDc prolongation was detected in the MM, MM + NP and MM + NP + ES groups (Fig. 5 e,f). Furthermore, we observed a more pronounced positive-chronotropic response of iPSC-CMs to isoprenaline in these three groups than in the B27 group, which correlates with FPDc-shortening (Fig. 5 g,h). The EC 50 of isoprenaline for the chronotropic effect was also much lower in these three groups than in the B27 group (Fig. 5 h). These experiments demonstrate the substantial impact of the maturation state of iPSC-CMs on their response to various cardioactive drugs. They emphasise the importance of utilising iPSC-CMs with more adult-like electrophysiological properties for accurate drug risk assessment. Combined approach downregulates MAPK/PI3K-AKT pathways Previous studies have shown that FA-enriched media induce iPSC-CM maturation by regulating key genes involved in FA metabolism, mitochondrial function, calcium cycling, ion channels and sarcomere 13 , 20 . To gain insight into the molecular mechanisms driving iPSC-CM maturation by NP and ES, we performed RNA sequencing (RNA-seq) analysis. Surprisingly, NP had little synergistic effect when combined with MM, whereas the addition of ES strongly influenced gene expression (Fig. 6 a-d; Extended Data Fig. 4 a,b). Comparing the MM and MM + NP groups, only 163 differentially expressed genes were identified, of which 56 were upregulated and 107 downregulated in the MM + NP group. In contrast, 1,370 significantly upregulated and 1,657 downregulated genes were identified in the MM + NP + ES group compared to the MM group, of which 747 significantly upregulated and 990 downregulated genes were also identified when compared to the MM + NP group, indicating the synergistic effects of NP and ES. Pathway enrichment analysis of the downregulated genes in the MM + NP + ES group mainly mapped to MAPK/PI3K-AKT, TNFR2-NFκB, G-protein-coupled receptor (GPCR), and cytokine/chemokine signalling (Fig. 6 e; Extended Data Fig. 4 d; Suppl. Table 2). Using the transcription factor target database, we identified the SRF cluster, which includes many genes involved in cell cycle regulation and cell proliferation (Fig. 6 f,g; Extended Data Fig. 4 c). We also found a decrease in SRF protein levels in CMs from the MM + NP + ES group compared to the other groups (Fig. 6 h). These findings encouraged us to evaluate the expression of genes that regulate cell cycle progression. Notably, activators of G2/M checkpoints including cyclins ( CCNB1-3 ), cyclin-dependent kinase 1 ( CDK1 ) were downregulated, whereas CDK inhibitors ( CDKN1A , CDC20 ) were upregulated in the MM + NP + ES group compared to the other two groups (Fig. 7 a,b). Interestingly, genes ( ANLN , SEPTIN7/2 ) encoding activators of cytokinesis were also downregulated. We did not observe any significant changes in the gene sets ( CCND1-3 , CCNE1/2 , CCNA1/2 , CDK2/4/6 , CDKN2A-D , CDKN1B/C , CDH1 ) controlling the G1 and S progression and the G1/S checkpoint (Fig. 7 a,b). These data suggest a cell cycle arrest after S phase and before exit from M phase in the MM + NP + ES group, which may lead to bi-nucleation or nuclear polyploidy. To confirm this, we examined DNA content and found that the number of diploid iPSC-CMs was significantly reduced and polyploid cells significantly increased in the MM + NP + ES group compared to the other three groups (Fig. 7 c,f). Interestingly, we observed no difference in the proportion of 5-ethynyl-2’-deoxyuridine (EdU)-incorporated iPSC-CMs between all four groups, which can detect DNA synthesis during S phase (Fig. 7 d,g). However, the proportion of Ki67 + iPSC-CMs and Ki67 − polyploid iPSC-CMs was higher in the MM + NP + ES group than in the other groups (Fig. 7 e,h,i). Ki67 is widely expressed throughout the entire cell cycle, except in G0, and reaches a maximum in S/G2 31 . These results indicate that the downregulation of MAPK/PI3K-AKT and SRF-related genes is involved in G2/M arrest and polyploidy development of iPSC-CMs. Changes in gene expression profile associated with metabolism and electrophysiology The pathway enrichment analysis of genes upregulated in the MM + NP + ES group revealed that the combined approach induced the upregulation of genes involved in electron transport chain (ETC), TCA cycle, mitochondrial biogenesis, NRF2 signalling, glucose metabolism, N-glycan biosynthesis, FA oxidation, tRNA aminoacylation, etc. (Fig. 8 a; Extended Data Fig. 4 e; Suppl. Table 3). These gene clusters were only slightly upregulated in the MM + NP group compared to the MM group (Extended Data Fig. 4 e). Using the transcription factor target database we identified two clusters, TFAM (Fig. 8 b) and HMCES (Fig. 8 c), which were enriched in the MM + NP + ES group (Extended Data Fig. 4 c). In these two clusters, mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunits ( MTND2-6 ) and pseudogenes ( MTND4P12 , MTND5P11 , MTND6P4 ), cytochrome c oxidase subunits ( MT-CO1/3 ) and pseudogenes ( MT-CO1P2 , MT-CO1P12 , MT-CO3P12 ), cytochrome b ( MTCYB ), 12S rRNA ( MT-RNR1 ) and tRNAs ( MT-TP/-TM/-TE/-TI/-TW/-TC/-TY/-TR/-TN/-TQ ) were upregulated in the MM + NP + ES group compared to the MM group. Subsequently, we found increased mitochondrial mass in CMs from the MM + NP + ES group compared to the other groups (Fig. 8 d). Furthermore, CMs from the MM + NP + ES group showed a higher expression of OPA1 , PPARGC1α and PPARα (Fig. 8 g), confirming an enhanced mitochondrial development in response to ES. Measurements of oxygen consumption rate as a surrogate for mitochondrial function showed an increased basal and maximal respiration, ATP production and spare capacity of CMs from the MM group compared to the B27 control. There is only a marginal additional increase in these parameters in the MM + NP + ES and MM + NP groups compared to the MM group (Fig. 8 e,f). These findings indicate that the combination of MM + NP + ES leads to upregulation of oxidative phosphorylation, activation of mtDNA-encoded components and an overall enhancement of mitochondrial development and function. Further examination of individual changes in key ion channel components revealed a clear correlation between gene expression and channel function (Fig. 8 h). We found upregulation of genes contributing to I to ( KCNA7 , KCNC3 , KCNC4 , KCND2 , KCND3 ), I K1 ( KCNJ2 , KCNJ4 , KCNJ6 , KCNJ11 , KCNJ12 ), I Kr ( KCNH2 , KCNE2 ) and I Ks ( KCNQ1 ) currents, as well as genes encoding calcium-activated ( KCNN1 ) and voltage-gated ( KCNS3 , KCNAB2 , KCNIP3 ) potassium channels in the MM + NP + ES group compared to the MM group. In contrast, genes encoding the LTCCs ( CACNA1C , CACNA1D , CACNA1A , CACNA2D3 , CACNA2D4 ) and regulating the LTCC activity ( CACNG7 ) were downregulated, whereas the genes encoding the T-type calcium channels ( CACNA1H , CACNA1I ) were upregulated. No significant differences in SCN5A expression were found between the three groups, but SCN1B was upregulated and SCN3B was downregulated in the MM + NP + ES group. In addition, the expression of the genes encoding calcium handling proteins were not significantly altered. Collectively, these results are consistent with the significantly increased I Na , I to , I K1 , and I Kr but decreased I Ca−L currents in iPSC-CMs from the MM + NP + ES group. Discussion In this study, we systematically investigated the collective impact of MM, NP and ES on the maturation of iPSC-CMs. Our findings demonstrate that the concurrent application of MM, NP and ES serves as an efficient strategy to enhance the structural, electrophysiological, metabolic and functional maturation of iPSC-CMs. This maturation process involves the modulation of MAPK/PI3K-AKT signalling as well as the regulation of TFAM/HMCES and SRF target genes, which is of significant relevance for the utilisation of iPSC-CMs in disease modelling and drug testing. The core of the maturation strategy is the application of FA-supplemented MM in iPSC-CMs, which induces the metabolic transition from glucose-based energy production to FA β-oxidation. This process is essential for enhanced maturation, reflected by an increase in cell size and mitochondrial network as well as enhanced respiratory, electrophysiological, and contractile activity. These findings align with previously reported data on the positive effects of FA supplementation 13 , 20 . By stepwise addition of NP and ES to MM, we found that the combination of NP with MM had only a limited impact on the metabolic maturation of iPSC-CMs, as demonstrated by subtle changes in the expression profile of gene clusters related to energy metabolism as well as mitochondrial development and function, such as ETC (also known as oxidative phosphorylation), TCA cycle, FA oxidation, and glucose metabolism (Extended Data Fig. 4 e). Strikingly, the addition of ES to MM + NP resulted in significant upregulation of these gene clusters, likely due to the increased FA β-oxidation in iPSC-CMs to meet the energy demand for the persistent beating activity at 2 Hz. An important finding of our study is that GSEA analysis of the upregulated genes in MM + NP + ES using the transcription factor target database maps to the enrichment of HMCES- and TFAM-related target gene sets. HMCES (5-hydroxymethylcytosine binding, embryonic stem cell-specific) may safeguard the genomic and mtDNA integrity of iPSC-CMs during oxidative stress responses triggered by elevated levels of reactive oxygen species due to the use of FA. This protective mechanism involves the formation of stable DNA-protein crosslinks with abasic DNA damage to prevent error-prone repair pathways 32 , 33 , 34 . TFAM (mitochondrial transcription factor A) is essential for the transcription, replication and packaging of mtDNA into nucleoids. It is indispensable for the meticulous regulation of mitochondrial biogenesis, ensuring the seamless adaptation of the mitochondrial population to precisely match the energy demand of the cell 35 . This is supported by our observation that most mtDNA-encoded essential components of the ETC as well as rRNAs and tRNAs required for the translation of mtDNA-encoded proteins were significantly upregulated by the ES stimulation (Fig. 8 ). PGC-1α ( PPARGC1α ) is the master regulator of mitochondrial energy metabolism, respiration and biogenesis through interaction with its various coactivators ERR, PPAR and NRF1/2 36 . Together with ERR, it controls mitochondrial dynamics via activation of genes involved in mitochondrial fission and fusion including OPA1 and MFN2 37 , which were found to be upregulated in the MM + NP-ES group. Together with PPARα, which is also upregulated in the MM + NP + ES, PGC-1α regulates genes involved in mitochondrial FA oxidation and many other cellular lipid metabolic pathways 35 , 36 . Furthermore, PGC-1α together with NRF1/2 promotes mitochondrial biogenesis by activating TFAM 35 , 36 . Another interesting discovery of our study revealed a significant downregulation of genes involved in MAPK/PI3K signalling pathways in the MM + NP + ES group. This aligns with the substantial downregulation of the MAPK and PI3K-AKT pathways in the postnatal heart when compared to the neonatal heart 38 . Thus, MAPK/PI3K-AKT inhibition promotes iPSC-CM maturation, partially mediated by the upregulation of PGC-1α 38 . Further research is needed to determine whether the downregulation of MAPK/PI3K signalling pathways in the MM + NP + ES group is involved in PGC-1α and TFAM activation through the control of AMPK 39 , 36 , AKT 40 and/or mTORC1 41 pathways, and is therefore essential for the metabolic maturation of iPSC-CMs. Most strikingly, our RNA-seq and cell cycle analysis data show that downregulated MAPK/PI3K-AKT signalling is involved in the downregulation of genes important for the G2/M transition and cytokinesis, leading to polyploidy (nuclear polyploidy and/or multinucleation) of iPSC-CMs. Human CMs are diploid during the first years of life and gradually become polyploid over time. By the second decade of life, approximately 25% of CM are multinucleated and 57% polyploid 42 . In CMs, the major cyclin-CDK complexes controlling cell cycle progression are CCND-CDK4/6 (G1 phase), CCNE-CDK2 (G1/S transition), CCNA-CDK2/1 (S and G2 phase and S/G2 transition) and CCNB-CDK1 (G2/M transition and M phase), the activity of which is inhibited by CDK inhibitors 31 , 43 , 44 . The CCNB-CDK1 complex is not expressed in cell cycle-arrested adult CMs, and it is also not required for CM hypertrophy 44 . In our study, we found a decrease in the expression of CCNB-CDK1 in iPSC-CMs from the MM + NP + ES group. Despite this decrease, these cells exhibited ongoing DNA synthesis, an increased proportion of polyploid CMs and higher cell volume and granularity. The downregulated CCNB-CDK gene expression is associated with the upregulation of CDK inhibitor p21 and MEIS1 that are negatively regulated by TBX20 45 . Better understanding of how MAPK/PI3K-AKT signalling controls cell cycle progression, including the expression of TBX20, MEIS1 and p21, may provide valuable mechanistic insights to promote iPSC-CM maturation or to stimulate adult CM regeneration. Another important finding of our study is that NP and ES synergistically induce the maturation of different ion channels. We found that NP combined with MM strongly increased I Kr and I K1 , but had little effect on I to , I Na and I Ca−L . However, the addition of ES to MM + NP led to significant changes in all currents (larger I Na , I to , I K1 , and I Kr , but smaller I Ca−L ) and maturation of electrophysiology (more negative RMP, shorter APD, higher Vmax, APA, CV and spike amplitude, and the ‘notch-and-dome’ AP morphology) in iPSC-CMs, similar to adult CMs 9 , 29 , 46 . The enhanced electrophysiological functionality of iPSC-CMs contributes to their improved predictive value for risk assessment of cardioactive drugs, especially in the case of multi-channel blockers such as verapamil, ranolazine or alfuzosin 15 , 16 . Several studies reported that verapamil inhibits the beating activity of iPSC-CMs 47 , 15 , 16 , probably because depolarisation in immature iPSC-CMs does not rely exclusively on I Na , as in adult CMs, but also on I Ca−L 15 . The RNA-seq data suggest that the increased I to , I K1 , and I Kr and the decreased I Ca−L in the MM + NP + ES group may be due to the upregulation of genes encoding different potassium channels and the downregulation of genes encoding LTCCs. In addition, the downregulation of PI3K-AKT signalling may contribute to the reduced I Ca−L densities 48 . Furthermore, the similar Ca 2+ transient amplitudes of all groups together with the decreased I Ca−L in iPSC-CMs from MM, MM + NP and MM + NP + ES groups in comparison to the B27 condition suggest an enhanced excitation-contraction coupling gain, an important indicator of improved calcium handling 22 . The colocalisation of RYR2 with α-actinin and the ability to adapt to high-frequency stimulation support an improved calcium handling, especially in CMs from the MM + NP + ES group. Interestingly, we observed no changes in the expression of SCN5A , coding for the pore-forming α-subunit of the sodium channel (Na V 1.5), but an upregulation of SCN1B and a downregulation of SCN3B , which encode the β-subunits (Na V -β1/3) of the sodium channel that interact with the α-subunits 49 . While SCN1B is highly expressed in adult CMs, SCN3B is highly expressed in the embryonic heart 50 . Previous studies have shown that co-expression of SCN1B with SCN5A increases the density of I Na 50 , and the β1-subunit modulates the cell surface localisation, gating, and kinetics of α-subunits 51 . In addition, Na V -β1 also regulates voltage-gated potassium channels, including K V 4.3 and associates with the cardiac intercalated disc proteins N-cadherin and Cx43 51 , contributing to the enhanced electrical signal conduction. Future studies should focus on whether/how the downregulation of MAPK/PI3K-AKT signalling in iPSC-CMs regulates the gene expression related to ion channel (for example, K + and Na + channels) maturation and function 52 . Taken together, we demonstrate that the combined application of MM, NP and ES synergistically induces structural, electrophysiological, metabolic and functional maturation of iPSC-CMs and provide first insights into the mechanism driving advanced maturation of iPSC-CMs. Cultivation in FA-enriched MM strongly improves mitochondrial development and electrophysiological functionality of iPSC-CMs. Although the addition of NP to MM had little effect on the gene expression profile, it induced a specific increase in I K1 and I Kr current