Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation

Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation | 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 Research Article Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation HyunJu Seo, Ju-young Kim, Hyun-Jai Cho, Joo-Eun Lee, Sang-Beom Bang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6392558/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ABSTRCT Background: Cardiovascular diseases remain the leading cause of death worldwide, and the limited efficiency of human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation hampers its potential in disease modeling and regenerative therapy. Calcium signaling plays a central role in cardiac maturation, and proper regulation of intracellular calcium dynamics is essential for activating transcriptional programs that drive cardiomyocyte differentiation. Recent studies have suggested that closure of the mitochondrial permeability transition pore (mPTP) enhances cardiomyocyte differentiation by modulating calcium homeostasis and reducing reactive oxygen species (ROS). Building on this concept, we investigated the effects of 7-aminoindole (7-AI), a novel compound that inhibits mitochondrial calcium influx via the mitochondrial calcium uniporter (MCU), on cardiomyocyte differentiation. Methods: Using both mouse embryonic stem cells and hiPSCs, we treated cells undergoing differentiation with 7-AI at the cardiac progenitor stage (day 4) to inhibit MCU activity. We assessed differentiation efficiency by measuring nuclear and cytosolic calcium levels, activation of Ca²⁺/calmodulin-dependent protein kinase (CaMK), and phosphorylation status of the transcription factor cAMP response element-binding protein (CREB). Cardiac-specific gene expression was evaluated by quantifying cardiac cTnT, α-SA, and MYH6/7. Structural and functional maturation of the derived cardiomyocytes was determined using immunostaining and contractility assays. Results: Treatment with 7-AI significantly increased nuclear calcium levels and activated both CaMK and CREB, leading to the enhanced expression of cardiac-specific genes. Both mouse embryonic and hiPSC-derived cardiomyocytes displayed improved structural organization and contractile properties after 7-AI treatment. Comparative analysis between wild-type and CREB-deficient cells confirmed that CREB is essential for proper cardiomyocyte maturation because CREB deficiency leads to reduced cardiac marker expression and impaired myofibrillar organization. Conclusions: Our study demonstrated that 7-AI enhanced cardiomyocyte differentiation by inhibiting MCU, thereby redistributing calcium from the mitochondria to the nucleus. This redistribution activates CaMK and CREB, which in turn upregulate cardiac-specific gene expression, ultimately promoting the structural and functional maturation of cardiomyocytes. Targeting calcium dynamics during the cardiac progenitor stage represents a novel strategy for improving the efficiency of cardiac differentiation. These findings provide valuable insights into the molecular mechanisms governing cardiac maturation and offer a promising approach to generate functional cardiomyocytes for therapeutic applications. 7-aminoindole mitochondrial calcium uniporter (MCU) CaMK–CREB signaling maturation human induced pluripotent stem cell derived cardiomyocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cardiovascular diseases are the leading cause of mortality worldwide, necessitating the exploration of underlying mechanisms and clinical manifestations and development of innovative therapeutic strategies for these diseases. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are valuable tools providing a robust platform for cardiac research, disease modeling, and drug screening[ 1 – 3 ]. However, improving the efficiency of hiPSC-CM differentiation is a major challenge. Therefore, in this study, we aimed to enhance the efficiency of hiPSC-CM differentiation using a novel compound to modulate the intracellular calcium dynamics. Closure of the mitochondrial permeability transition pore (mPTP), which promotes mitochondrial maturation and CM differentiation, reduces the reactive oxygen species (ROS) levels and modulates the calcium dynamics in early embryonic CMs.[ 4 ] As mPTP closure influences calcium homeostasis, we hypothesized that the initial inhibition of calcium influx is the primary mechanism protecting mitochondria and driving stem cell differentiation, whereas the observed reduction in ROS levels is a downstream effect of altered calcium signaling, as suggested in a previous report.[ 5 ] Therefore, calcium signaling, rather than ROS, is the key regulator of CM maturation.[ 6 , 7 ] Ca²⁺/calmodulin-dependent protein kinase (CaMK) integrates intracellular calcium signals to regulate various cellular processes, such as cell differentiation.[ 8 – 10 ] During hiPSC-CM maturation, CaMK coordinates calcium signaling among the cytosol, endoplasmic/sarcoplasmic reticulum, and mitochondria, influencing various CM-specific functions.[ 11 , 12 ] We previously demonstrated that 7-aminoindole (7-AI), a compound inhibiting mPTP opening and mitochondrial calcium influx, provides cardioprotection by preserving the mitochondrial membrane potential and suppressing ROS production[ 13 ]. Based on these findings, we hypothesized that 7-AI enhances hiPSC-CM differentiation by modulating the mitochondrial calcium uptake and that treatment with 7-AI during the cardiac progenitor cell stage increases the cytosolic and nuclear calcium levels by inhibiting the mitochondrial calcium uniporter (MCU), as suggested in previous reports.[ 14 , 15 ] Calcium redistribution possibly activates CaMK and the transcription factor, cAMP response element-binding protein (CREB), which together promote the expression of cardiac-specific genes, including those encoding ryanodine receptors, inositol 1,4,5-trisphosphate receptors, and sarco/endoplasmic reticulum calcium ATPase (SERCA).[ 16 , 17 ] Consistent with the proposed mechanism, our comparative analyses of wild-type and CREB-deficient cells revealed that functional CREB was essential for CM maturation by increasing the CM marker (cardiac troponin T [cTnT], α-actinin, and MYH6/7) levels and supporting proper myofibrillar organization. This study showed that targeting calcium dynamics via MCU inhibition during the cardiac progenitor cell stage (day 4) significantly improved the CM differentiation efficiency, providing insights into the fundamental mechanisms governing cardiac differentiation and presenting a novel strategy to enhance cardiac differentiation. Comprehensive understanding of the association between calcium distribution and cardiac differentiation will facilitate the efficient generation of functional CMs for various therapeutic applications. Methods Material: Necrosis inhibitor A novel necrosis inhibitor—a 7-Amino-indole chemical—was developed by LG Chem at the LG Chem Life Science R&D Campus in Daejeon, Korea ( http://www.rnd.lgchem.com/global/main ). The study material, referred to as NecroX, is available upon reasonable request. Based on preliminary experiments with various compounds in the NecroX series, we selected NecroX-7 ((tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxothiomorpholin-4-yl) methyl-1H-indol-7-yl]amine; C 25 H 32 N 4 O 4 S 2 ) for further evaluation. mESC culture and differentiation mESCs (ES-C57BL/6; ATCC number: SCRC-1002; ATCC, Manassas, USA) were