densities and cell alignment. The MM + NP + ES combination induces molecular changes that occur during cardiac development, leading to increased structural maturation, polyploidy, improved mitochondrial development, and current patterns of I Na , I to , I K1 , I Kr and I Ca−L more similar to adult human CMs. These changes translated into an altered sensitivity of iPSC-CMs to cardioactive drugs, suggesting the efficacy of our maturation approach to improve the predictive power of iPSC-CMs in drug screening. Furthermore, the improved maturation of iPSC-CMs highlights the potential of our combined approach to recapitulate clinical phenotypes that require an advanced development state of iPSC-CMs. Methods Directed differentiation of iPSCs into iPSC-CMs, and pro-maturation culture of iPSC-CMs In this study, three human iPSC lines were used, which were reprogrammed from somatic cells of three healthy individuals previously. The cell lines iWTD2.1 (UMGi001-A clone 1) and iBM76.3 (UMGi005-A clone 3) were generated from dermal fibroblasts and mesenchymal stem cells, respectively, using STEMCCA lentivirus 27 , 53 . The cell line isWT7.22 (UMGi020-B clone 22) was generated from dermal fibroblasts using the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit 54 . The iPSC generation and application in research were approved by the Ethics Committee of the University Medical Center Göttingen (approval number: 21/1/11 and 10/9/15) and TU Dresden (EK 422092019). All iPSCs were cultured on Geltrex (Thermo Fisher Scientific) coated 6-well plates in Essential 8 (E8) medium (Thermo Fisher Scientific). E8 medium was changed daily and cells were passaged or differentiated when they were ~ 85% confluent. To initiate differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with Glutamax and HEPES, 0.5 mg/mL human recombinant albumin, and 0.2 mg/mL L-ascorbic acid 2-phosphate and treated with 4 µM CHIR99021 (Merck Millipore), an inhibitor of GSK3β. After 48 h, CHIR99021 was removed and the cells were treated with 5 µM IWP2 (Wnt antagonist II, Merck Millipore) for another two days. The first beating cells were detected on day 8 post differentiation. From day 8, cells were cultivated in B27 medium containing RPMI 1640 with Glutamax and HEPES, supplemented with 1x B27 with insulin (Thermo Fisher Scientific). On day 15 after differentiation, the cells were digested and passaged. Cells were first incubated with 1 mg/mL collagenase B (Worthington Biochemical) for 1 h in an incubator, then detached iPSC-CM clusters were gently collected in a 15-mL Falcon tube and dissociated with 0.25% trypsin/EDTA (Thermo Fisher Scientific) for 8 min. Dissociated iPSC-CMs were resuspended in cardio-digestion medium (80% B27 medium, 20% fetal calf serum, and 2 µM thiazovivin (Merck Millipore)). The resuspended cells were seeded into Geltrex-coated 6-well plates for the B27 and MM groups, or onto Geltrex-coated ø25 mm nanopatterned (NP) coverslips (NanoSurface Coverglass, Curi Bio) in 6-well plates for the MM + NP and MM + NP + ES groups at a density of 300,000-500,000 cells per well. Cells were maintained in B27 medium for 6 days with medium changes every 2 days. On day 21 post differentiation, all the non-B27 groups were switched from B27 medium to maturation medium (MM, Suppl. Table 1) for 7 days, with medium changes every 2 days. On day 28 post differentiation, the MM + NP + ES group was subjected to 2 Hz electric field stimulation (2 ms pulse duration, 8 V) using a C-Pace EP (IonOptix) together with a 6-well C-dish (IonOptix) for 14 days. On day 42 post differentiation, cells from all four groups were harvested directly for analysis or dissociated, replated and cultured for another 7 days for further analysis (Fig. 1 a). Patch clamp analysis iPSC-CMs at day 42 from the four groups were dissociated using a previously described method 55 , 56 . For MM + NP and MM + NP + ES groups, NP coverslips with cells were transferred to a 3.5-cm dish and then treated for 10 min with 2 mL of 20 U/mL papain (Sigma-Aldrich) dissolved in 1.1 mM EDTA-buffered B27 medium containing 2.5 µM blebbistatin (dissolved in DMSO). The cells were gently centrifuged at 50 g for 1 min. After aspirating the supernatant, the cell pellet was gently resuspended in B27 medium containing 2.5 µM blebbistatin and stored at 4℃ until measured. All automated patch-clamp experiments were performed at room temperature using Patchliner Quattro (Nanion technologies GmbH) with low resistance (for I Na , I K1 , and I Ca−L recordings) and medium resistance (for I to recordings) NPC-16 chips. The intracellular pipette and extracellular bath solutions for I Na , I K1 , I Ca−L , and I to are listed in Suppl. Table 4. To record I Na , cells were depolarised from a holding potential of -100 mV using voltage steps from − 80 to + 70 mV for 20 ms in 5 mV steps. The sweep interval was 2 s. Nifedipine (10 µM) was used to block I Ca−L . I K1 was recorded using test potentials of 2 s duration between − 130 and 10 mV from a holding potential of -40 mV. The sweep interval was 10 s. The protocol was repeated in the presence of 0.5 mM BaCl 2 and the Ba 2+ -sensitive current was calculated as I K1 . To record I Ca−L , cells were depolarised for 100 ms to voltages between − 80 and + 60 mV from a holding potential of -90 mV, and the sweep interval was 3 s. I to was recorded by increasing the test potential stepwise from − 40 mV to + 60 mV in 10 mV steps from a holding potential of -90 mV with a 20 ms pre-pulse to -35 mV to inactivate I Na . Each pulse lasted for 600 ms, and the sweep interval was 10 s. CdCl 2 (0.5 mM) was used to block sodium and calcium currents. For action potential (AP) recordings and I Kr measurements using manual patch clamp technique, CMs from all four groups were used directly after overnight recovery in cardio-digestion medium in order to minimise the time that CMs from the MM + NP and MM + NP + ES groups spent in non-NP and non-ES conditions. The pipette and extracellular solutions used for AP and I Kr recordings are listed in Suppl. Table 4. All manual patch-clamp experiments were performed at room temperature using a ruptured whole-cell patch clamp with a HEKA EPC10 amplifier and Patchmaster (HEKA Elektronik). To assess resting membrane potential (RMP), maximum upstroke velocity (Vmax), and action potential amplitude (APA), spontaneous APs were recorded in Tyrode’s solution without current injection. To assess AP duration (APD), a negative current was injected into the CMs to maintain the RMP at approximately − 80 mV prior to application of 0.5 Hz pacing stimulation. Signals were filtered with 2.9 and 10 kHz Bessel filters. At least 5 consecutive stable spontaneous APs and paced APs were averaged to determine RMP, Vmax, APA and APD at 90% repolarisation (APD 90 ) using LabChart 8 software (ADInstruments). The holding potential of the I Kr recording was set at -50 mV. I Kr was elicited by 2.5 s depolarisation steps from − 40 mV to potentials from + 40 to -40 mV in 10 mV decrements. This was followed by a 4 s repolarisation phase to -40 mV to elicit the I Kr tail current. The pulse interval for each sweep was 10 s. I Kr was defined as the E-4031-sensitive current by subtracting the current recorded after application of 1 µM E-4031 from the current recorded before application. Multi-electrode array All multi-electrode array (MEA) recordings were performed using a Maestro Edge equipped with AxIS Navigator software (Axion BioSystems) at a sampling rate of 12,500 Hz, a temperature of 37°C, and 5% CO 2 . To assess the field potential (FP) properties, the digested iPSC-CMs were seeded onto Geltrex-coated CytoView 6-well MEA plates (Axion BioSystems). Approximately 200,000 cells were resuspended in 20 µL of cardio-digestion medium and seeded onto the electrode distribution area of the MEA plates. To evaluate the drug response, the digested iPSC-CMs were seeded into Geltrex-coated CytoView 24-well MEA plates (Axion BioSystems) at a density of 25,000 cells per well. Around one hour after seeding, an additional 1 mL of cardio-digestion medium was gently added into every well. The digested iPSC-CMs were recovered for 7 days in the same medium used during the maturation period. To avoid the influence of different media during recording, iPSC-CMs of all conditions were incubated in MM for one hour before starting measurements. MEA drug testing was performed using a sequential addition protocol with concentration increments after baseline activity recording in each well (Extended Data Fig. 3 ). Vehicle controls for all conditions were performed on each assay plate. After drug addition, cells were incubated at 37°C, 5% CO 2 for 12 min, and the response was recorded for 2 min. The main metrics including conduction velocity (CV), spike amplitude, spike slope, inter-beat interval, and corrected field potential duration (FPD C , corrected by Fridericia’s formula) were further analysed using AxIS Navigator, Cardiac Analysis Tool and AxIS Metric Plotting tool (Axion BioSystems). Spontaneous beating frequency was defined as the reciprocal of the averaged inter-beat interval. The mainstream CV values were averaged for CV quantification. Calcium transient measurement For calcium transient measurement 57 , iPSC-CMs at day 42 from all four groups were dissociated and replated onto Geltrex-coated ø25 mm coverslips at a density of 200,000 cells per well of a 6-well plate. After a 7-day recovery period, cells were loaded with 2.5 µM Fura-2 (Thermo Fisher Scientific) in B27 medium (B27 group) and MM medium (the other three groups) at 37℃ for 30 min and washed twice with the corresponding medium. Cells were incubated for 10 min to achieve complete de-esterification of intracellular Fura-2. Intracellular calcium was recorded at 35°C using a 40x objective on an Olympus IX70 microscope equipped with the IonOptix system (IonOptix). Samples were excited at 340 and 380 nm with a switching frequency of 200 Hz and the emitted fluorescence was collected at 510 nm. Cytosolic calcium levels were defined as the ratio of fluorescence intensity at 340 and 380 nm (340/380 nm). Ca 2+ transients were recorded in Tyrode’s solution containing (in mM): NaCl 138, KCl 4, CaCl 2 1.8, MgCl 2 1, NaH 2 PO 4 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). To normalise Ca 2+ transient frequency, iPSC-CMs from all four groups were field stimulated (6 V, 10 ms) at a pacing rate of 0.5 Hz using a MyoPacer (IonOptix). To assess the calcium content, 10 mM caffeine was applied to the CMs under 0.5 Hz pacing. Monotonic transient analysis was performed using LabChart 8 software (ADInstruments) and the following parameters were determined: diastolic Ca 2+ level, systolic Ca 2+ level, Ca 2+ transient amplitude (systolic Ca 2+ level minus diastolic Ca 2+ level), and decay rate (tau) of Ca 2+ transients. Video-based contraction analysis Video-based contraction analysis was performed as previously described 5 . Briefly, videos (1024x1024 pixel, 60 FPS, length 30 s, 2 videos per culture, 3 cultures per batch) were recorded using a Hamamatsu Orca Flash 4.0 V3 camera (Hamamatsu). Videos were exported as MPEG4 files and analysed using Maia software using a block size of 10.7 µm (16 pixels), frameshift of 67 ms, and maximum distance shift of 4.69 µm (7 pixels). Video recording was performed on day 42. Contraction and relaxation peaks in the raw beating traces were assessed manually. Western blot On day 42, iPSC-CMs were scraped off, pelleted, snap-frozen in liquid nitrogen and stored at -80°C. NP coverslips with cells were transferred into new plates to selectively collect cells from NP surfaces. Cells were lysed by homogenisation in RIPA buffer (150 mM NaCl, 50 mM Tris, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, and 1 mM PMSF) supplemented with inhibitors of proteases (cOmplete mini, EDTA-free, Roche) and phosphatases (PhosSTOP, Roche) and incubated for 30 min at 4°C with gentle rotation. Lysates were centrifuged at 14,000 rpm for 20 min at 4°C and protein concentration was determined by BCA assay following the manufacturer’s instruction. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in TBS-T overnight at 4°C. Afterwards, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies for 1 h at room temperature (antibodies are listed in Suppl. Table 5). Proteins were visualised by chemiluminescence using the Super Signal West Dura Chemiluminescent Substrate kit in combination with the Fusion FX Spectra Imaging System (Peqlab). Flow cytometry Cells were detached and singularised using collagenase and trypsin, fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature and stored in PBS containing 1% BSA at 4°C. For staining, iPSC-CMs were permeabilised in PBS containing 1% BSA and 0.1% Triton-X for 10 min at room temperature. Staining was performed with specific antibodies as described in Suppl. Table 5. cTNT was detected using either the primary antibody mouse anti-cTNT (Thermo Fisher Scientific, MS-295-P1) or directly coupled cTNT-APC (Miltenyi Biotec, 130-120-543). For cTnT and Tom20 staining, cTNT-APC and anti-Tom20 (Santa Cruz, sc-17764) antibodies were used. Negative controls were performed using either the respective secondary antibodies (for samples detected with the non-coupled primary antibodies) or isotype controls (for samples detected with the directly coupled primary antibodies). After incubation with primary antibodies, cells were washed with PBS containing 1% BSA, followed by incubation with secondary antibodies, and Hoechst 33342 (5 µg/mL). To assess EdU-incorporation, PFA-fixed iPSC-CMs were incubated with mouse anti-cTNT antibody in Click-iT™ permeabilisation and wash reagent (Thermo Fischer Scientific, C10645) overnight, EdU click reaction was performed according to manufacturer’s instructions, and DNA was stained with Draq5 (abcam, ab108410, 10 µM). To determine the activity of Ki67, iPSC-CMs were stained with fluorophore-conjugated antibodies cTNT-APC (Miltenyi Biotec, 130-120-543) and Ki67-FITC (Miltenyi Biotec, 130-117-691) for 1 hour at 4°C. DNA was stained with Hoechst 33342. Afterwards, cells were resuspended in PBS containing 1% BSA and analysed on an LSRII or FACS Canto II flow cytometer. At least 10,000 events were recorded for each sample. Immunofluorescence staining For staining of RYR2, α-actinin and Hoechst 33342, cells were fixed with 4% PFA for 20 min and permeabilised with PBS containing 1% BSA and 0.1% triton-X 100 for 10 min at room temperature. For detection of Cx43 and Hoechst 33342, iPSC-CMs were fixed in methanol-acetone (7:3, v/v , 10 min at -20°C). After fixation, cells were washed with PBS and incubated in PBS containing 1% BSA at 4°C for at least 2 h. Incubation with primary antibodies was performed overnight at 4°C in PBS containing 1% BSA. After washing the coverslips in PBS, samples were incubated with secondary antibodies and Hoechst 33342 in PBS containing 1% BSA for 1 h at room temperature. Coverslips were washed with PBS, deionised water and mounted onto glass slides using Fluoromount-G mounting medium (Thermo Fisher Scientific). Imaging was performed using Keyence BZ-X700E fluorescence microscope (Keyence) or LSM880 confocal microscope (Carl Zeiss). Antibodies were used as indicated in Suppl. Table 5. To analyse the colocalisation of RYR2 and α-actinin, samples were imaged using an LSM880 confocal microscope (at 60x magnification) with fixed settings (gain, laser intensity, offsets) for all conditions within an experiment. Colocalisation was quantified using Cell Profiler software. For this, composite images were loaded, single channels were separated using the ColorToGray module, colocalisation of the red (RYR2) and green (α-actinin) channels was measured for the entire image covering 2–4 cells using the MeasureColocalization module, and data were exported using the ExportToSpreadsheet function. Colocalisation was measured for 3 independent experiments with 6 images per experiment and conditions. Seahorse measurements The Seahorse system was used to determine the metabolic activity of iPSC-CMs. Cells were dissociated and singularised using collagenase B and trypsin on day 42 and replated in 96-well Seahorse assay plates (Agilent, 103792-100) at a density of 15,000 cells per well in cardio-digestion medium. The next day, the medium was changed to the appropriate culture medium (B27 medium for the B27 group, MM for three other groups) and cells were recovered for 7 days with medium changes every other day. Seahorse recordings were performed using Seahorse Agilent XF Analyzer (Agilent) according to the manufacturer’s protocols. The Seahorse XF Cell Mito Stress Test Kit (Agilent, 103010-100) with sequential addition of oligomycin, FCCP and rotenone/antimycin A was used to study mitochondrial respiration. All data were normalised to the total amount of protein per well after lysis of cells in RIPA buffer. Real-time PCR On day 42, the cells were washed with ice-cold PBS, scraped from the culture plate or NP coverslips and centrifuged at 2,000 g at 4°C. Cell pellets were lysed in TRIzol™ (Thermo Fisher Scientific), chloroform was added, samples were mixed thoroughly and centrifuged for 15 min at 4°C and 15,000 g to separate phases. RNA was extracted, diluted with 95% EtOH and purified using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA was eluted in RNase/DNase-free water. Concentration was determined using a Nanodrop™ spectrophotometer (Thermo Fisher Scientific). Synthesis of cDNA was performed using the iScript cDNA Synthesis Kit (Bio-Rad) with 200 ng total RNA input per reaction (20 µL) according to the manufacturer’s protocol. For the detection of gene expression, samples and specific primers (Suppl. Table 6) were prepared using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and real-time PCR was performed in Hard-Shell Optical 96-well plates using the CFX96 Real-time PCR system (Bio-Rad). Initial denaturation was done at 95°C for 30 s, followed by 45 amplification cycles (15 s at 95°C, 1 min elongation at 60°C). A melting curve was obtained over the temperature range 65–95°C. HPRT was used as a reference gene for calculation of normalised relative expression (2 ΔΔCt ). RNA sequencing and DEG analysis mRNA was isolated from an average of 600 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer’s instructions. Samples were then directly subjected to the strand-specific RNA-seq library preparation workflow (Ultra II Directional RNA Library Prep, NEB). Ligation was performed using the NEB Next Adapter from the NEB Next Multiplex Oligos for Illumina Kit. After ligation, the adapters were depleted by XP bead purification (Beckman Coulter), where the bead solution was added to the samples in a ratio of 0.9:1. Unique dual indexing was done during the subsequent PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 index (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, Primer 2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two further XP bead purifications (0.9:1), the libraries were quantified using the Fragment Analyzer (Agilent). Libraries were sequenced on an Illumina NovaSeq 6000 in 100 bp paired-end mode with an average of 50 million fragments per library. FastQC ( http://www.bioinformatics.babraham.ac.uk/ ) was used to perform a basic quality control of the resulting sequencing data. Fragments were aligned to the human reference genome hg38 with the support of the Ensembl 104 splice sites using the aligner STAR (2.7.10b). Counts per gene and sample were obtained based on the overlap of the uniquely mapped fragments with the same Ensembl annotation using featureCounts (v2.0.1). Normalisation of raw fragments based on library size and testing for differential expression between the different conditions was performed using the DESeq R package (v1.38.3). Sample to sample Euclidean distance, Pearson' and Spearman correlation coefficient (r) and PCA based on the top 500 genes with the highest variance were computed to explore the correlation between biological replicates and different libraries. To identify differentially expressed genes (DEGs), counts were fitted to the negative binomial distribution and genes were tested between conditions using the Wald test of DESeq2. Resulting p-values were corrected for multiple testing using the Independent Hypothesis Weighting package (IHW 1.26.0). Genes with a maximum false discovery rate (FDR) of 10% were considered to be significantly differentially expressed. TPM values were generated using Kallisto (v0.46.1). Gene set enrichment analysis was performed based on normalised count data using GSEA software v.4.3.2 58 and canonical pathway collection (c2.cp.v2023.2.Hs) or transcription factor target collection (c3.tft.v2023.2.Hs) with following parameters: weighted scoring, meandiv normalisation, max_probe mode, maximum gene set size 500 genes, minimum set size 15 genes, and 1000 permutations. GSEA results were applied for the creation of enrichment maps using Cytoscape software according to workflow of Reimand et al. 59 . Briefly, nodes were included based on FDR q-value < 0.2 and NES ≥ 1.5 for upregulated and ≤ -1.9 for downregulated genes. Edges cut-off was set to 0.375 and pathway cluster names were determined after manual examination of all individual nodes in clusters. Sequencing data have been uploaded (EGAD00001011289) and are available on request from the European Genome-Phenome Archive (EGA, https://ega-archive.org/ ). Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9, with different comparisons indicated in figure legends. Results were considered statistically significant when the p-value was < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Declarations Competing interests The authors declare no conflict of interest. Author Contributions W.L., M.S, and K.G. conceived the study, managed the project progress and coordinated the experiments and analysis; W.L., X.L., A.S., S.A., O.G., M.S.P., M.H., R-P.S., K.F., J.P., Y.U., G.T., P.M., M.S. performed experiments; W.L., X.L., A.S., G.T., M.H., M.L., A.D., M.S. and K.G. contributed to data analysis and visualisation was performed by W.L. and M.S.. P.M., A.E-A. and K.G. provided resources. The paper was prepared by W.L., M.S. and K.G. with all authors providing feedback. All authors have read and agreed to the current version of the manuscript. Acknowledgments We thank Susann Hoefner from the Flow Cytometry Core Facility and the Core Facility Cellular Imaging (CFCI) of the TU Dresden for excellent technical support. This work was funded by the Free State of Saxony and the European Union (SAB EFRE projects “PhenoCor” with project number 100387678 to A.E.-A. and K. Guan and “CardioEpiX” with project number 100685417 to K. 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J Bone Min Res 36:1621–1635 Li W et al (2019) Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs. Stem Cell Res 41:101662 Li W, Luo X, Ulbricht Y, Guan K (2021) Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study. Stem Cell Res 56:102565 Luo X et al (2020) IP3R-mediated compensatory mechanism for calcium handling in human induced pluripotent stem cell-derived cardiomyocytes with cardiac ryanodine receptor deficiency. Front Cell Dev Biol 8:772 Subramanian A et al (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 Reimand J et al (2019) Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc 14:482–517 Additional Declarations There is NO Competing Interest. Supplementary Files SupplInfoNatCommun.docx SupplTable2GSEAcoreenrichmentgenesofdownregulatedclustersFigure6ePDFExport.pdf SupplTable3GSEAcoreenrichmentgenesofupregulatedclustersFigure8aPDFExport.pdf SupplVideo1.mp4 Suppl Video 1 Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3973784","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289261299,"identity":"1591b654-dd68-4bb9-9d24-13ca15b789d3","order_by":0,"name":"Kaomei 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Trimaglio","email":"","orcid":"","institution":"Institute of Clinical Chemistry and Laboratory Medicine, Department of Clinical Pathobiochemistry, University Hospital Dresden","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Trimaglio","suffix":""},{"id":289261313,"identity":"04972a47-63ee-4bbe-ba45-e879848e289a","order_by":14,"name":"Ali El-Armouche","email":"","orcid":"https://orcid.org/0000-0003-2514-9429","institution":"Institute of Pharmacology and Toxicology, Technische Universität Dresden","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"El-Armouche","suffix":""},{"id":289261314,"identity":"28583119-a5fd-4262-b25b-fd6a733f3c04","order_by":15,"name":"Andreas Dahl","email":"","orcid":"https://orcid.org/0000-0002-2668-8371","institution":"DRESDEN-concept Genome Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Dahl","suffix":""},{"id":289261315,"identity":"a883397c-2059-4a16-877e-7a7bdc2f312a","order_by":16,"name":"Peter Mirtschink","email":"","orcid":"","institution":"Institute of Clinical Chemistry and Laboratory Medicine, Department of Clinical Pathobiochemistry, University Hospital Dresden","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Mirtschink","suffix":""},{"id":289261316,"identity":"da2c0df8-12ed-4541-98d3-4a1d2745f2cb","order_by":17,"name":"Mario Schubert","email":"","orcid":"https://orcid.org/0000-0002-8375-8233","institution":"Technische Universität Dresden","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Schubert","suffix":""}],"badges":[],"createdAt":"2024-02-20 20:21:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3973784/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3973784/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58044-6","type":"published","date":"2025-03-21T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54446755,"identity":"8f0aa559-db6a-43d0-b52e-f0cdc96f224b","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":549790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy design and structural characterisation of iPSC-CMs\u003c/strong\u003e.\u003cstrong\u003e a\u003c/strong\u003e, Schematic overview of the study design.\u003cstrong\u003e \u003c/strong\u003eDifferentiated iPSC-CMs were digested on day 15-16 (d15-16) and randomly divided into 4 experimental groups designed to investigate the effects of MM, NP and ES. Extensive characterisation of the cells was performed on day 42 (d42). For some experiments, including immunostaining, calcium imaging, seahorse assays and MEA measurements, iPSC-CMs were replated into the corresponding assay plates on d42 and allowed to recover for another 7 days. \u003cstrong\u003eb\u003c/strong\u003e, Representative morphology of iPSC-CMs under different conditions at d42. Scale bar, 200 µm for all four groups. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Quantification of cell volume (FSC-A; \u003cstrong\u003ec\u003c/strong\u003e) and granularity (SSC-A; \u003cstrong\u003ed\u003c/strong\u003e) in the cTNT-positive CM population using flow cytometry analysis. \u003cstrong\u003ee\u003c/strong\u003e, Proportion of cTNT-positive cells. \u003cstrong\u003ef\u003c/strong\u003e, Quantification of mean cTNT fluorescence intensity. The gating strategy used to analyse the flow cytometry data is shown in Extended Data Fig. 1. \u003cstrong\u003eg\u003c/strong\u003e, Representative immunostaining for α-actinin and RYR2. Cells were replated at d42 and allowed to recover for 7 days in their respective media.\u003cstrong\u003e h\u003c/strong\u003e, Colocalisation of RYR2 with α-actinin quantified from microscopy images (as in \u003cstrong\u003eg\u003c/strong\u003e; n = 18 for each group, from 3 independent batches, 6 images/batch). \u003cstrong\u003ei\u003c/strong\u003e, Representative images of iPSC-CMs stained for Cx43 (red), lower panels represent the region of interest.\u003cstrong\u003e \u003c/strong\u003eSquares, circles and triangles represent different iPSC lines. Statistical analysis was performed by one-way ANOVA with Tukey’s post-test: * p \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001; **** p \u0026lt; 0.0001. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/378edb810ef938580b4aa01b.jpeg"},{"id":54446757,"identity":"19564a76-187f-4f6c-b225-3773fc2a24ea","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1228629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of action potential and field potential parameters in iPSC-CMs\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSpontaneous AP traces from the four groups. A notch event (red arrow) is only present in the MM+NP+ES group. \u003cstrong\u003eb\u003c/strong\u003e, Quantification of spontaneous AP metrics: RMP, Vmax and APA (n = 21, 25, 21 and 17 cells from 4 independent differentiations for the four groups, respectively). \u003cstrong\u003ec\u003c/strong\u003e, APD\u003csub\u003e90\u003c/sub\u003e quantification in APs paced at 0.5 Hz (n = 13, 24, 22 and 20 cells from 4 independent differentiations for the four groups, respectively). \u003cstrong\u003ed\u003c/strong\u003e, Representative \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e traces recorded in iPSC-CMs from the four groups. \u003cstrong\u003ee\u003c/strong\u003e, Statistical analysis of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e from three independent differentiations of two iPSC lines. The stimulation protocol is shown as an inset. \u003cstrong\u003ef\u003c/strong\u003e, Representative heatmaps of FP propagation. \u003cstrong\u003eg\u003c/strong\u003e, Quantification of FP parameters: conduction velocity, spike amplitude and spike slope (n = 24 cultures per group from 4 independent differentiations). A schematic diagram of FP traces is included in Fig. 5a, showing how the data for spike amplitude and slope were analysed. Squares and circles represent different iPSC lines. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) and two-way ANOVA with Sidak’s multiple comparison test (\u003cstrong\u003ee\u003c/strong\u003e): * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/83e5cbf8867240aa3c0d7513.jpeg"},{"id":54446756,"identity":"23adf5c5-629a-4693-b189-88a9e11b3351","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1548639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eK1\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eKr\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e recordings in iPSC-CMs\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Representative \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e traces recorded under 25 mM extracellular Na\u003csup\u003e+\u003c/sup\u003e concentration. \u003cstrong\u003eb\u003c/strong\u003e, Statistical analysis of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e for the four groups B27, MM, MM+NP, and MM+NP+ES (n = 19, 27, 26, and 24 cells from three independent differentiations of two iPSC lines, respectively). The stimulation protocol is shown as an inset. \u003cstrong\u003ec\u003c/strong\u003e, Representative traces of 0.5 mM BaCl\u003csub\u003e2\u003c/sub\u003e-sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e for different groups. \u003cstrong\u003ed\u003c/strong\u003e, Statistical analysis of BaCl\u003csub\u003e2\u003c/sub\u003e-sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e for the four groups (n = 19, 17, 18, and 15 cells from 3 independent differentiations of two iPSC lines, respectively). The stimulation protocol is shown as an inset. \u003cstrong\u003ee\u003c/strong\u003e, Shown are traces of 1 µM E-4031-sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e in the four groups. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e, Averaged E-4031-sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e step currents (\u003cstrong\u003ef\u003c/strong\u003e) and tail currents (\u003cstrong\u003eg\u003c/strong\u003e) in the four groups (n = 14, 16, 13, and 15 cells from 3 independent differentiation experiments, respectively). The \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e pulse stimulation is shown as an inset. Two-way ANOVA with Sidak’s multiple comparison test was used for statistical analysis: * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/23fc308bb466453ebe17c888.jpg"},{"id":54446750,"identity":"dbdf3466-0169-4104-9449-193b673d1232","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1435171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of calcium handling and beating properties of iPSC-CMs\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Representative \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa-L\u003c/em\u003e\u003c/sub\u003e recordings using automated patch-clamp technique. \u003cstrong\u003eb\u003c/strong\u003e, Statistical analysis of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa-L\u003c/em\u003e\u003c/sub\u003e for the four groups (n = 18, 15, 15, and 9 cells from 3 independent differentiations of two iPSC lines, respectively). \u003cstrong\u003ec\u003c/strong\u003e, Representative Ca\u003csup\u003e2+\u003c/sup\u003e transient traces recorded at 0.5 Hz followed by application of 10 mM caffeine to induce the release of total sarcoplasmic reticulum calcium. \u003cstrong\u003ed\u003c/strong\u003e, Statistical analysis of calcium transient parameters: diastolic Ca\u003csup\u003e2+\u003c/sup\u003e level, systolic Ca\u003csup\u003e2+\u003c/sup\u003e level, transient amplitude and decay constant tau measured in iPSC-CMs paced at 0.5 Hz. \u003cstrong\u003ee\u003c/strong\u003e, Sarcoplasmic reticulum calcium release induced by 10 mM caffeine. \u003cstrong\u003ef\u003c/strong\u003e, Analysis of beating rate. CMs of the MM+NP+ES group contracted with a beating rate of 120 BPM (beats per minute) during the presence of ES but regained spontaneous beating rate after ES was discontinued (dashed bar). \u003cstrong\u003eg\u003c/strong\u003e, Statistical analysis of beating properties: contraction time, relaxation time and beating duration of iPSC-CMs under 0.5 Hz field stimulation. A schematic diagram of a beat trace showing how the parameters were analysed is shown in Extended Data Fig. 2a-b. \u003cstrong\u003eh\u003c/strong\u003e, Heatmap of the ability of iPSC-CMs to adapt to increasing pacing frequencies from n = 9 cultures of 3 independent batches of iPSC-CMs. Representative beating traces are shown in Extended Data Fig. 2c. \u003cstrong\u003ei\u003c/strong\u003e, Expression of denoted marker genes for structural maturation.