cultured using mouse embryonic fibroblasts (CF-1, ATCC number: SCRC-1040) on an mESC medium with the recombinant mouse leukemia inhibitory factor (ESG1107; Merck Millipore, Darmstadt, Germany). Briefly, 2.2 × 10 6 mESCs were incubated in an Aggrewell (#27845/27945; STEMCELL Technologies, Vancouver, Canada) in an embryoid body medium with the recombinant bone morphogenetic protein-4 (5020-BP; R&D Systems, Minneapolis, USA) for one day to form embryoid bodies. ESCs were incubated in a suspension culture for two days in an embryoid body medium with bone morphogenetic protein-4, activin A (recombinant human/mouse/rat activin A 338-AC; R&D Systems), and recombinant human basic fibroblast growth factor (bFGF; 13256029; Thermo Fisher Scientific, MA, USA). On CM differentiation day 3, embryoid bodies were attached to a 6-well plate with CM differentiation medium supplemented with bFGF, recombinant human epidermal growth factor (236-EG; R&D Systems), recombinant human cardiotrophin-1 (612-CD; R&D Systems), and recombinant mouse vascular endothelial growth factor (493-MV; R&D Systems), which was changed every two days. Then, 5 and 100 nM 7-AI was added on CM differentiation days 4–8. hiPSC culture and differentiation hiPSCs reprogramed from newborn foreskin fibroblasts (GSC-3006G; AMS Biotechnology [GlobalStem], Abingdon, UK) using the four Yamanaka factors were cultured using STO (SIM; ATCC number: CRL-1503) on the Dulbecco's modified Eagle’s medium/nutrient mixture F12 Glutamax (10565-018; Thermo Fisher Scientific) supplemented with knockout serum replacement, 10 mM non-essential amino acids, 200 mM L-glutamine, 55 mM β-mercaptoethanol, and 10 ng/mL human recombinant bFGF. CM differentiation of iPSCs was performed as described by Lian et al., with some modifications. Directed CM differentiation from human pluripotent stem cells by modulating Wnt/b-catenin signaling under fully defined conditions; Nature protocols, 2013, 162, VOL.8 NO.1). hiPSC colonies were detached using dispase (17105-041; Thermo Fisher Scientific) and dissociated into single cells. Then, 1.5 × 10 6 hiPSCs were seeded on matrigel (354277; Corning, NY, USA)-coated 35-mm dishes, grown on mTeSR1 (#85851; STEMCELL Technologies) until reaching 100% confluency, and subjected to cardiac differentiation. The following chemicals were sequentially administered: 6 µM CHIR99021 (252917-06-9; Cayman, MI, USA) for the first two days, followed by 10 µM recombinant human/mouse/rat activin A (338-AC; R&D Systems) and 20 µM recombinant human bFGF (13256029; Thermo Fisher Scientific) the next day, and 5 µM IWR1 (I0161; Sigma-Aldrich, St. Louis, USA) three days after that. The medium was replaced with the Roswell Park Memorial Institute-1640 medium (11875-085; Thermo Fisher Scientific) supplemented with B27 supplement (minus insulin) once every two days until the CMs contracted. Subsequently, 7-AI (5 and 100 nM) was administered one day before cell differentiation initiation. MCU inhibitor RuR (5 µM) was also administered at the same time as 7-AI. Flow cytometry mESC-CM and hiPSC-CM differentiation was assessed via flow cytometry using the cTnT marker. Cultured mESC-CMs were detached from the dish using trypsin and washed with phosphate-buffered saline (PBS) via centrifugation at 1800 rpm for 5 min. hiPSC-CMs were dissociated into single cells using accutase and washed with PBS via centrifugation at 1200 rpm for 5 min. These cells were resuspended in 1 mL of fluorescence-activated cell sorting buffer before staining. After permeabilization with the permeabilization buffer (Gibco) at 4°C for 10 min, the cells were incubated with primary antibodies at room temperature for 1 h, washed with the fluorescence-activated cell sorting buffer, incubated again with the Alexa-488-conjugated secondary antibodies at room temperature for 1 h, and subjected to flow cytometry. Immunofluorescence assay To assess the differentiation efficiency of mESC-CMs and hiPSC-CMs after 7-AI treatment, immunostaining was performed using the α-SA Tom20 antibody to visualize the mitochondria. The cells were incubated overnight with primary antibodies at 4°C, followed by incubation with the secondary Alexa-488- and Alexa-555-conjugated antibodies at room temperature for 1 h. Images were acquired using a confocal microscope (Leica). Cell size was calculated using the mean cell area with the ImageJ software, and cell length was measured in pixels and converted to actual length (µm) for quantitative analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Next, mRNA expression levels were quantified via real-time RT-PCR using the 7500 real-time PCR system, according to the manufacturer’s protocol. PCR was performed using a 96-well plate with three replicates at a final volume of 20 µL, and expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase RNA levels. mRNA expression analysis was performed according to the TOYOBO protocol. Quantitative real-time PCR for binding analysis was performed using the SYBR Green protocol. Western blotting Three experimental groups (vehicle, 5 nM 7-AI-treated, and 100 nM 7-AI-treated hiPSC CMs) were lysed using the radioimmunoprecipitation assay buffer containing protease/phosphatase inhibitors and centrifuged at 15,000 rpm for 30 min at 4°C. The isolated proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk at room temperature for 30 min, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with the respective secondary antibodies. Immunoblot signals were detected using Amersham 680 and quantified using the ImageJ software. FLUO-4 assay hiPSC-CMs cultured in 35-mm dishes were stained with 1 µM FLUO-4 (F14201; Thermo Fisher Scientific, Invitrogen) and 2 mM probenecid for 15 min in a 37°C in a 5% CO 2 incubator. After FLUO-4 staining, the medium was replaced with PBS containing 1% fetal bovine serum (FBS), and the cells were incubated at room temperature for 20 min. Subsequently, the medium was switched to Tyrode’s solution containing glucose and calcium to measure the calcium changes. To determine the calcium levels in CMs, 1 mM caffeine was applied and calcium signal changes were observed using the A1 confocal laser microscope (Nikon, Melville, NY) via time-lapse confocal imaging. Images were captured every 2 s for 10 min. Fluorescence intensity data for each cell was acquired using the NIS-Elements C software (Nikon), and ΔF/F was calculated from this data. ΔF/F of calcium signals were graphed, and maximum intensity of calcium signals, signal rise rate, and time to reach 90% of the maximum calcium signal were quantified. To assess the intracellular calcium signaling changes during CM differentiation, iPSCs were seeded in a 35-mm µ-Dish (ibidi) and cultured until they reached the desired confluency. After incubating with 1 µM FLUO-4 and 2 mM probenecid in a 5% CO₂ incubator at 37°C for 15 min, the cells were incubated again at room temperature for 15 min. Then, the cells were washed with PBS, and the medium was replaced with PBS containing 1% FBS. To assess the intracellular calcium flux, 6 µM CHIR99021, a compound used in the early stage of CM differentiation, was applied to stimulate calcium movement in cells. EP study Spontaneous action potentials (APs) were recorded from single hiPSC-CMs using current-clamp mode at 37°C. Cells exhibiting stable spontaneous beating activity were selected for analysis. Once AP waveforms stabilized, the average of five consecutive AP traces was analyzed under each test condition. AP