\u003cstrong\u003e \u003c/strong\u003eStatistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison test (\u003cstrong\u003eb\u003c/strong\u003e) and one-way ANOVA with Tukey’s post-test (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/800bb90a4785b42545f21f41.jpg"},{"id":54446754,"identity":"7db6054c-2ad5-43ac-a75d-c8e22bb2ff51","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1197585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaturation status of iPSC-CMs affects their drug response. a\u003c/strong\u003e, Schematic diagram of FP traces showing how the data were analysed. \u003cstrong\u003eb\u003c/strong\u003e, Quantitative analysis of the effect of verapamil on spike amplitude. Data show box and whisker plot of n = 17-18 replicates/wells from 3 independent experiments (from 2 different iPSC lines). Experimental design is shown in Extended Data Fig. 3. \u003cstrong\u003ec\u003c/strong\u003e, Representative averaged FP traces of verapamil-treated iPSC-CMs showing the FPDc shortening. \u003cstrong\u003ed\u003c/strong\u003e, Quantitative analysis of the effect of verapamil on FPDc shortening. n = 17-18 replicates/wells from 3 independent experiments (from 2 different iPSC lines). \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eRepresentative traces illustrating the FPDc prolongation induced by increasing concentrations of E-4031. \u003cstrong\u003ef\u003c/strong\u003e, Quantitative analysis of the effect of E-4031 on FPDc. n = 10-12 wells from 2 independent experiments. \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e, Quantitative analysis of concentration-dependent effect of isoprenaline on FPDc (\u003cstrong\u003eg\u003c/strong\u003e) and beating rate (\u003cstrong\u003eh\u003c/strong\u003e). n = 18-24 replicates/wells from 3-4 independent experiments. Statistical analysis using two-way ANOVA with Dunnett’s post-test (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/74354e2f541b382f4b600a46.jpg"},{"id":54446762,"identity":"195a47a9-78fb-4bcc-a903-afa833f583cb","added_by":"auto","created_at":"2024-04-10 16:22:57","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":358650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA sequencing of iPSC-CMs cultivated under MM, MM+NP and MM+NP+ES conditions. a-d\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eVolcano plots (\u003cstrong\u003ea-c\u003c/strong\u003e) and Venn analyses (\u003cstrong\u003ed\u003c/strong\u003e) of significantly differentially expressed genes (p \u0026lt; 0.01) between iPSC-CMs from MM, MM+NP and MM+NP+ES groups. \u003cstrong\u003ee\u003c/strong\u003e, Enrichment map illustrating clustered pathways identified in gene set enrichment analysis (GSEA) based on canonical pathway database. Pathways were filtered based on max. size of 500 genes, NES ≤ -1.9, FDR q-value ≤ 0.2 and cluster size of ≥ 2 pathways. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eEnrichment plot of SRF_Q4 gene sets obtained with GSEA. \u003cstrong\u003eg\u003c/strong\u003e, Heatmaps of the expression of SRF target genes downregulated in MM+NP+ES. \u003cstrong\u003eh\u003c/strong\u003e, Western blot of total SRF.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/ee27b2875043ea141bb9f599.jpeg"},{"id":54446759,"identity":"5ca809c8-0583-46c5-bfd8-bd938f1ca60f","added_by":"auto","created_at":"2024-04-10 16:22:57","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":355119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell cycle regulation of iPSC-CMs cultivated under MM, MM+NP and MM+NP+ES conditions. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eScheme illustrating the expression of cyclin-CDK complexes and respective inhibitors during cell cycle. \u003cstrong\u003eb\u003c/strong\u003e, Heatmaps of cyclin-CDK complexes, CDK inhibitors and genes relevant in cytokinesis. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e,Flow cytometry plots showing cellular DNA content (\u003cstrong\u003ec\u003c/strong\u003e), DNA synthesis activity (\u003cstrong\u003ed\u003c/strong\u003e), and Ki67 activity (\u003cstrong\u003ee\u003c/strong\u003e). \u003cstrong\u003ef\u003c/strong\u003e,Quantification of diploid and polyploid iPSC-CMs (cTNT-positive population). Data from 15 independent experiments. \u003cstrong\u003eg\u003c/strong\u003e, Proportion of EdU-incorporated iPSC-CMs. \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e, Quantification of diploid and polyploid CMs in Ki67-positive (\u003cstrong\u003eh\u003c/strong\u003e) and negative (\u003cstrong\u003ei\u003c/strong\u003e) population. Data from 6 independent experiments. Data from 4 (\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e) and 6 (\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e) independent experiments. Statistical analysis using two-way ANOVA (\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) or one-way ANOVA (\u003cstrong\u003eg\u003c/strong\u003e) with Tukey’s post-test (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/3519774e19c096546a39d5f7.jpeg"},{"id":54446752,"identity":"043d624d-eee4-4f35-adf9-0cf3363a5af7","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":454683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulation of TFAM and HMCES target genes contributes to mitochondrial development induced by ES. a\u003c/strong\u003e, Enrichment maps illustrating clustered pathways identified in gene set enrichment analysis (GSEA) based on canonical pathway database. Pathways were filtered based on max. size of 500 genes, NES ≥ 1.5, FDR q-value ≤ 0.2 and cluster size of ≥ 2 pathways. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, Enrichment plots and most regulated gene in TFAM and HMCES clusters. \u003cstrong\u003ed\u003c/strong\u003e, Quantification of Tom20 intensity detected in cTNT-positive CM population. \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSeahorse mean traces (\u003cstrong\u003ee\u003c/strong\u003e) and determined parameters (\u003cstrong\u003ef\u003c/strong\u003e) were performed with sequential addition of oligomycin (ATP synthase inhibitor), carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP; mitochondrial uncoupler) and rotenone/antimycin A (complex 1 and 2 inhibitor) (n = 4 independent experiments for B27, MM+NP+ES; n = 5 for MM and MM+NP). \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eExpression levels of \u003cem\u003eOPA1\u003c/em\u003e, \u003cem\u003ePPARGC1α\u003c/em\u003eand \u003cem\u003ePPARα\u003c/em\u003e determined using real-time PCR. Data from 4 (\u003cem\u003ePPARα\u003c/em\u003e) or 5 (\u003cem\u003eOPA1\u003c/em\u003e, \u003cem\u003ePPARGC1α\u003c/em\u003e) independent experiments. \u003cstrong\u003eh\u003c/strong\u003e, Heatmaps of selected genes encoding ion channels. Statistical analysis was performed using one-way ANOVA and Tukey’s post-test, * p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/bfd7c8c96cf50f68a547aefb.jpeg"},{"id":79004261,"identity":"6532644b-100b-4b8e-934c-3bd419e07cf9","added_by":"auto","created_at":"2025-03-22 07:08:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8626302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/98e1db67-64e3-4bfa-b2d5-e2c5c72a73ab.pdf"},{"id":54446753,"identity":"f4476276-a948-4c0d-aeb9-db0e061d2525","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47037,"visible":true,"origin":"","legend":"","description":"","filename":"SupplInfoNatCommun.docx","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/9aa93df166f05ea069989002.docx"},{"id":54446758,"identity":"ff4c7569-9afa-4182-862c-d26f6de0c70c","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":123934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplTable2GSEAcoreenrichmentgenesofdownregulatedclustersFigure6ePDFExport.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/28bfbab24ed58c6c281a9233.pdf"},{"id":54446751,"identity":"3990fdf2-c60e-4aa3-8881-5d5931a7994e","added_by":"auto","created_at":"2024-04-10 16:22:56","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":163519,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable3GSEAcoreenrichmentgenesofupregulatedclustersFigure8aPDFExport.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/90a540fe9b104c415a3d6874.pdf"},{"id":54446761,"identity":"9b04a62d-10ff-49a5-9102-cadfe6e474c2","added_by":"auto","created_at":"2024-04-10 16:22:57","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14675521,"visible":true,"origin":"","legend":"\u003cp\u003eSuppl Video 1\u003c/p\u003e","description":"","filename":"SupplVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3973784/v1/2353aad4dd335a8f65c9373b.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe discovery of human induced pluripotent stem cells (iPSCs) represents a breakthrough for medical research and clinical application. As an unlimited source of cardiomyocytes (CMs) with a patient-specific genetic background, iPSC-derived CMs (iPSC-CMs) can be employed to explore disease mechanisms and drug effects. Additionally, they hold the potential for regenerating lost myocardium in patients with heart failure\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated the ability of iPSC-CMs to recapitulate clinical features of inherited cardiomyopathies\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, arrhythmias\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and cardiotoxic drug responses\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, even with patient-specific sensitivities\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite these achievements, their immaturity remains a significant limitation. In comparison to adult CMs, iPSC-CMs exhibit considerable differences in their morphology, gene expression patterns, metabolism and functionality\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, which may explain their low sensitivity to hypoxia(-reperfusion) injury\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, or the lack of features expected from a clinical phenotype of an inherited disease\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The use of iPSC-CMs to predict the pro-arrhythmic activity of drugs in the Comprehensive \u003cem\u003ein vitro\u003c/em\u003e Proarrhythmia Assay (CiPA) has shown good correlation with the clinical risk of torsade de pointes or QTc prolongation. However, discrepancies have been reported for some multichannel blockers. For example, verapamil has a good safety profile in the clinic, but abolishes the beating activity of iPSC-CMs at clinically relevant concentrations\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, probably due to differences in the expression of genes encoding ion channels, such as \u003cem\u003eSCN5A\u003c/em\u003e (Na\u003csub\u003eV\u003c/sub\u003e1.5), \u003cem\u003eCACNA1C\u003c/em\u003e (Ca\u003csub\u003eV\u003c/sub\u003e1.2), \u003cem\u003eKCNH2\u003c/em\u003e (hERG) and \u003cem\u003eKCNQ1\u003c/em\u003e (K\u003csub\u003eV\u003c/sub\u003e7.1) in iPSC-CMs compared to adult CMs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Whether the establishment of more adult-like current patterns in iPSC-CMs affects their drug response remains elusive\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, several approaches have been developed to improve the maturation of iPSC-CMs. The integration of fibroblasts and/or endothelial cells into cardiac tissue models derived from iPSC-CMs significantly enhanced both structural and functional development, as demonstrated in various studies\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, it is worth noting that 3D-tissue generation is challenging and the experimental throughput is lower compared to 2D-cultures\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Other approaches to enhance iPSC-CM maturation include supplementation of the culture medium with fatty acids (FA)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, hormones or small molecules\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, micro- or nanopatterning (NP) of culture surfaces\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and electrostimulation (ES)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. As these stimuli have been investigated independently, the most effective factor for enhancing iPSC-CM maturation remains unclear. It is uncertain whether combined approaches could produce synergistic effects, and the underlying mechanisms driving the advanced maturation of iPSC-CMs remain to be elucidated.\u003c/p\u003e \u003cp\u003eHere, we demonstrate that the combination of FA-enriched maturation medium (MM), NP and ES is an efficient approach to generate iPSC-CMs with an advanced maturation state and pharmacological response more closely resembling that of adult CMs. By systematically combining MM, NP and ES and using in-depth functional and molecular analyses, our data provide mechanistic insights into how these different stimuli influence the cellular structure, metabolism and electrophysiology of iPSC-CMs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe used the directed differentiation protocol to generate ventricular-like CMs from iPSCs derived from 3 healthy individuals\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. On day 15, iPSC-CMs were digested and distributed to 4 experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). B27 medium, routinely used for iPSC-CM culture, served as a control. To unravel the synergistic effects of MM, NP, and ES on the maturation of iPSC-CMs, we systematically applied NP and ES to MM in a stepwise parallel manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). MM was designed based on a published FA-supplemented medium that enhances the metabolic maturation of iPSC-CMs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, with some modifications (Suppl. Table\u0026nbsp;1). NP was used to induce cell alignment and ES was applied to induce a beating frequency of 2 Hz (Suppl. Video 1).\u003c/p\u003e\n\u003ch3\u003eCombined approach enhances structural maturation of iPSC-CMs\u003c/h3\u003e\n\u003cp\u003eWe observed that NP application induced changes in cell shape and a significant increase in the alignment of iPSC-CMs in the MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups compared to the B27 and MM groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In all three groups (MM, MM\u0026thinsp;+\u0026thinsp;NP, and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES) there was a noticeable increase in cell volume and granularity of iPSC-CMs compared to the B27 control. Compared to the MM group, NP did not induce an additional increase in cell volume and granularity, but the combination of MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES induced further significant increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d), suggesting that ES plays an important role in the hypertrophic growth of iPSC-CMs. A comparable proportion of cardiac troponin T (cTNT)-positive cells was found in all conditions, with the highest cTNT mean fluorescence intensity in iPSC-CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f). Co-immunostaining for the sarcomeric protein α-actinin and cardiac ryanodine receptor (RYR2) revealed well-organised sarcomeric structures in iPSC-CMs under all conditions, but marked differences for the RYR2 localisation and the α-actinin/RYR2 colocalisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h). In B27-cultured CMs, robust RYR2 staining was observed in the nucleus and punctate staining in the cytosol, with a low degree of α-actinin/RYR2 colocalisation. In contrast, CMs from the other three groups revealed reduced nuclear RYR2 staining and an augmented presence of striated patterns. The α-actinin/RYR2 colocalisation was enhanced in both the MM and MM\u0026thinsp;+\u0026thinsp;NP groups, and this effect was further augmented by ES. In the B27 and MM groups, the gap junction protein connexin 43 (Cx43) was partially localised to perinuclear regions rather than the plasma membrane, whereas Cx43 membrane localisation was increased in CMs of the MM\u0026thinsp;+\u0026thinsp;NP group and further significantly improved by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). These results provide evidence for the additive effects of NP and ES to MM on the structural maturation of iPSC-CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCombined approach improves electrophysiological maturation of iPSC-CMs\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of MM, NP and ES on electrophysiological properties, we performed patch-clamp and multi-electrode array (MEA) studies to investigate the action potential (AP) and field potential (FP) parameters of single and monolayer iPSC-CMs, respectively. We observed the \u0026lsquo;notch-and-dome\u0026rsquo; AP morphology only in iPSC-CMs (43%) of the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The resting membrane potential (RMP) was found to be progressively more negative in CMs from the MM (-49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 mV), MM\u0026thinsp;+\u0026thinsp;NP (-58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mV) and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES (-65.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mV) groups compared to the B27 group (-44.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mV), while the maximum AP upstroke velocity (Vmax) was gradually increased in iPSC-CMs from the MM (5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 V/s), MM\u0026thinsp;+\u0026thinsp;NP (6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 V/s) and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES (11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 V/s) groups compared to the B27 control (4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 V/s). Similarly, a gradual increase in AP amplitude (APA) was observed in the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The AP duration at 90% repolarisation (APD\u003csub\u003e90\u003c/sub\u003e) was significantly shorter in iPSC-CMs paced at 0.5 Hz in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group than in the B27 control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). As the transient outward K\u003csup\u003e+\u003c/sup\u003e current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e) underlies the prominent phase 1 repolarisation of cardiac APs and the \u0026lsquo;notch-and-dome\u0026rsquo; AP morphology, we measured \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e and found a significantly higher \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e density in iPSC-CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group, but only a slight increase in MM and MM\u0026thinsp;+\u0026thinsp;NP conditions compared to the B27 group; and NP itself has less effect on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e when comparing the MM\u0026thinsp;+\u0026thinsp;NP group with the MM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e).\u003c/p\u003e \u003cp\u003eIntercellular electrotonic coupling and conduction velocity (CV) across CMs are largely dependent on Cx43 expression at the gap junction\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Consistent with the Cx43 localisation data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), heatmaps of electrical signal propagation analysed by MEA illustrate the stepwise increase in CV in iPSC-CM monolayers from the MM (22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 cm/s), MM\u0026thinsp;+\u0026thinsp;NP (25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 cm/s) and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES (27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 cm/s) groups, compared to the B27 condition (12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 cm/s). Similar stepwise changes in spike amplitude and slope were observed in the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef,g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo analyse which changes in specific ion currents underlie the improved electrophysiological functionality, we recorded \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e using the patch-clamp technique. We found that \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e density was significantly higher in MM-cultured iPSC-CMs with a mean peak current density of -87.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 pA/pF at -20 mV compared to the B27 group (-40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 pA/pF). Notably, NP induced only a small increase in \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e density with a mean peak of -100.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6 pA/pF at -25 mV when compared to the MM group, but \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e density was further significantly induced by ES in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group with a mean peak of -136.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5 pA/pF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). These data are consistent with the AP- (Vmax, APA) and FP-metrics (CV, spike amplitude and slope) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe electrophysiological immaturity of iPSC-CMs compared to adult CMs is partly attributed to the low density of the hyperpolarising K\u003csup\u003e+\u003c/sup\u003e current \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, which is important for stabilising the RMP\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We found a very low \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e density in B27-cultured iPSC-CMs (-2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 pA/pF at -130 mV), and only a slight increase in \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e in the MM group (-6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 pA/pF). Interestingly, NP induced a significant increase in \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e in the MM\u0026thinsp;+\u0026thinsp;NP group (-13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 pA/pF), which was further induced by ES (-15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 pA/pF in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). HERG channels conducting the rapid delayed rectifier K\u003csup\u003e+\u003c/sup\u003e current \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e are involved in phase 3 repolarisation of cardiac APs. Similar to \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, both E-4031-sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e step and tail current densities were only slightly induced by MM, but significantly induced by NP and further enhanced by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). These data are consistent with the most negative RMP and the shortest APD\u003csub\u003e90\u003c/sub\u003e in iPSC-CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003eTaken together, these findings highlight the distinct effects of the three stimuli on specific ion currents. This is evidenced by the strong influence of NP on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e, with less or no effect on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, and the robust effect of MM on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, but less on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e. Importantly, the data underline that the combined approach significantly enhanced electrophysiological maturation of iPSC-CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCombined approach improves calcium handling and contractility of iPSC-CMs\u003c/h2\u003e \u003cp\u003eSince excitation-contraction coupling in CMs involves calcium cycling to convert electrical signals into mechanical output (contraction), we next examined L-type calcium channel (LTCC) current \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e and calcium transients in iPSC-CMs. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e densities exhibited similar reductions in both the MM and MM\u0026thinsp;+\u0026thinsp;NP groups when compared to the B27 group, which were further reduced by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). To quantify intracellular Ca\u003csup\u003e2+\u003c/sup\u003e dynamics, we performed Fura-2-based calcium imaging in iPSC-CMs paced at 0.5 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-e). Significantly reduced diastolic and systolic Ca\u003csup\u003e2+\u003c/sup\u003e levels were observed in the MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups compared to the B27 group, but Ca\u003csup\u003e2+\u003c/sup\u003e transient amplitudes were comparable between all groups despite the reduced \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e density in the MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups. The Ca\u003csup\u003e2+\u003c/sup\u003e transient decay time constant (tau) is also significantly shortened in the MM group compared to the control, whereas no further shortening was observed in the MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups. Application of 10 mM caffeine resulted in significantly increased Ca\u003csup\u003e2+\u003c/sup\u003e release from the sarcoplasmic reticulum (SR) in the MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups compared to the B27 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These data suggest a more efficient coupling between \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e and SR Ca\u003csup\u003e2+\u003c/sup\u003e release, enhanced Ca\u003csup\u003e2+\u003c/sup\u003e decay kinetics and a higher SR calcium content in these three groups.\u003c/p\u003e \u003cp\u003eVideo-based analysis of iPSC-CM beating properties\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e showed that the changes in calcium handling were associated with improved contractile function. Stopping ES in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group resulted in cessation of beating, followed by regaining of spontaneous beating activity within 15\u0026ndash;30 minutes at a rate comparable to the other three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Significantly shorter beating duration, contraction and relaxation times were observed in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). We found a similar trend in the MM and MM\u0026thinsp;+\u0026thinsp;NP groups compared to the B27 control, but no significant difference between the two groups. Consistent with this observation, all cultures in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group successfully captured high-frequency (2 Hz) field stimulation, whereas none of the B27 cultures demonstrated this capability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This improved contractile function was accompanied by an increased gene expression ratio of \u003cem\u003eTNNI3\u003c/em\u003e/\u003cem\u003eTNNI1\u003c/em\u003e and \u003cem\u003eMYL2/MYL7\u003c/em\u003e, whereas the expression of \u003cem\u003eMYH6\u003c/em\u003e, encoding the fast-twitch MHC isoform, was upregulated in response to sustained ES, leading to a reduced \u003cem\u003eMYH7\u003c/em\u003e/\u003cem\u003eMYH6\u003c/em\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These results demonstrate that MM, NP and ES individually and synergistically induce electrophysiological and functional maturation of iPSC-CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCombined approach improves drug response of iPSC-CMs\u003c/h2\u003e \u003cp\u003eTo investigate whether the maturation state of iPSC-CMs influences their drug response, we chose verapamil (calcium-channel blocker), E-4031 (hERG-channel blocker) and isoprenaline (β-adrenergic stimulus) as model substances to detect pro-arrhythmic activity based on changes in FP parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). We observed beating arrest in cultures from the B27 (all cultures), MM (9/17) and MM\u0026thinsp;+\u0026thinsp;NP (7/18) groups at 1 \u0026micro;M verapamil. Concentration-dependent reductions in spike amplitude were observed in the B27 group and, to a lesser extent, in the MM and MM\u0026thinsp;+\u0026thinsp;NP groups. In contrast, no effect of verapamil on beating activity and spike amplitude was observed in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Verapamil-induced shortening of FP duration (FPDc), which corresponds to QT-shortening in the clinic, was comparable in iPSC-CMs from the MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups. However, this effect was more pronounced compared to iPSC-CMs cultured in B27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d). Previous studies showed that immature iPSC-CMs failed to produce APD prolongation after E-4031 treatment, even at high concentrations\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Similarly, we found that E-4031 induced only minor changes in FPDc in B27-cultured iPSC-CMs, whereas significant concentration-dependent FPDc prolongation was detected in the MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f). Furthermore, we observed a more pronounced positive-chronotropic response of iPSC-CMs to isoprenaline in these three groups than in the B27 group, which correlates with FPDc-shortening (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg,h). The EC\u003csub\u003e50\u003c/sub\u003e of isoprenaline for the chronotropic effect was also much lower in these three groups than in the B27 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). These experiments demonstrate the substantial impact of the maturation state of iPSC-CMs on their response to various cardioactive drugs. They emphasise the importance of utilising iPSC-CMs with more adult-like electrophysiological properties for accurate drug risk assessment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCombined approach downregulates MAPK/PI3K-AKT pathways\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that FA-enriched media induce iPSC-CM maturation by regulating key genes involved in FA metabolism, mitochondrial function, calcium cycling, ion channels and sarcomere\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To gain insight into the molecular mechanisms driving iPSC-CM maturation by NP and ES, we performed RNA sequencing (RNA-seq) analysis. Surprisingly, NP had little synergistic effect when combined with MM, whereas the addition of ES strongly influenced gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-d; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Comparing the MM and MM\u0026thinsp;+\u0026thinsp;NP groups, only 163 differentially expressed genes were identified, of which 56 were upregulated and 107 downregulated in the MM\u0026thinsp;+\u0026thinsp;NP group. In contrast, 1,370 significantly upregulated and 1,657 downregulated genes were identified in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the MM group, of which 747 significantly upregulated and 990 downregulated genes were also identified when compared to the MM\u0026thinsp;+\u0026thinsp;NP group, indicating the synergistic effects of NP and ES.