recordings were conducted in an extracellular solution containing (mM) 145 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 5 glucose, and 1.8 CaCl2 (pH 7.4). The internal pipette solution consisted of (mM) 120 K-Asp, 20 KCl, 5 NaCl, 2 CaCl2, 10 HEPES, 5 EGTA, and 5 Mg-ATP (pH 7.25). Cells were monitored to ensure stable electrophysiological properties before data acquisition. AP subtypes in hiPSC-CMs were classified based on action potential duration at 90% repolarization (APD90), the time required for the membrane potential to return to 90% of its resting level following depolarization. Nodal-type APs were defined as APD90 < 100 ms, atrial-type APs as 100 ms ≤ APD90 < 250 ms, and ventricular-type APs as APD90 ≥ 250 ms. Further classification between atrial- and nodal-type APs was based on differences in amplitude (TA) and upstroke velocity (dV/dtmax) Nuclear and cytoplasmic protein extraction Vehicle and hiPSC-CM groups treated with 7-AI (5 and 100 nM) from day 1 of differentiation were harvested on day 6. To extract the nuclear and cytoplasmic proteins from differentiated CMs, the cells were washed with PBS, transferred to a 15-mL tube, and centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was aspirated, and the cells were resuspended in 1 mL of cold PBS in a 1.5-mL tube, followed by centrifugation at 3500 rpm for 5 min. The pellet was resuspended in 200 µL (per 100 mm dish) of cold buffer A and vortexed. After allowing the lysate to swell on ice for 20 min, it was passed through a 1-mL syringe fitted with an 18-21-gauge needle 3–4 times, and cell lysis was confirmed via trypan blue staining. The lysate was further centrifuged at 9000 rpm for 15 min, and the resulting supernatant containing the cytosolic proteins was carefully collected. The nuclear pellet was recovered, resuspended in 20 µL of cold buffer B, and incubated on ice for 20 min. The lysate was centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatant containing the nuclear proteins was transferred to a new tube. Western blotting was performed to analyze the proteins, and concentrations of the nuclear/cytosolic proteins were determined using the BCA protein assay kit. Buffer A contained 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl, 10 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol (freshly added), and 0.2 mM phenylmethylsulfonyl fluoride (freshly added). Buffer B contained 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl₂, 25% glycerol, 0.5 mM dithiothreitol (freshly added), and 0.2 mM phenylmethylsulfonyl fluoride (freshly added). Western blotting analysis was performed to determine the transcription factor expression levels using the phospho-CREB (Ser133; 87G3) rabbit monoclonal (#9198; Cell Signaling Technology), CREB (48H2) rabbit monoclonal (#9197; Cell Signaling Technology), anti-SERCA2 (#2861; Abcam), anti-NFATc1 (#MA3024; Thermo Fisher), and anti-lamin A/C (#2032S; Cell Signaling technology) antibodies. Lentiviral transduction of hiPSC-CMs Lentiviral transduction was performed on day 4 of hiPSC-CM differentiation. Lentivirus, prepared according to the manufacturer’s protocol, and polybrene (1 µg/mL) were used for transduction over 2–3 d. TRC CREB1 shRNA vector was constructed by Horizon (USA). Transfection of the calcium indicator vector into hiPSC-CMs Calcium indicator vectors CMV-NLS-R-GECO and CMV-mito-GEM-GECO1 were kindly gifted by R. E. Campbell (plasmids #32462 and #32461; Addgene). 13 On day 12 of hiPSC-CM differentiation, the cells were washed with PBS, and the medium was replaced with the Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific). Plasmid transfection was performed using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer’s recommended protocol. Plasmid DNA (5 µg) was diluted in 125 µL Opti-MEM and gently mixed. In a separate tube, 12.5 µL of Lipofectamine 2000 Reagent was added to 125 µL Opti-MEM, mixed gently, and incubated at room temperature for 5 min. Then, the plasmid DNA dilution was added to the Lipofectamine 2000 dilution, mixed gently, and incubated at room temperature for 20 min to allow the formation of the plasmid DNA–Lipofectamine complex. After 20 min, the complex was applied dropwise to the cells, gently mixed by rocking the dish, and incubated in at 37°C. After 48 h, the cells were first incubated with 1 µM FLUO-4 and 2 mM probenecid in a 5% CO₂ incubator at 37°C for 15 min and then at room temperature for 15 min. After washing with PBS, the medium was replaced with PBS containing 1% FBS. The cells were treated with 2 µM thapsigargin and 100 nM 7-AI or PBS as a vehicle, and calcium signaling was examined. The cells were treated with 50 µM tBH (Sigma-Aldrich) for strong calcium stimulation. Finally, calcium signaling and fluorescence intensity analyses were conducted as described above. Statistical analyses Data are represented as the mean ± standard error of the mean. Statistical analyses were conducted via one-way analysis of variance using the GraphPad Prism 5 software. Statistical significance was set at P < 0.05. Results 7-AI enhances cardiac differentiation efficiency in mouse embryonic stem cells We investigated the effects of 7-AI on CM differentiation using mESCs, which are relatively easy to differentiate. mESCs were treated with 7-AI (5 and 100 nM) during CM differentiation, and differentiation efficiency was compared with that of the vehicle group. Notably, 7-AI-treated groups exhibited significantly higher beating rates and more than twice the contractile area than the vehicle group (Fig. 1 A). Furthermore, on day 10 post-differentiation, CM-specific marker cTnT levels were significantly elevated in the 7-AI-treated groups, indicating that 7-AI increased the CM differentiation efficiency (Fig. 1 B). 7-AI promotes the maturation of mESC-derived cardiomyocytes Next, genetic and functional maturation of differentiated CMs treated with 7-AI was evaluated using α-sarcomeric actin (α-SA), a key sarcomeric structural protein. α-SA is specifically expressed in striated muscle tissues, such as CMs, making it a valuable marker to assess the functional and structural maturation of CMs. Immunofluorescence staining for α-SA, followed by length measurement revealed that the average length of α-SA was significantly longer in the 7-AI-treated groups than in the control group. Furthermore, CM size increased in a concentration-dependent manner in the 7-AI-treated groups relative to that in the vehicle group (Fig. 2 A). Expression levels of ion channel genes playing key roles in the electrical and mechanical functions of mature CMs were also examined. Levels of ion channel genes KV4.3 , NCX1 , and NaV1.5 were higher in the 7-AI-treated groups than in the vehicle group, although no significant differences were observed in the expression levels of mERG , KV2.2 , and CaV1.2 (Fig. 2 B). Calcium kinetics analysis using Fluo-4 revealed that differentiated CMs treated with 100 nM 7-AI exhibited more than double the amplitude (ΔF/F) of the vehicle CMs during beating. Additionally, the treated group exhibited Vmax upstroke (ΔF/F/sec) more than three-fold higher than that of the vehicle group. Therefore, 7-AI-treated CMs exhibited high intracellular calcium fluctuations and rapid calcium release and uptake, showing enhanced contraction speed and efficiency. Furthermore, time to 90% peak(s) in the 7-AI-treated CM group was reduced by more than three-fold compared to that in the control group, indicating efficient calcium release in the 7-AI-treated CM group (Fig. 2 C). Collectively, these results suggest that 7-AI treatment during CM differentiation promotes the activation of ion channels regulating calcium release and uptake in calcium storage compartments, thereby supporting CM differentiation into functionally mature cells. 