\u003c/p\u003e \u003cp\u003ePathway enrichment analysis of the downregulated genes in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group mainly mapped to MAPK/PI3K-AKT, TNFR2-NFκB, G-protein-coupled receptor (GPCR), and cytokine/chemokine signalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed; Suppl. Table\u0026nbsp;2). Using the transcription factor target database, we identified the SRF cluster, which includes many genes involved in cell cycle regulation and cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef,g; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). We also found a decrease in SRF protein levels in CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). These findings encouraged us to evaluate the expression of genes that regulate cell cycle progression. Notably, activators of G2/M checkpoints including cyclins (\u003cem\u003eCCNB1-3\u003c/em\u003e), cyclin-dependent kinase 1 (\u003cem\u003eCDK1\u003c/em\u003e) were downregulated, whereas CDK inhibitors (\u003cem\u003eCDKN1A\u003c/em\u003e, \u003cem\u003eCDC20\u003c/em\u003e) were upregulated in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). Interestingly, genes (\u003cem\u003eANLN\u003c/em\u003e, \u003cem\u003eSEPTIN7/2\u003c/em\u003e) encoding activators of cytokinesis were also downregulated. We did not observe any significant changes in the gene sets (\u003cem\u003eCCND1-3\u003c/em\u003e, \u003cem\u003eCCNE1/2\u003c/em\u003e, \u003cem\u003eCCNA1/2\u003c/em\u003e, \u003cem\u003eCDK2/4/6\u003c/em\u003e, \u003cem\u003eCDKN2A-D\u003c/em\u003e, \u003cem\u003eCDKN1B/C\u003c/em\u003e, \u003cem\u003eCDH1\u003c/em\u003e) controlling the G1 and S progression and the G1/S checkpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). These data suggest a cell cycle arrest after S phase and before exit from M phase in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group, which may lead to bi-nucleation or nuclear polyploidy. To confirm this, we examined DNA content and found that the number of diploid iPSC-CMs was significantly reduced and polyploid cells significantly increased in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the other three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec,f). Interestingly, we observed no difference in the proportion of 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU)-incorporated iPSC-CMs between all four groups, which can detect DNA synthesis during S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed,g). However, the proportion of Ki67\u003csup\u003e+\u003c/sup\u003e iPSC-CMs and Ki67\u003csup\u003e\u0026minus;\u003c/sup\u003e polyploid iPSC-CMs was higher in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group than in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee,h,i). Ki67 is widely expressed throughout the entire cell cycle, except in G0, and reaches a maximum in S/G2\u003csup\u003e31\u003c/sup\u003e. These results indicate that the downregulation of MAPK/PI3K-AKT and SRF-related genes is involved in G2/M arrest and polyploidy development of iPSC-CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChanges in gene expression profile associated with metabolism and electrophysiology\u003c/h2\u003e \u003cp\u003eThe pathway enrichment analysis of genes upregulated in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group revealed that the combined approach induced the upregulation of genes involved in electron transport chain (ETC), TCA cycle, mitochondrial biogenesis, NRF2 signalling, glucose metabolism, N-glycan biosynthesis, FA oxidation, tRNA aminoacylation, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee; Suppl. Table\u0026nbsp;3). These gene clusters were only slightly upregulated in the MM\u0026thinsp;+\u0026thinsp;NP group compared to the MM group (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eUsing the transcription factor target database we identified two clusters, TFAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb) and HMCES (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec), which were enriched in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In these two clusters, mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunits (\u003cem\u003eMTND2-6\u003c/em\u003e) and pseudogenes (\u003cem\u003eMTND4P12\u003c/em\u003e, \u003cem\u003eMTND5P11\u003c/em\u003e, \u003cem\u003eMTND6P4\u003c/em\u003e), cytochrome c oxidase subunits (\u003cem\u003eMT-CO1/3\u003c/em\u003e) and pseudogenes (\u003cem\u003eMT-CO1P2\u003c/em\u003e, \u003cem\u003eMT-CO1P12\u003c/em\u003e, \u003cem\u003eMT-CO3P12\u003c/em\u003e), cytochrome b (\u003cem\u003eMTCYB\u003c/em\u003e), 12S rRNA (\u003cem\u003eMT-RNR1\u003c/em\u003e) and tRNAs (\u003cem\u003eMT-TP/-TM/-TE/-TI/-TW/-TC/-TY/-TR/-TN/-TQ\u003c/em\u003e) were upregulated in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the MM group.\u003c/p\u003e \u003cp\u003eSubsequently, we found increased mitochondrial mass in CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Furthermore, CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group showed a higher expression of \u003cem\u003eOPA1\u003c/em\u003e, \u003cem\u003ePPARGC1α\u003c/em\u003e and \u003cem\u003ePPARα\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg), confirming an enhanced mitochondrial development in response to ES. Measurements of oxygen consumption rate as a surrogate for mitochondrial function showed an increased basal and maximal respiration, ATP production and spare capacity of CMs from the MM group compared to the B27 control. There is only a marginal additional increase in these parameters in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES and MM\u0026thinsp;+\u0026thinsp;NP groups compared to the MM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee,f). These findings indicate that the combination of MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES leads to upregulation of oxidative phosphorylation, activation of mtDNA-encoded components and an overall enhancement of mitochondrial development and function.\u003c/p\u003e \u003cp\u003eFurther examination of individual changes in key ion channel components revealed a clear correlation between gene expression and channel function (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh). We found upregulation of genes contributing to \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e (\u003cem\u003eKCNA7\u003c/em\u003e, \u003cem\u003eKCNC3\u003c/em\u003e, \u003cem\u003eKCNC4\u003c/em\u003e, \u003cem\u003eKCND2\u003c/em\u003e, \u003cem\u003eKCND3\u003c/em\u003e), \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e (\u003cem\u003eKCNJ2\u003c/em\u003e, \u003cem\u003eKCNJ4\u003c/em\u003e, \u003cem\u003eKCNJ6\u003c/em\u003e, \u003cem\u003eKCNJ11\u003c/em\u003e, \u003cem\u003eKCNJ12\u003c/em\u003e), \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e (\u003cem\u003eKCNH2\u003c/em\u003e, \u003cem\u003eKCNE2\u003c/em\u003e) and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKs\u003c/em\u003e\u003c/sub\u003e (\u003cem\u003eKCNQ1\u003c/em\u003e) currents, as well as genes encoding calcium-activated (\u003cem\u003eKCNN1\u003c/em\u003e) and voltage-gated (\u003cem\u003eKCNS3\u003c/em\u003e, \u003cem\u003eKCNAB2\u003c/em\u003e, \u003cem\u003eKCNIP3\u003c/em\u003e) potassium channels in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group compared to the MM group. In contrast, genes encoding the LTCCs (\u003cem\u003eCACNA1C\u003c/em\u003e, \u003cem\u003eCACNA1D\u003c/em\u003e, \u003cem\u003eCACNA1A\u003c/em\u003e, \u003cem\u003eCACNA2D3\u003c/em\u003e, \u003cem\u003eCACNA2D4\u003c/em\u003e) and regulating the LTCC activity (\u003cem\u003eCACNG7\u003c/em\u003e) were downregulated, whereas the genes encoding the T-type calcium channels (\u003cem\u003eCACNA1H\u003c/em\u003e, \u003cem\u003eCACNA1I\u003c/em\u003e) were upregulated. No significant differences in \u003cem\u003eSCN5A\u003c/em\u003e expression were found between the three groups, but \u003cem\u003eSCN1B\u003c/em\u003e was upregulated and \u003cem\u003eSCN3B\u003c/em\u003e was downregulated in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group. In addition, the expression of the genes encoding calcium handling proteins were not significantly altered. Collectively, these results are consistent with the significantly increased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e but decreased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e currents in iPSC-CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we systematically investigated the collective impact of MM, NP and ES on the maturation of iPSC-CMs. Our findings demonstrate that the concurrent application of MM, NP and ES serves as an efficient strategy to enhance the structural, electrophysiological, metabolic and functional maturation of iPSC-CMs. This maturation process involves the modulation of MAPK/PI3K-AKT signalling as well as the regulation of TFAM/HMCES and SRF target genes, which is of significant relevance for the utilisation of iPSC-CMs in disease modelling and drug testing.\u003c/p\u003e \u003cp\u003eThe core of the maturation strategy is the application of FA-supplemented MM in iPSC-CMs, which induces the metabolic transition from glucose-based energy production to FA β-oxidation. This process is essential for enhanced maturation, reflected by an increase in cell size and mitochondrial network as well as enhanced respiratory, electrophysiological, and contractile activity. These findings align with previously reported data on the positive effects of FA supplementation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. By stepwise addition of NP and ES to MM, we found that the combination of NP with MM had only a limited impact on the metabolic maturation of iPSC-CMs, as demonstrated by subtle changes in the expression profile of gene clusters related to energy metabolism as well as mitochondrial development and function, such as ETC (also known as oxidative phosphorylation), TCA cycle, FA oxidation, and glucose metabolism (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Strikingly, the addition of ES to MM\u0026thinsp;+\u0026thinsp;NP resulted in significant upregulation of these gene clusters, likely due to the increased FA β-oxidation in iPSC-CMs to meet the energy demand for the persistent beating activity at 2 Hz.\u003c/p\u003e \u003cp\u003eAn important finding of our study is that GSEA analysis of the upregulated genes in MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES using the transcription factor target database maps to the enrichment of HMCES- and TFAM-related target gene sets. HMCES (5-hydroxymethylcytosine binding, embryonic stem cell-specific) may safeguard the genomic and mtDNA integrity of iPSC-CMs during oxidative stress responses triggered by elevated levels of reactive oxygen species due to the use of FA. This protective mechanism involves the formation of stable DNA-protein crosslinks with abasic DNA damage to prevent error-prone repair pathways\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. TFAM (mitochondrial transcription factor A) is essential for the transcription, replication and packaging of mtDNA into nucleoids. It is indispensable for the meticulous regulation of mitochondrial biogenesis, ensuring the seamless adaptation of the mitochondrial population to precisely match the energy demand of the cell\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This is supported by our observation that most mtDNA-encoded essential components of the ETC as well as rRNAs and tRNAs required for the translation of mtDNA-encoded proteins were significantly upregulated by the ES stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePGC-1α (\u003cem\u003ePPARGC1α\u003c/em\u003e) is the master regulator of mitochondrial energy metabolism, respiration and biogenesis through interaction with its various coactivators ERR, PPAR and NRF1/2\u003csup\u003e36\u003c/sup\u003e. Together with ERR, it controls mitochondrial dynamics via activation of genes involved in mitochondrial fission and fusion including \u003cem\u003eOPA1\u003c/em\u003e and \u003cem\u003eMFN2\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e, which were found to be upregulated in the MM\u0026thinsp;+\u0026thinsp;NP-ES group. Together with PPARα, which is also upregulated in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES, PGC-1α regulates genes involved in mitochondrial FA oxidation and many other cellular lipid metabolic pathways\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, PGC-1α together with NRF1/2 promotes mitochondrial biogenesis by activating TFAM\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother interesting discovery of our study revealed a significant downregulation of genes involved in MAPK/PI3K signalling pathways in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group. This aligns with the substantial downregulation of the MAPK and PI3K-AKT pathways in the postnatal heart when compared to the neonatal heart\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Thus, MAPK/PI3K-AKT inhibition promotes iPSC-CM maturation, partially mediated by the upregulation of PGC-1α\u003csup\u003e38\u003c/sup\u003e. Further research is needed to determine whether the downregulation of MAPK/PI3K signalling pathways in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group is involved in PGC-1α and TFAM activation through the control of AMPK\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, AKT\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and/or mTORC1\u003csup\u003e41\u003c/sup\u003e pathways, and is therefore essential for the metabolic maturation of iPSC-CMs.\u003c/p\u003e \u003cp\u003eMost strikingly, our RNA-seq and cell cycle analysis data show that downregulated MAPK/PI3K-AKT signalling is involved in the downregulation of genes important for the G2/M transition and cytokinesis, leading to polyploidy (nuclear polyploidy and/or multinucleation) of iPSC-CMs. Human CMs are diploid during the first years of life and gradually become polyploid over time. By the second decade of life, approximately 25% of CM are multinucleated and 57% polyploid\u003csup\u003e42\u003c/sup\u003e. In CMs, the major cyclin-CDK complexes controlling cell cycle progression are CCND-CDK4/6 (G1 phase), CCNE-CDK2 (G1/S transition), CCNA-CDK2/1 (S and G2 phase and S/G2 transition) and CCNB-CDK1 (G2/M transition and M phase), the activity of which is inhibited by CDK inhibitors\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The CCNB-CDK1 complex is not expressed in cell cycle-arrested adult CMs, and it is also not required for CM hypertrophy\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In our study, we found a decrease in the expression of CCNB-CDK1 in iPSC-CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group. Despite this decrease, these cells exhibited ongoing DNA synthesis, an increased proportion of polyploid CMs and higher cell volume and granularity. The downregulated CCNB-CDK gene expression is associated with the upregulation of CDK inhibitor p21 and MEIS1 that are negatively regulated by TBX20\u003csup\u003e45\u003c/sup\u003e. Better understanding of how MAPK/PI3K-AKT signalling controls cell cycle progression, including the expression of TBX20, MEIS1 and p21, may provide valuable mechanistic insights to promote iPSC-CM maturation or to stimulate adult CM regeneration.\u003c/p\u003e \u003cp\u003eAnother important finding of our study is that NP and ES synergistically induce the maturation of different ion channels. We found that NP combined with MM strongly increased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, but had little effect on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e. However, the addition of ES to MM\u0026thinsp;+\u0026thinsp;NP led to significant changes in all currents (larger \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e, but smaller \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e) and maturation of electrophysiology (more negative RMP, shorter APD, higher Vmax, APA, CV and spike amplitude, and the \u0026lsquo;notch-and-dome\u0026rsquo; AP morphology) in iPSC-CMs, similar to adult CMs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The enhanced electrophysiological functionality of iPSC-CMs contributes to their improved predictive value for risk assessment of cardioactive drugs, especially in the case of multi-channel blockers such as verapamil, ranolazine or alfuzosin\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Several studies reported that verapamil inhibits the beating activity of iPSC-CMs\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, probably because depolarisation in immature iPSC-CMs does not rely exclusively on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, as in adult CMs, but also on \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e15\u003c/sup\u003e. The RNA-seq data suggest that the increased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e and the decreased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e in the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group may be due to the upregulation of genes encoding different potassium channels and the downregulation of genes encoding LTCCs. In addition, the downregulation of PI3K-AKT signalling may contribute to the reduced \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e densities\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Furthermore, the similar Ca\u003csup\u003e2+\u003c/sup\u003e transient amplitudes of all groups together with the decreased \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e in iPSC-CMs from MM, MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups in comparison to the B27 condition suggest an enhanced excitation-contraction coupling gain, an important indicator of improved calcium handling\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The colocalisation of RYR2 with α-actinin and the ability to adapt to high-frequency stimulation support an improved calcium handling, especially in CMs from the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group.\u003c/p\u003e \u003cp\u003eInterestingly, we observed no changes in the expression of \u003cem\u003eSCN5A\u003c/em\u003e, coding for the pore-forming α-subunit of the sodium channel (Na\u003csub\u003eV\u003c/sub\u003e1.5), but an upregulation of \u003cem\u003eSCN1B\u003c/em\u003e and a downregulation of \u003cem\u003eSCN3B\u003c/em\u003e, which encode the β-subunits (Na\u003csub\u003eV\u003c/sub\u003e-β1/3) of the sodium channel that interact with the α-subunits\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eSCN1B\u003c/em\u003e is highly expressed in adult CMs, \u003cem\u003eSCN3B\u003c/em\u003e is highly expressed in the embryonic heart\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that co-expression of \u003cem\u003eSCN1B\u003c/em\u003e with \u003cem\u003eSCN5A\u003c/em\u003e increases the density of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and the β1-subunit modulates the cell surface localisation, gating, and kinetics of α-subunits\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In addition, Na\u003csub\u003eV\u003c/sub\u003e-β1 also regulates voltage-gated potassium channels, including K\u003csub\u003eV\u003c/sub\u003e4.3 and associates with the cardiac intercalated disc proteins N-cadherin and Cx43\u003csup\u003e51\u003c/sup\u003e, contributing to the enhanced electrical signal conduction. Future studies should focus on whether/how the downregulation of MAPK/PI3K-AKT signalling in iPSC-CMs regulates the gene expression related to ion channel (for example, K\u003csup\u003e+\u003c/sup\u003e and Na\u003csup\u003e+\u003c/sup\u003e channels) maturation and function\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, we demonstrate that the combined application of MM, NP and ES synergistically induces structural, electrophysiological, metabolic and functional maturation of iPSC-CMs and provide first insights into the mechanism driving advanced maturation of iPSC-CMs. Cultivation in FA-enriched MM strongly improves mitochondrial development and electrophysiological functionality of iPSC-CMs. Although the addition of NP to MM had little effect on the gene expression profile, it induced a specific increase in \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e current densities and cell alignment. The MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES combination induces molecular changes that occur during cardiac development, leading to increased structural maturation, polyploidy, improved mitochondrial development, and current patterns of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e more similar to adult human CMs. These changes translated into an altered sensitivity of iPSC-CMs to cardioactive drugs, suggesting the efficacy of our maturation approach to improve the predictive power of iPSC-CMs in drug screening. Furthermore, the improved maturation of iPSC-CMs highlights the potential of our combined approach to recapitulate clinical phenotypes that require an advanced development state of iPSC-CMs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDirected differentiation of iPSCs into iPSC-CMs, and pro-maturation culture of iPSC-CMs\u003c/h2\u003e \u003cp\u003eIn this study, three human iPSC lines were used, which were reprogrammed from somatic cells of three healthy individuals previously. The cell lines iWTD2.1 (UMGi001-A clone 1) and iBM76.3 (UMGi005-A clone 3) were generated from dermal fibroblasts and mesenchymal stem cells, respectively, using STEMCCA lentivirus\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The cell line isWT7.22 (UMGi020-B clone 22) was generated from dermal fibroblasts using the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The iPSC generation and application in research were approved by the Ethics Committee of the University Medical Center G\u0026ouml;ttingen (approval number: 21/1/11 and 10/9/15) and TU Dresden (EK 422092019).\u003c/p\u003e \u003cp\u003eAll iPSCs were cultured on Geltrex (Thermo Fisher Scientific) coated 6-well plates in Essential 8 (E8) medium (Thermo Fisher Scientific). E8 medium was changed daily and cells were passaged or differentiated when they were ~\u0026thinsp;85% confluent. To initiate differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with Glutamax and HEPES, 0.5 mg/mL human recombinant albumin, and 0.2 mg/mL L-ascorbic acid 2-phosphate and treated with 4 \u0026micro;M CHIR99021 (Merck Millipore), an inhibitor of GSK3β. After 48 h, CHIR99021 was removed and the cells were treated with 5 \u0026micro;M IWP2 (Wnt antagonist II, Merck Millipore) for another two days. The first beating cells were detected on day 8 post differentiation. From day 8, cells were cultivated in B27 medium containing RPMI 1640 with Glutamax and HEPES, supplemented with 1x B27 with insulin (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eOn day 15 after differentiation, the cells were digested and passaged. Cells were first incubated with 1 mg/mL collagenase B (Worthington Biochemical) for 1 h in an incubator, then detached iPSC-CM clusters were gently collected in a 15-mL Falcon tube and dissociated with 0.25% trypsin/EDTA (Thermo Fisher Scientific) for 8 min. Dissociated iPSC-CMs were resuspended in cardio-digestion medium (80% B27 medium, 20% fetal calf serum, and 2 \u0026micro;M thiazovivin (Merck Millipore)). The resuspended cells were seeded into Geltrex-coated 6-well plates for the B27 and MM groups, or onto Geltrex-coated \u0026oslash;25 mm nanopatterned (NP) coverslips (NanoSurface Coverglass, Curi Bio) in 6-well plates for the MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups at a density of 300,000-500,000 cells per well. Cells were maintained in B27 medium for 6 days with medium changes every 2 days. On day 21 post differentiation, all the non-B27 groups were switched from B27 medium to maturation medium (MM, Suppl. Table\u0026nbsp;1) for 7 days, with medium changes every 2 days. On day 28 post differentiation, the MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES group was subjected to 2 Hz electric field stimulation (2 ms pulse duration, 8 V) using a C-Pace EP (IonOptix) together with a 6-well C-dish (IonOptix) for 14 days. On day 42 post differentiation, cells from all four groups were harvested directly for analysis or dissociated, replated and cultured for another 7 days for further analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePatch clamp analysis\u003c/h2\u003e \u003cp\u003eiPSC-CMs at day 42 from the four groups were dissociated using a previously described method\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. For MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups, NP coverslips with cells were transferred to a 3.5-cm dish and then treated for 10 min with 2 mL of 20 U/mL papain (Sigma-Aldrich) dissolved in 1.1 mM EDTA-buffered B27 medium containing 2.5 \u0026micro;M blebbistatin (dissolved in DMSO). The cells were gently centrifuged at 50\u003cem\u003eg\u003c/em\u003e for 1 min. After aspirating the supernatant, the cell pellet was gently resuspended in B27 medium containing 2.5 \u0026micro;M blebbistatin and stored at 4℃ until measured.\u003c/p\u003e \u003cp\u003eAll automated patch-clamp experiments were performed at room temperature using Patchliner Quattro (Nanion technologies GmbH) with low resistance (for \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e recordings) and medium resistance (for \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e recordings) NPC-16 chips. The intracellular pipette and extracellular bath solutions for \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eand I\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e are listed in Suppl. Table\u0026nbsp;4. To record \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, cells were depolarised from a holding potential of -100 mV using voltage steps from \u0026minus;\u0026thinsp;80 to +\u0026thinsp;70 mV for 20 ms in 5 mV steps. The sweep interval was 2 s. Nifedipine (10 \u0026micro;M) was used to block \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e was recorded using test potentials of 2 s duration between \u0026minus;\u0026thinsp;130 and 10 mV from a holding potential of -40 mV. The sweep interval was 10 s. The protocol was repeated in the presence of 0.5 mM BaCl\u003csub\u003e2\u003c/sub\u003e and the Ba\u003csup\u003e2+\u003c/sup\u003e-sensitive current was calculated as \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e. To record \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e, cells were depolarised for 100 ms to voltages between \u0026minus;\u0026thinsp;80 and +\u0026thinsp;60 mV from a holding potential of -90 mV, and the sweep interval was 3 s. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e was recorded by increasing the test potential stepwise from \u0026minus;\u0026thinsp;40 mV to +\u0026thinsp;60 mV in 10 mV steps from a holding potential of -90 mV with a 20 ms pre-pulse to -35 mV to inactivate \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e. Each pulse lasted for 600 ms, and the sweep interval was 10 s. CdCl\u003csub\u003e2\u003c/sub\u003e (0.5 mM) was used to block sodium and calcium currents.\u003c/p\u003e \u003cp\u003eFor action potential (AP) recordings and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e measurements using manual patch clamp technique, CMs from all four groups were used directly after overnight recovery in cardio-digestion medium in order to minimise the time that CMs from the MM\u0026thinsp;+\u0026thinsp;NP and MM\u0026thinsp;+\u0026thinsp;NP\u0026thinsp;+\u0026thinsp;ES groups spent in non-NP and non-ES conditions. The pipette and extracellular solutions used for AP and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e recordings are listed in Suppl. Table\u0026nbsp;4. All manual patch-clamp experiments were performed at room temperature using a ruptured whole-cell patch clamp with a HEKA EPC10 amplifier and Patchmaster (HEKA Elektronik).\u003c/p\u003e \u003cp\u003eTo assess resting membrane potential (RMP), maximum upstroke velocity (Vmax), and action potential amplitude (APA), spontaneous APs were recorded in Tyrode\u0026rsquo;s solution without current injection. To assess AP duration (APD), a negative current was injected into the CMs to maintain the RMP at approximately \u0026minus;\u0026thinsp;80 mV prior to application of 0.5 Hz pacing stimulation. Signals were filtered with 2.9 and 10 kHz Bessel filters. At least 5 consecutive stable spontaneous APs and paced APs were averaged to determine RMP, Vmax, APA and APD at 90% repolarisation (APD\u003csub\u003e90\u003c/sub\u003e) using LabChart 8 software (ADInstruments).\u003c/p\u003e \u003cp\u003eThe holding potential of the \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e recording was set at -50 mV. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e was elicited by 2.5 s depolarisation steps from \u0026minus;\u0026thinsp;40 mV to potentials from +\u0026thinsp;40 to -40 mV in 10 mV decrements. This was followed by a 4 s repolarisation phase to -40 mV to elicit the \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e tail current. The pulse interval for each sweep was 10 s. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e was defined as the E-4031-sensitive current by subtracting the current recorded after application of 1 \u0026micro;M E-4031 from the current recorded before application.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMulti-electrode array\u003c/h2\u003e \u003cp\u003eAll multi-electrode array (MEA) recordings were performed using a Maestro Edge equipped with AxIS Navigator software (Axion BioSystems) at a sampling rate of 12,500 Hz, a temperature of 37\u0026deg;C, and 5% CO\u003csub\u003e2\u003c/sub\u003e. To assess the field potential (FP) properties, the digested iPSC-CMs were seeded onto Geltrex-coated CytoView 6-well MEA plates (Axion BioSystems). Approximately 200,000 cells were resuspended in 20 \u0026micro;L of cardio-digestion medium and seeded onto the electrode distribution area of the MEA plates. To evaluate the drug response, the digested iPSC-CMs were seeded into Geltrex-coated CytoView 24-well MEA plates (Axion BioSystems) at a density of 25,000 cells per well. Around one hour after seeding, an additional 1 mL of cardio-digestion medium was gently added into every well. The digested iPSC-CMs were recovered for 7 days in the same medium used during the maturation period. To avoid the influence of different media during recording, iPSC-CMs of all conditions were incubated in MM for one hour before starting measurements. MEA drug testing was performed using a sequential addition protocol with concentration increments after baseline activity recording in each well (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Vehicle controls for all conditions were performed on each assay plate. After drug addition, cells were incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 min, and the response was recorded for 2 min. The main metrics including conduction velocity (CV), spike amplitude, spike slope, inter-beat interval, and corrected field potential duration (FPD\u003csub\u003eC\u003c/sub\u003e, corrected by Fridericia\u0026rsquo;s formula) were further analysed using AxIS Navigator, Cardiac Analysis Tool and AxIS Metric Plotting tool (Axion BioSystems). Spontaneous beating frequency was defined as the reciprocal of the averaged inter-beat interval. The mainstream CV values were averaged for CV quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCalcium transient measurement\u003c/h2\u003e \u003cp\u003eFor calcium transient measurement\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, iPSC-CMs at day 42 from all four groups were dissociated and replated onto Geltrex-coated \u0026oslash;25 mm coverslips at a density of 200,000 cells per well of a 6-well plate. After a 7-day recovery period, cells were loaded with 2.5 \u0026micro;M Fura-2 (Thermo Fisher Scientific) in B27 medium (B27 group) and MM medium (the other three groups) at 37℃ for 30 min and washed twice with the corresponding medium. Cells were incubated for 10 min to achieve complete de-esterification of intracellular Fura-2. Intracellular calcium was recorded at 35\u0026deg;C using a 40x objective on an Olympus IX70 microscope equipped with the IonOptix system (IonOptix). Samples were excited at 340 and 380 nm with a switching frequency of 200 Hz and the emitted fluorescence was collected at 510 nm. Cytosolic calcium levels were defined as the ratio of fluorescence intensity at 340 and 380 nm (340/380 nm). Ca\u003csup\u003e2+\u003c/sup\u003e transients were recorded in Tyrode\u0026rsquo;s solution containing (in mM): NaCl 138, KCl 4, CaCl\u003csub\u003e2\u003c/sub\u003e 1.8, MgCl\u003csub\u003e2\u003c/sub\u003e 1, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). To normalise Ca\u003csup\u003e2+\u003c/sup\u003e transient frequency, iPSC-CMs from all four groups were field stimulated (6 V, 10 ms) at a pacing rate of 0.5 Hz using a MyoPacer (IonOptix). To assess the calcium content, 10 mM caffeine was applied to the CMs under 0.5 Hz pacing. Monotonic transient analysis was performed using LabChart 8 software (ADInstruments) and the following parameters were determined: diastolic Ca\u003csup\u003e2+\u003c/sup\u003e level, systolic Ca\u003csup\u003e2+\u003c/sup\u003e level, Ca\u003csup\u003e2+\u003c/sup\u003e transient amplitude (systolic Ca\u003csup\u003e2+\u003c/sup\u003e level minus diastolic Ca\u003csup\u003e2+\u003c/sup\u003e level), and decay rate (tau) of Ca\u003csup\u003e2+\u003c/sup\u003e transients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVideo-based contraction analysis\u003c/h2\u003e \u003cp\u003eVideo-based contraction analysis was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Briefly, videos (1024x1024 pixel, 60 FPS, length 30 s, 2 videos per culture, 3 cultures per batch) were recorded using a Hamamatsu Orca Flash 4.0 V3 camera (Hamamatsu). Videos were exported as MPEG4 files and analysed using Maia software using a block size of 10.7 \u0026micro;m (16 pixels), frameshift of 67 ms, and maximum distance shift of 4.69 \u0026micro;m (7 pixels). Video recording was performed on day 42. Contraction and relaxation peaks in the raw beating traces were assessed manually.