7-AI enhances cardiac differentiation efficiency in human induced pluripotent stem cells Next, we examined the effects of 7-AI on CM differentiation using hiPSC-CMs. During differentiation, human fibroblast-derived iPSCs were treated with 7-AI (5 and 100 nM). Notably, 7-AI-treated groups exhibited significantly stronger contractions and larger beating areas than the vehicle group (Fig. 3 A). Furthermore, cTnT levels were elevated in the 7-AI-treated groups, indicating that 7-AI increased the CM differentiation efficiency (Fig. 3 B). 7-AI promotes the maturation of hiPSC-derived cardiomyocytes Structural maturity of hiPSC-CMs was examined via α-SA staining. Total cell size was significantly larger and sarcomere length was significantly longer in the 7-AI-treated group than in the vehicle group (Fig. 4 A). To evaluate genetic maturation, expression levels of the CM-specific markers, such as cTnT and MHC6 , and genes encoding ion channels characteristic of mature CMs were analyzed. Upon treatment with 7-AI (5, 100, and 500 nM), highest increase in cTnT and MHC6 levels was observed in the 100 nM 7-AI-treated group. Analysis of ion channel gene expression revealed that the 7-AI-treated hiPSC-CMs exhibited significantly higher levels of Kv4.3 and NCX1 (similar to mESC-CMs) as well as hERG , Kv2.2 , and CaV1.2 (different from mESC-CMs) than the vehicle CMs (Fig. 4 B). In the calcium kinetics analysis of hiPSC-CMs, 100 nM 7-AI-treated group exhibited a higher amplitude (ΔF/F) and Vmax upstroke (ΔF/F/sec) than the vehicle group, indicating enhanced contraction speed and calcium handling efficiency. Furthermore, time to 90% peak(s) was reduced in the 7-AI-treated group, indicating rapid calcium release. Overall, 7-AI-treated groups exhibited more stable and efficient excitation–contraction coupling than the control group (Fig. 4 C). Electrophysiological (EP) studies were also conducted to classify the hiPSC-CM cell types AP subtypes in hiPSC-CMs were classified as nodal-type (APD90 < 100 ms), atrial-type (100 ms ≤ APD90 < 250 ms), and ventricular-type (APD90 ≥ 250 ms) based on AP duration. Pie charts show the distribution of AP subtypes in vehicle- and 7-AI-treated cells. Representative AP traces illustrate differences in AP morphology among the subtypes. 7-AI treatment increased the proportion of ventricular-type APs while reducing atrial-type populations, suggesting an influence on cardiomyocyte electrophysiological maturation. APD90, action potential duration at 90% repolarization (Fig. 4 D). 7-AI treatment upregulates cardiomyocyte-specific genes and proteins Mature CMs efficiently regulate the calcium dynamics. Therefore, we analyzed the expression levels of genes and proteins involved in calcium release, uptake, and storage. Levels of CaV1.2, an L-type calcium channel responsible for calcium influx from the extracellular space into the cytoplasm, and ryanodine receptor 2 and IP3R2, receptors facilitating calcium release from the sarcoplasmic reticulum into the cytoplasm, were significantly higher in the 7-AI-treated groups than in the vehicle group. Levels of SERCA2, which promotes calcium reuptake from the cytoplasm into the sarcoplasmic reticulum, were also elevated. However, no significant difference in the expression levels of sequestrin, a key protein associated with calcium storage, was observed among the groups. Expression levels of phospholamban, which regulates SERCA2 activity and controls the calcium reuptake rate, were lower in the 7-AI-treated groups (Fig. 5 A). Protein expression analysis revealed significantly increased cTnT and ryanodine receptor levels, slightly increased SERCA2 and IP3R2 levels, and no change in phospholamban levels in the 7-AI-treated groups (Fig. 5 B). We examined the nuclear translocation of transcription factors responding to calcium signaling to determine the mechanisms underlying the increased expression levels of calcium-related proteins involved in intracellular calcium transport and CM differentiation. To confirm the activation of specific transcription factors during CM differentiation, nuclear and cytoplasmic proteins were separated and analyzed. cTnT transcription factors, including Nkx2.5[ 18 ] and GATA4, rely on CREB phosphorylation in response to calcium signaling.[ 19 ] Therefore, we evaluated whether 7-AI treatment induces CREB phosphorylation. Compared to the vehicle, 7-AI increased the nuclear translocation of phosphorylated CREB, even at a low concentration of 5 nM. However, total amount of CREB was unaffected by 7-AI treatment, with most CREB remaining in the cytoplasm. In contrast, NFATc1, another calcium-responsive transcription factor, did not show any increase in nuclear translocation following 7-AI treatment. Relative amounts of nuclear and cytoplasmic proteins were quantified using the nuclear housekeeping protein, lamin A/C, and cytoplasmic housekeeping protein, β-actin, respectively (Fig. 5 C). To further assess the effect of increased nuclear translocation of phosphorylated CREB on CM differentiation of hiPSCs, we used a short hairpin RNA (shRNA) to inhibit CREB expression in the early differentiation stage and subsequently differentiated the cells with or without 7-AI treatment. Flow cytometric analysis was used to count the cTnT-positive CMs and evaluate their differentiation efficiency. In the control group (without CREB knockdown), 7-AI treatment significantly promoted CM differentiation. However, 7-AI treatment did not enhance CM differentiation in the cells treated with the shRNA, which inhibited CREB expression. Notably, vehicle cells showed no significant reduction in differentiation efficiency after CREB knockdown (Fig. 5 D). Correlation between intranuclear calcium transport and cardiomyocyte differentiation and maturation We further investigated the effect of 7-AI treatment on calcium transport to clarify the mechanisms underlying calcium redistribution during the CM differentiation of hiPSCs. In the early differentiation stage, hiPSCs were treated with CHIR99021, a glycogen synthase kinase-3β inhibitor promoting mesoderm progenitor cell differentiation, and calcium movement was monitored using FLUO-4, a calcium-sensitive fluorescent dye. CHIR99021 treatment rapidly increased the cytoplasmic calcium levels. In the 7-AI-treated group, cytoplasmic calcium was transported into the nucleus, resulting in a three-fold increase in nuclear calcium levels compared to those in the vehicle group (Fig. 6 A). This increase in nuclear calcium levels was unexpectedly large, prompting us to investigate the underlying mechanisms. Although 7-AI inhibits mPTP, this effect alone cannot explain the excessive increase in nuclear calcium levels. An earlier version of the 7-AI compound acted as an MCU inhibitor.[ 20 ] Therefore, we hypothesized that MCU inhibition by 7-AI blocks mitochondrial calcium uptake, leading to the accumulation of excess cytoplasmic calcium, which translocates to the nucleus, considerably increasing the nuclear calcium levels. To verify this hypothesis, we used genetically encoded calcium indicators (GECOs), fluorescent indicators changing their fluorescence intensity based on the intracellular calcium levels, to monitor calcium transport over time.[ 21 , 22 ] These indicators were targeted to specific cellular organelles. Using vectors obtained from Addgene, we monitored the real-time calcium fluctuations in specific subcellular compartments (Fig. 6 A). Differentiated CMs were transfected with the CMV-NLS-R-GECO and CMV-mito-GEM-GECO1 vectors to measure the nuclear and mitochondrial calcium levels, respectively. CMs transfected with these GECO vectors were pretreated with thapsigargin, a SERCA inhibitor blocking calcium reuptake by ER. Next, we added tert-butylhydroquinone (tBH) to rapidly increase the intracellular calcium levels and monitored the calcium dynamics in the presence and absence of 7-AI. Upon tBH treatment, vehicle cells showed calcium influx into both the nucleus and mitochondria, followed by a gradual decrease in calcium levels. In contrast, 7-AI-treated cells exhibited minimal calcium uptake by the mitochondria, and calcium remained concentrated in the nucleus at significantly high levels (Fig. 6 B). Consistently, quantitative analysis showed that nuclear calcium levels and time to peak intensity were higher in the 7-AI-treated group than in the vehicle group (Fig. 6 C). These results suggest that 7-AI inhibits mitochondrial calcium uptake, leading to the accumulation of excess cytoplasmic calcium, which translocates to the nucleus, causing nuclear calcium overload. To validate the hypothesis that MCU inhibition enhances CM differentiation by blocking mitochondrial calcium uptake and promoting nuclear calcium accumulation, we tested whether the effect of 7-AI is replicated by the MCU inhibitor, ruthenium red (RuR). hiPSCs were treated with RuR or 7-AI, followed by the induction of CM differentiation. Flow cytometric analysis of cTnT-positive cells revealed that both the RuR-treated and 7-AI-treated groups showed similar increases in the proportions of cTnT-positive cells, which were significantly higher than that in the vehicle group. These findings suggest that inhibiting mitochondrial calcium uptake increases nuclear calcium accumulation, thereby promoting the differentiation and maturation of hiPSC-CMs. Discussion This study provides novel insights into the mechanisms by which calcium signaling modulates cardiomyocyte (CM) differentiation and maturation. Generally, 7-aminoindole (7-AI) was known to promote muscle cell maturation by inhibiting mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) accumulation. Here, we found that 7-AI enhances CM differentiation via an alternative pathway based on intracellular calcium redistribution. We demonstrated that 7-AI treatment significantly enhanced the differentiation efficiency and functional maturation of both mESC-CMs and hiPSC-CMs. Specifically, 7-AI treatment increased the contractile area, beating rate, and proportion of cardiac troponin T (cTnT)-expressing cells, as revealed by flow cytometry. Additionally, 7-AI-treated groups exhibited morphological changes, including increased cell size and sarcomere length, as well as upregulation of CM-specific markers and ion channel levels. Further investigation into the underlying mechanisms revealed that 7-AI primarily inhibited MCU, which facilitates cytosolic calcium uptake by mitochondria. This inhibition prevents excessive mitochondrial calcium accumulation and promotes calcium redistribution to the nucleus. Consequently, nuclear calcium activates the transcription factor cAMP response element-binding protein (CREB), which in turn promotes the expression of key genes involved in CM differentiation and maturation. Similar effects were observed with RuR, another MCU inhibitor, supporting our hypothesis. Our findings underscore the importance of calcium dynamics in the CM development. While calcium is essential for normal CM function, its dysregulation can lead to various pathological conditions, such as hypertrophy and fibrosis. Interestingly, our study demonstrates that controlled nuclear calcium influx promotes CM differentiation and maturation in progenitor cells without inducing pathological remodeling. Our findings provide valuable insights for improving the cardiac differentiation protocols. By targeting the MCU and modulating intracellular calcium distribution during the critical cardiac lineage commitment phase, 7-AI enhanced the differentiation efficiency and functional maturation of CMs. These effects should be considered when developing and optimizing functional CM development protocols for applications in regenerative medicine and disease modeling. Nevertheless, a notable limitation of our study was the lack of in vivo validation experiments. Future studies are essential to confirm the therapeutic potential and safety profile of 7-AI in animal models, thereby bridging the gap between our in vitro findings and the clinical applications in myocardial regeneration. Future studies should optimize the dosage and timing of 7-AI treatment to maximize its beneficial effects and delineate its impact on other calcium-dependent transcription factors such as NFATc1. Moreover, investigating the effects of 7-AI on the differentiation of other cell types to expand its application as a versatile regulator of calcium dynamics in developmental biology. Conclusion In summary, our study revealed new roles of 7-AI in modulating intracellular calcium distribution to significantly enhance the differentiation efficiency and functional maturation of CMs, highlighting a new avenue for refining cardiac differentiation protocols and advancing myocardial regeneration strategies. Importantly, this study introduces 7-AI as a promising small molecule modulator of nuclear calcium signaling, offering a novel strategy to fine-tune cardiomyocyte development. Given the simplicity of chemical modulation, these findings have great translational potential for scalable stem cell-based cardiac therapy applications. Declarations RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hyun-Jai Cho( [email protected] ) Availability of data and materials The datasets used during the current study are available from the corresponding authors on reasonable request. Ethics approval and consent to participate The human induced pluripotent stem cells (hiPSCs) used in this study were generated from Nuff (newborn foreskin fibroblast) cells (cat. no. AMS.GSC-3006G, AMS Biotechnology), a commercially available cell line. According to the supplier, the Nuff cells were obtained with informed consent from donors and in compliance with ethical guidelines. The reprogramming factors OCT4, SOX2, KLF4, and cMYC were introduced using lentiviruses(Takahashi et al.,2007). No additional ethics approval was required for this study, as no new human samples were collected. ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF)& funded by the Korean government (MSIT) (No. RS-2022-NR067329). National Research Foundation of Korea (NRF) grant (2019R1F1A1063542) funded by the Korea Government (MSIT). The authors declare that they have not used AI-generated work in this manuscript. AUTHOR CONTRIBUTIONS Conceptualization, JY Kim, HM Yang; Experiments, JY Kim, HJ Seo, JE Lee, SB Bang, MK Jeon ; visualization, JY Kim, HJ Seo; AP study, HA Lee; supervision, HJ Cho. .; writing – original draft, JY Kim, HJ Seo; writing – review & editing, JY Kim, YJ Shin DECLARATION OF INTERESTS The authors declare no competing interests. 