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eOn day 42, iPSC-CMs were scraped off, pelleted, snap-frozen in liquid nitrogen and stored at -80\u0026deg;C. NP coverslips with cells were transferred into new plates to selectively collect cells from NP surfaces. Cells were lysed by homogenisation in RIPA buffer (150 mM NaCl, 50 mM Tris, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, and 1 mM PMSF) supplemented with inhibitors of proteases (cOmplete mini, EDTA-free, Roche) and phosphatases (PhosSTOP, Roche) and incubated for 30 min at 4\u0026deg;C with gentle rotation. Lysates were centrifuged at 14,000 rpm for 20 min at 4\u0026deg;C and protein concentration was determined by BCA assay following the manufacturer\u0026rsquo;s instruction. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in TBS-T overnight at 4\u0026deg;C. Afterwards, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies for 1 h at room temperature (antibodies are listed in Suppl. Table\u0026nbsp;5). Proteins were visualised by chemiluminescence using the Super Signal West Dura Chemiluminescent Substrate kit in combination with the Fusion FX Spectra Imaging System (Peqlab).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCells were detached and singularised using collagenase and trypsin, fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature and stored in PBS containing 1% BSA at 4\u0026deg;C. For staining, iPSC-CMs were permeabilised in PBS containing 1% BSA and 0.1% Triton-X for 10 min at room temperature. Staining was performed with specific antibodies as described in Suppl. Table\u0026nbsp;5. cTNT was detected using either the primary antibody mouse anti-cTNT (Thermo Fisher Scientific, MS-295-P1) or directly coupled cTNT-APC (Miltenyi Biotec, 130-120-543). For cTnT and Tom20 staining, cTNT-APC and anti-Tom20 (Santa Cruz, sc-17764) antibodies were used. Negative controls were performed using either the respective secondary antibodies (for samples detected with the non-coupled primary antibodies) or isotype controls (for samples detected with the directly coupled primary antibodies). After incubation with primary antibodies, cells were washed with PBS containing 1% BSA, followed by incubation with secondary antibodies, and Hoechst 33342 (5 \u0026micro;g/mL). To assess EdU-incorporation, PFA-fixed iPSC-CMs were incubated with mouse anti-cTNT antibody in Click-iT\u0026trade; permeabilisation and wash reagent (Thermo Fischer Scientific, C10645) overnight, EdU click reaction was performed according to manufacturer\u0026rsquo;s instructions, and DNA was stained with Draq5 (abcam, ab108410, 10 \u0026micro;M). To determine the activity of Ki67, iPSC-CMs were stained with fluorophore-conjugated antibodies cTNT-APC (Miltenyi Biotec, 130-120-543) and Ki67-FITC (Miltenyi Biotec, 130-117-691) for 1 hour at 4\u0026deg;C. DNA was stained with Hoechst 33342.\u003c/p\u003e \u003cp\u003eAfterwards, cells were resuspended in PBS containing 1% BSA and analysed on an LSRII or FACS Canto II flow cytometer. At least 10,000 events were recorded for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eFor staining of RYR2, α-actinin and Hoechst 33342, cells were fixed with 4% PFA for 20 min and permeabilised with PBS containing 1% BSA and 0.1% triton-X 100 for 10 min at room temperature. For detection of Cx43 and Hoechst 33342, iPSC-CMs were fixed in methanol-acetone (7:3, \u003cem\u003ev/v\u003c/em\u003e, 10 min at -20\u0026deg;C). After fixation, cells were washed with PBS and incubated in PBS containing 1% BSA at 4\u0026deg;C for at least 2 h. Incubation with primary antibodies was performed overnight at 4\u0026deg;C in PBS containing 1% BSA. After washing the coverslips in PBS, samples were incubated with secondary antibodies and Hoechst 33342 in PBS containing 1% BSA for 1 h at room temperature. Coverslips were washed with PBS, deionised water and mounted onto glass slides using Fluoromount-G mounting medium (Thermo Fisher Scientific). Imaging was performed using Keyence BZ-X700E fluorescence microscope (Keyence) or LSM880 confocal microscope (Carl Zeiss). Antibodies were used as indicated in Suppl. Table\u0026nbsp;5.\u003c/p\u003e \u003cp\u003eTo analyse the colocalisation of RYR2 and α-actinin, samples were imaged using an LSM880 confocal microscope (at 60x magnification) with fixed settings (gain, laser intensity, offsets) for all conditions within an experiment. Colocalisation was quantified using Cell Profiler software. For this, composite images were loaded, single channels were separated using the ColorToGray module, colocalisation of the red (RYR2) and green (α-actinin) channels was measured for the entire image covering 2\u0026ndash;4 cells using the MeasureColocalization module, and data were exported using the ExportToSpreadsheet function. Colocalisation was measured for 3 independent experiments with 6 images per experiment and conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSeahorse measurements\u003c/h2\u003e \u003cp\u003eThe Seahorse system was used to determine the metabolic activity of iPSC-CMs. Cells were dissociated and singularised using collagenase B and trypsin on day 42 and replated in 96-well Seahorse assay plates (Agilent, 103792-100) at a density of 15,000 cells per well in cardio-digestion medium. The next day, the medium was changed to the appropriate culture medium (B27 medium for the B27 group, MM for three other groups) and cells were recovered for 7 days with medium changes every other day. Seahorse recordings were performed using Seahorse Agilent XF Analyzer (Agilent) according to the manufacturer\u0026rsquo;s protocols. The Seahorse XF Cell Mito Stress Test Kit (Agilent, 103010-100) with sequential addition of oligomycin, FCCP and rotenone/antimycin A was used to study mitochondrial respiration. All data were normalised to the total amount of protein per well after lysis of cells in RIPA buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR\u003c/h2\u003e \u003cp\u003eOn day 42, the cells were washed with ice-cold PBS, scraped from the culture plate or NP coverslips and centrifuged at 2,000\u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C. Cell pellets were lysed in TRIzol\u0026trade; (Thermo Fisher Scientific), chloroform was added, samples were mixed thoroughly and centrifuged for 15 min at 4\u0026deg;C and 15,000\u003cem\u003eg\u003c/em\u003e to separate phases. RNA was extracted, diluted with 95% EtOH and purified using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer\u0026rsquo;s protocol. RNA was eluted in RNase/DNase-free water. Concentration was determined using a Nanodrop\u0026trade; spectrophotometer (Thermo Fisher Scientific). Synthesis of cDNA was performed using the iScript cDNA Synthesis Kit (Bio-Rad) with 200 ng total RNA input per reaction (20 \u0026micro;L) according to the manufacturer\u0026rsquo;s protocol. For the detection of gene expression, samples and specific primers (Suppl. Table\u0026nbsp;6) were prepared using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and real-time PCR was performed in Hard-Shell Optical 96-well plates using the CFX96 Real-time PCR system (Bio-Rad). Initial denaturation was done at 95\u0026deg;C for 30 s, followed by 45 amplification cycles (15 s at 95\u0026deg;C, 1 min elongation at 60\u0026deg;C). A melting curve was obtained over the temperature range 65\u0026ndash;95\u0026deg;C. HPRT was used as a reference gene for calculation of normalised relative expression (2\u003csup\u003eΔΔCt\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing and DEG analysis\u003c/h2\u003e \u003cp\u003emRNA was isolated from an average of 600 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer\u0026rsquo;s instructions. Samples were then directly subjected to the strand-specific RNA-seq library preparation workflow (Ultra II Directional RNA Library Prep, NEB). Ligation was performed using the NEB Next Adapter from the NEB Next Multiplex Oligos for Illumina Kit. After ligation, the adapters were depleted by XP bead purification (Beckman Coulter), where the bead solution was added to the samples in a ratio of 0.9:1. Unique dual indexing was done during the subsequent PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 index (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, Primer 2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two further XP bead purifications (0.9:1), the libraries were quantified using the Fragment Analyzer (Agilent). Libraries were sequenced on an Illumina NovaSeq 6000 in 100 bp paired-end mode with an average of 50\u0026nbsp;million fragments per library.\u003c/p\u003e \u003cp\u003eFastQC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.babraham.ac.uk/\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.babraham.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to perform a basic quality control of the resulting sequencing data. Fragments were aligned to the human reference genome hg38 with the support of the Ensembl 104 splice sites using the aligner STAR (2.7.10b). Counts per gene and sample were obtained based on the overlap of the uniquely mapped fragments with the same Ensembl annotation using featureCounts (v2.0.1). Normalisation of raw fragments based on library size and testing for differential expression between the different conditions was performed using the DESeq R package (v1.38.3). Sample to sample Euclidean distance, Pearson' and Spearman correlation coefficient (r) and PCA based on the top 500 genes with the highest variance were computed to explore the correlation between biological replicates and different libraries. To identify differentially expressed genes (DEGs), counts were fitted to the negative binomial distribution and genes were tested between conditions using the Wald test of DESeq2. Resulting p-values were corrected for multiple testing using the Independent Hypothesis Weighting package (IHW 1.26.0). Genes with a maximum false discovery rate (FDR) of 10% were considered to be significantly differentially expressed. TPM values were generated using Kallisto (v0.46.1).\u003c/p\u003e \u003cp\u003eGene set enrichment analysis was performed based on normalised count data using GSEA software v.4.3.2\u003csup\u003e58\u003c/sup\u003e and canonical pathway collection (c2.cp.v2023.2.Hs) or transcription factor target collection (c3.tft.v2023.2.Hs) with following parameters: weighted scoring, meandiv normalisation, max_probe mode, maximum gene set size 500 genes, minimum set size 15 genes, and 1000 permutations. GSEA results were applied for the creation of enrichment maps using Cytoscape software according to workflow of Reimand et al.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Briefly, nodes were included based on FDR q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.2 and NES\u0026thinsp;\u0026ge;\u0026thinsp;1.5 for upregulated and \u0026le; -1.9 for downregulated genes. Edges cut-off was set to 0.375 and pathway cluster names were determined after manual examination of all individual nodes in clusters.\u003c/p\u003e \u003cp\u003eSequencing data have been uploaded (EGAD00001011289) and are available on request from the European Genome-Phenome Archive (EGA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ega-archive.org/\u003c/span\u003e\u003cspan address=\"https://ega-archive.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9, with different comparisons indicated in figure legends. Results were considered statistically significant when the p-value was \u0026lt;\u0026thinsp;0.05 (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eW.L., M.S, and K.G. conceived the study, managed the project progress and coordinated the experiments and analysis; W.L., X.L., A.S., S.A., O.G., M.S.P., M.H., R-P.S., K.F., J.P., Y.U., G.T., P.M., M.S. performed experiments; W.L., X.L., A.S., G.T., M.H., M.L., A.D., M.S. and K.G. contributed to data analysis and visualisation was performed by W.L. and M.S.. P.M., A.E-A. and K.G. provided resources. The paper was prepared by W.L., M.S. and K.G. with all authors providing feedback. All authors have read and agreed to the current version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Susann Hoefner from the Flow Cytometry Core Facility and the Core Facility Cellular Imaging (CFCI) of the TU Dresden for excellent technical support. This work was funded by the Free State of Saxony and the European Union (SAB EFRE projects \u0026ldquo;PhenoCor\u0026rdquo; with project number 100387678 to A.E.-A. and K. Guan and \u0026ldquo;CardioEpiX\u0026rdquo; with project number 100685417 to K. Guan as well as ESF Plus project \u0026ldquo;MultiMOD\u0026rdquo; with project number 100649621). X. Luo was financially supported by the ESF Plus project \u0026ldquo;MultiMOD\u0026rdquo;. M. Schubert was supported by a MeDDrive grant from the Medical Faculty of the TU Dresden. A. Strano and M. Hasse were financially supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under project number 288034826 \u0026ndash;IRTG 2251: \u0026ldquo;Immunological and Cellular Strategies in Metabolic Disease\u0026rdquo; for project 10 to K. Guan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKolanowski TJ, Antos CL, Guan K (2017) Making human cardiomyocytes up to date: Derivation, maturation state and perspectives. Int J Cardiol 241:379\u0026ndash;386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStreckfuss-Bomeke K et al (2017) Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes. 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Nat Protoc 14:482\u0026ndash;517\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"induced pluripotent stem cell-derived cardiomyocytes, maturation, nanopatterning, electrostimulation, drug testing","lastPublishedDoi":"10.21203/rs.3.rs-3973784/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3973784/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules, thus hindering their potential role in guiding personalised drug selection for patients. Here, we demonstrate an approach that combines lipid-enriched maturation medium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with an advanced electrophysiological, structural and metabolic phenotype. Through a systematic, stepwise parallel testing of the three stimuli, electrostimulation emerged as the pivotal factor to enhance mitochondrial development and to improve the electrophysiological properties of iPSC-CMs. The combined approach brought a substantial modification in their current composition by increasing \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNa\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eto\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eK1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eKr\u003c/em\u003e\u003c/sub\u003e but decreasing \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eCa\u0026minus;L\u003c/em\u003e\u003c/sub\u003e, resulting in a significant change in their sensitivity to cardioactive drugs. Transcriptome analysis revealed that activation of HMCES and TFAM targets played a role in mitochondrial development, whereas the downregulation of MAPK/PI3K signalling pathways and SRF targets were associated with polyploidy of iPSC-CMs. Taken together, our study provides mechanistic insights into the maturation of iPSC-CMs with a more adult-like drug response.\u003c/p\u003e","manuscriptTitle":"Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 16:22:49","doi":"10.21203/rs.3.rs-3973784/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9132f89a-560c-4d2f-97dc-8c2e9e438665","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30458395,"name":"Biological sciences/Stem cells/Stem-cell differentiation"},{"id":30458396,"name":"Health sciences/Cardiology/Cardiovascular biology/Heart development"}],"tags":[],"updatedAt":"2025-03-22T07:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-3973784","link":"https://doi.org/10.1038/s41467-025-58044-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-21 04:00:00","publishedOnDateReadable":"March 21st, 2025"},"versionCreatedAt":"2024-04-10 16:22:49","video":"","vorDoi":"10.1038/s41467-025-58044-6","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58044-6","workflowStages":[]},"version":"v1","identity":"rs-3973784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3973784","identity":"rs-3973784","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}