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Hom JR, Quintanilla RA, Hoffman DL, de Mesy Bentley KL, Molkentin JD, Sheu SS, Porter GA, Jr.: The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation. Dev Cell 2011, 21(3):469-478. Cho SW, Park JS, Heo HJ, Park SW, Song S, Kim I, Han YM, Yamashita JK, Youm JB, Han J et al : Dual modulation of the mitochondrial permeability transition pore and redox signaling synergistically promotes cardiomyocyte differentiation from pluripotent stem cells. J Am Heart Assoc 2014, 3(2):e000693. Park J, Park E, Ahn BH, Kim HJ, Park JH, Koo SY, Kwak HS, Park HS, Kim DW, Song M et al : NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats. Toxicol Appl Pharmacol 2012, 263(1):1-6. Kim HJ, Koo SY, Ahn BH, Park O, Park DH, Seo DO, Won JH, Yim HJ, Kwak HS, Park HS et al : NecroX as a novel class of mitochondrial reactive oxygen species and ONOO(-) scavenger. Arch Pharm Res 2010, 33(11):1813-1823. Li B, Dedman JR, Kaetzel MA: Nuclear Ca2+/calmodulin-dependent protein kinase II in the murine heart. Biochim Biophys Acta 2006, 1763(11):1275-1281. Sheng M, Greenberg ME: The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990, 4(4):477-485. Ghosh A, Greenberg ME: Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 1995, 268(5208):239-247. Puceat M, Jaconi M: Ca2+ signalling in cardiogenesis. Cell Calcium 2005, 38(3-4):383-389. Knollmann BC, Roden DM: A genetic framework for improving arrhythmia therapy. Nature 2008, 451(7181):929-936. Hwang IC, Kim JY, Kim JH, Lee JE, Seo JY, Lee JW, Park J, Yang HM, Kim SH, Cho HJ et al : Therapeutic Potential of a Novel Necrosis Inhibitor, 7-Amino-Indole, in Myocardial Ischemia-Reperfusion Injury. Hypertension 2018, 71(6):1143-1155. Kon N, Murakoshi M, Isobe A, Kagechika K, Miyoshi N, Nagayama T: DS16570511 is a small-molecule inhibitor of the mitochondrial calcium uniporter. Cell Death Discov 2017, 3:17045. Kamer KJ, Mootha VK: The molecular era of the mitochondrial calcium uniporter. Nat Rev Mol Cell Biol 2015, 16(9):545-553. Li B, Kaetzel MA, Dedman JR: Signaling pathways regulating murine cardiac CREB phosphorylation. Biochem Biophys Res Commun 2006, 350(1):179-184. Blayney LM, Lai FA: Ryanodine receptor-mediated arrhythmias and sudden cardiac death. Pharmacol Ther 2009, 123(2):151-177. Keren-Politansky A, Keren A, Bengal E: Neural ectoderm-secreted FGF initiates the expression of Nkx2.5 in cardiac progenitors via a p38 MAPK/CREB pathway. Dev Biol 2009, 335(2):374-384. Ma H, Groth RD, Cohen SM, Emery JF, Li B, Hoedt E, Zhang G, Neubert TA, Tsien RW: gammaCaMKII shuttles Ca(2)(+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression. Cell 2014, 159(2):281-294. Thu VT, Kim HK, Long le T, Lee SR, Hanh TM, Ko TH, Heo HJ, Kim N, Kim SH, Ko KS et al : NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter. Cardiovasc Res 2012, 94(2):342-350. Wu J, Prole DL, Shen Y, Lin Z, Gnanasekaran A, Liu Y, Chen L, Zhou H, Chen SR, Usachev YM et al : Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum. Biochem J 2014, 464(1):13-22. Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T et al : An expanded palette of genetically encoded Ca(2)(+) indicators. Science 2011, 333(6051):1888-1891. Supplementary Files 20250409NecroXSupplementaryfigureSRT.pdf hiPSC7A15nM.mp4 hiPSC7AI100nM.mp4 hiPSCVehicle.mp4 mESC7AI100nM.mp4 mESC7AI5nM.mp4 mESCVehicle.mp4 Cite Share Download PDF Status: Posted 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-6392558","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456125669,"identity":"30b4e275-fbe9-44f3-96db-cac5bf9ea4a7","order_by":0,"name":"HyunJu Seo","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"HyunJu","middleName":"","lastName":"Seo","suffix":""},{"id":456125670,"identity":"3166176d-69b0-4dd3-ab63-22e6fed5c3b1","order_by":1,"name":"Ju-young Kim","email":"","orcid":"","institution":"Seoul National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ju-young","middleName":"","lastName":"Kim","suffix":""},{"id":456125671,"identity":"451b4295-4d5d-4ed1-b450-ddd20bef36bf","order_by":2,"name":"Hyun-Jai Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACAxAhAYQGDMwHoGJsRGthSyBBC4TBA2MT0GIukWP2wKLGQt6cvefz58I2Bnn+Bra0D/i0WM7IMTeQOCZhuLPn7DbpmW0MhjMOsB2egddhN3LMJCTYJBIMbuRuY+ZtY2DcwMDejN8vYC3/QFpyHn8GarEnTotkG1gLgzRQS+IGBrbD+LWceVYmIdknYbjhzDEzaZ5zEskzDrMl49dyPHmbtMS3OnmD482PP/OU2dj2t7cZ49XCIJDAwCyB4AKZzPg1MDDwH2BgxBsNo2AUjIJRMAoAEF9Abfcam0AAAAAASUVORK5CYII=","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hyun-Jai","middleName":"","lastName":"Cho","suffix":""},{"id":456125672,"identity":"654a5b91-963c-4062-ba47-c1983cbb6000","order_by":3,"name":"Joo-Eun Lee","email":"","orcid":"","institution":"Seoul National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Joo-Eun","middleName":"","lastName":"Lee","suffix":""},{"id":456125673,"identity":"6a99a533-f261-4f3f-9466-42540af810a5","order_by":4,"name":"Sang-Beom Bang","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Sang-Beom","middleName":"","lastName":"Bang","suffix":""},{"id":456125674,"identity":"2143be32-7321-4ee2-b32d-b194ac0dcdd4","order_by":5,"name":"Mika Jeon","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Mika","middleName":"","lastName":"Jeon","suffix":""},{"id":456125675,"identity":"fb3e7570-2a21-4f0b-a618-4f434d78c76c","order_by":6,"name":"Hyang-Ae Lee","email":"","orcid":"","institution":"Daejeon Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hyang-Ae","middleName":"","lastName":"Lee","suffix":""},{"id":456125676,"identity":"6de4e45f-7a75-4fb6-b360-ee8ccfcf4792","order_by":7,"name":"Yoo-Jeong Shin","email":"","orcid":"","institution":"The University of Sydney Faculty of Medicine and Health","correspondingAuthor":false,"prefix":"","firstName":"Yoo-Jeong","middleName":"","lastName":"Shin","suffix":""},{"id":456125677,"identity":"fc3f5599-7908-4dc2-a9d1-bcede5f11e1f","order_by":8,"name":"Han-Mo Yang","email":"","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Han-Mo","middleName":"","lastName":"Yang","suffix":""},{"id":456125678,"identity":"dc9abe36-fc48-43a4-9de4-7cc36897d77a","order_by":9,"name":"Hyo-Soo Kim","email":"","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hyo-Soo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-04-07 09:33:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6392558/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6392558/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82792125,"identity":"19dbdebb-8963-40b1-a978-f8f9f6f177bd","added_by":"auto","created_at":"2025-05-15 10:18:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":928992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e7-aminoindole(7-AI) treatment enhances the mES cardiac-derived differentiation efficiency.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-AI treatment increased the beating area and duration of mESC-derived cardiomyocytes. (B) Flow cytometry analysis showing an increase in cardiac Troponin T-expressing cells in mESC-derived cardiomyocytes through 7-AI treatment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/638a1a73e858dddf0ca6b4db.png"},{"id":82793820,"identity":"9a204037-5bb2-4112-b6b3-ecada85b7ef3","added_by":"auto","created_at":"2025-05-15 10:26:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4487887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e7-aminoindole (7-AI) plays a role in increasing the maturity of cardiomyocytes during mESC-derived differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-AI treatment enhanced cardiomyocyte size by increasing the length of α-sarcomeric actinin (α-SA). (B) Real-time PCR analysis demonstrated increased expression of cardiomyocyte markers and ion channels associated with mature cardiomyocytes upon 7-AI treatment. (C) Calcium kinetics of mature cardiomyocytes, assessed using FLUO-4, revealed enhanced calcium-handling properties in 7-AI-treated cardiomyocytes.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/30583ee6febc32c42cea853b.png"},{"id":82792127,"identity":"8e2cb6e5-7451-4832-a675-83918070b8e3","added_by":"auto","created_at":"2025-05-15 10:18:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":829713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e7-aminoindole(7-AI) treatment enhances the hiPSC cardiac-derived differentiation efficiency.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-AI treatment increased the beating area and contraction time of hiPSC-derived cardiomyocytes. (B) Flow cytometry analysis showing an increase in cTnT-expressing cells in iPSC-derived cardiomyocytes following 7-AI treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/589bc5c23c5410ef0ac8c8a5.png"},{"id":82792133,"identity":"267b0fb4-dbf1-40e6-8b3f-618c048a795f","added_by":"auto","created_at":"2025-05-15 10:18:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7382935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e7-aminoindole (7-AI) plays a role in increasing the maturity of cardiomyocytes during hiPSC-derived cardiomyocyte differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-AI treatment increases cardiomyocyte size by elongating α-SA. (B) Real-time PCR analysis showing the upregulation of cardiomyocyte markers and ion channels associated with mature cardiomyocytes in response to 7-AI treatment. (C) FLUO-4-based calcium imaging indicates enhanced calcium kinetics in 7-AI-treated cardiomyocytes. (D) Effects of 7-AI on action potential subtypes in hiPSC-CMs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/f0cdc7a250f8733c0d4dad75.png"},{"id":82793823,"identity":"1a9d39cb-e953-47d0-9d79-28762966369f","added_by":"auto","created_at":"2025-05-15 10:26:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4210495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased expression of genes and proteins related to cardiomyocyte differentiation and maturation through 7-AI treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-AI treatment increases the expression of genes involved in calcium handling during cardiomyocyte differentiation. (B) Western blot analysis revealed an increased expression of calcium-handling proteins following 7-AI treatment. Full-length blots are presented in Supplementary Figure 1A. (C) Immunofluorescence imaging showing the intranuclear migration of CREB, a transcription factor associated with calcium channel regulation, in 7-AI-treated cardiomyocytes. Full-length blots are presented in Supplementary Figure 1B. (D) Knockdown of CREB using shCREB inhibited cardiomyocyte differentiation despite 7-AI treatment, indicating the role of CREB in the 7-AI-mediated differentiation process.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/30396825cf92d8ce7dea9b40.png"},{"id":82792131,"identity":"13f72819-d4c6-4f7d-96fe-530103b9962d","added_by":"auto","created_at":"2025-05-15 10:18:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8326566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between increased intranuclear calcium transport and cardiomyocyte differentiation and maturity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Treatment with 7-AI caused an increase in intracellular calcium levels by small molecules during cardiomyocyte differentiation. (B) The movement of the calcium during 7-AI treatment was tracked using a vector that binds to calcium present in the mitochondria and nucleus. In the 7-AI treatment group, mitochondrial calcium migration was inhibited and intranuclear calcium was increased. (C) Treatment with 7-AI enhances intranuclear calcium transport during cardiomyocyte differentiation. (D) Inhibition of calcium influx into the mitochondria by treatment with the MCU blocker Ruthenium Red(RuR) showed almost the same cardiomyocyte differentiation efficiency as 7-AI treatment.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/9e741dcef6944f9992e427b5.png"},{"id":82793821,"identity":"896a1ff2-9bc9-429b-b36d-41f7ddb6a443","added_by":"auto","created_at":"2025-05-15 10:26:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":584677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic figure. Effect of the increase of intranuclear calcium by MCU inhibition on the differentiation and maturation of iPSC-derived cardiomyocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic illustration showing how 7-AI–mediated inhibition of the mitochondrial Ca2+ uniporter (MCU) increases intranuclear Ca2+, thereby promoting the differentiation and maturation of iPSC-derived cardiomyocytes. Reduced mitochondrial Ca2+ uptake elevates cytosolic/nuclear Ca2+ levels and activates CaMK/CREB signaling, which upregulates calcium-handling proteins (e.g., RyR, SERCA). This process enhances the transition from cardiac progenitors to immature cardiomyocytes and eventually mature cardiomyocytes. ER/SR, endoplasmic/sarcoplasmic reticulum; IP3R, inositol 1,4,5-trisphosphate receptor; MCU, mitochondrial Ca2+ uniporter; CaMK, Ca2+/calmodulin-dependent protein kinase; CREB, cAMP response element-binding protein.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/1020148dad7c662e7013f191.png"},{"id":93671096,"identity":"b11d216a-14d0-44cd-9781-16ea2f7123ec","added_by":"auto","created_at":"2025-10-16 09:58:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28054598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/e5af8e96-042d-451d-95fd-d7be5f3e91ca.pdf"},{"id":82793825,"identity":"be3165c6-3eff-4964-b77f-eb0549c7ea11","added_by":"auto","created_at":"2025-05-15 10:26:50","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":509940,"visible":true,"origin":"","legend":"","description":"","filename":"20250409NecroXSupplementaryfigureSRT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/399eb3d15abc630060a68a91.pdf"},{"id":82792143,"identity":"4a6abb17-e0e7-4f45-ba62-d4b69608d9c7","added_by":"auto","created_at":"2025-05-15 10:18:50","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24070919,"visible":true,"origin":"","legend":"","description":"","filename":"hiPSC7A15nM.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/0893374a2eaf8fab387d4ce6.mp4"},{"id":82792139,"identity":"da317aac-a890-4b89-b7e7-2f2bcc6f1e60","added_by":"auto","created_at":"2025-05-15 10:18:50","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":24327770,"visible":true,"origin":"","legend":"","description":"","filename":"hiPSC7AI100nM.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/f3edc9441cd9a5fdf74eda5e.mp4"},{"id":82792141,"identity":"810ccbbb-b247-46fa-9fb0-3b704dcc35da","added_by":"auto","created_at":"2025-05-15 10:18:50","extension":"mp4","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":24357788,"visible":true,"origin":"","legend":"","description":"","filename":"hiPSCVehicle.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/2c72ce6a9243f7b44007db2c.mp4"},{"id":82793826,"identity":"85c00366-ec02-4028-a0aa-79ddf94a5c83","added_by":"auto","created_at":"2025-05-15 10:26:50","extension":"mp4","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":26264297,"visible":true,"origin":"","legend":"","description":"","filename":"mESC7AI100nM.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/404af5612795cbcb702f5738.mp4"},{"id":82793827,"identity":"38231628-c0bb-42af-9f35-4bd88209221a","added_by":"auto","created_at":"2025-05-15 10:26:50","extension":"mp4","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":26324985,"visible":true,"origin":"","legend":"","description":"","filename":"mESC7AI5nM.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/33fa364fa488f5d050203ed2.mp4"},{"id":82792140,"identity":"4a266c33-6eac-4944-a765-5a375962c7e5","added_by":"auto","created_at":"2025-05-15 10:18:50","extension":"mp4","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":26509416,"visible":true,"origin":"","legend":"","description":"","filename":"mESCVehicle.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6392558/v1/94431692dd5e910ad3bed4a9.mp4"}],"financialInterests":"","formattedTitle":"Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases are the leading cause of mortality worldwide, necessitating the exploration of underlying mechanisms and clinical manifestations and development of innovative therapeutic strategies for these diseases. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are valuable tools providing a robust platform for cardiac research, disease modeling, and drug screening[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, improving the efficiency of hiPSC-CM differentiation is a major challenge. Therefore, in this study, we aimed to enhance the efficiency of hiPSC-CM differentiation using a novel compound to modulate the intracellular calcium dynamics.\u003c/p\u003e \u003cp\u003eClosure of the mitochondrial permeability transition pore (mPTP), which promotes mitochondrial maturation and CM differentiation, reduces the reactive oxygen species (ROS) levels and modulates the calcium dynamics in early embryonic CMs.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] As mPTP closure influences calcium homeostasis, we hypothesized that the initial inhibition of calcium influx is the primary mechanism protecting mitochondria and driving stem cell differentiation, whereas the observed reduction in ROS levels is a downstream effect of altered calcium signaling, as suggested in a previous report.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Therefore, calcium signaling, rather than ROS, is the key regulator of CM maturation.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCa\u0026sup2;⁺/calmodulin-dependent protein kinase (CaMK) integrates intracellular calcium signals to regulate various cellular processes, such as cell differentiation.[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] During hiPSC-CM maturation, CaMK coordinates calcium signaling among the cytosol, endoplasmic/sarcoplasmic reticulum, and mitochondria, influencing various CM-specific functions.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] We previously demonstrated that 7-aminoindole (7-AI), a compound inhibiting mPTP opening and mitochondrial calcium influx, provides cardioprotection by preserving the mitochondrial membrane potential and suppressing ROS production[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Based on these findings, we hypothesized that 7-AI enhances hiPSC-CM differentiation by modulating the mitochondrial calcium uptake and that treatment with 7-AI during the cardiac progenitor cell stage increases the cytosolic and nuclear calcium levels by inhibiting the mitochondrial calcium uniporter (MCU), as suggested in previous reports.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Calcium redistribution possibly activates CaMK and the transcription factor, cAMP response element-binding protein (CREB), which together promote the expression of cardiac-specific genes, including those encoding ryanodine receptors, inositol 1,4,5-trisphosphate receptors, and sarco/endoplasmic reticulum calcium ATPase (SERCA).[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Consistent with the proposed mechanism, our comparative analyses of wild-type and CREB-deficient cells revealed that functional CREB was essential for CM maturation by increasing the CM marker (cardiac troponin T [cTnT], α-actinin, and MYH6/7) levels and supporting proper myofibrillar organization.\u003c/p\u003e \u003cp\u003eThis study showed that targeting calcium dynamics via MCU inhibition during the cardiac progenitor cell stage (day 4) significantly improved the CM differentiation efficiency, providing insights into the fundamental mechanisms governing cardiac differentiation and presenting a novel strategy to enhance cardiac differentiation. Comprehensive understanding of the association between calcium distribution and cardiac differentiation will facilitate the efficient generation of functional CMs for various therapeutic applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eMaterial: Necrosis inhibitor\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eA novel necrosis inhibitor\u0026mdash;a 7-Amino-indole chemical\u0026mdash;was developed by LG Chem at the LG Chem Life Science R\u0026amp;D Campus in Daejeon, Korea (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rnd.lgchem.com/global/main\u003c/span\u003e\u003cspan address=\"http://www.rnd.lgchem.com/global/main\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The study material, referred to as NecroX, is available upon reasonable request. Based on preliminary experiments with various compounds in the NecroX series, we selected NecroX-7 ((tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxothiomorpholin-4-yl) methyl-1H-indol-7-yl]amine; C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e) for further evaluation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003emESC culture and differentiation\u003c/h3\u003e\n\u003cp\u003emESCs (ES-C57BL/6; ATCC number: SCRC-1002; ATCC, Manassas, USA) were cultured using mouse embryonic fibroblasts (CF-1, ATCC number: SCRC-1040) on an mESC medium with the recombinant mouse leukemia inhibitory factor (ESG1107; Merck Millipore, Darmstadt, Germany). Briefly, 2.2 × 10\u003csup\u003e6\u003c/sup\u003e mESCs were incubated in an Aggrewell (#27845/27945; STEMCELL Technologies, Vancouver, Canada) in an embryoid body medium with the recombinant bone morphogenetic protein-4 (5020-BP; R\u0026amp;D Systems, Minneapolis, USA) for one day to form embryoid bodies. ESCs were incubated in a suspension culture for two days in an embryoid body medium with bone morphogenetic protein-4, activin A (recombinant human/mouse/rat activin A 338-AC; R\u0026amp;D Systems), and recombinant human basic fibroblast growth factor (bFGF; 13256029; Thermo Fisher Scientific, MA, USA). On CM differentiation day 3, embryoid bodies were attached to a 6-well plate with CM differentiation medium supplemented with bFGF, recombinant human epidermal growth factor (236-EG; R\u0026amp;D Systems), recombinant human cardiotrophin-1 (612-CD; R\u0026amp;D Systems), and recombinant mouse vascular endothelial growth factor (493-MV; R\u0026amp;D Systems), which was changed every two days. Then, 5 and 100 nM 7-AI was added on CM differentiation days 4–8.\u003c/p\u003e\n\u003ch3\u003ehiPSC culture and differentiation\u003c/h3\u003e\n\u003cp\u003ehiPSCs reprogramed from newborn foreskin fibroblasts (GSC-3006G; AMS Biotechnology [GlobalStem], Abingdon, UK) using the four Yamanaka factors were cultured using STO (SIM; ATCC number: CRL-1503) on the Dulbecco's modified Eagle’s medium/nutrient mixture F12 Glutamax (10565-018; Thermo Fisher Scientific) supplemented with knockout serum replacement, 10 mM non-essential amino acids, 200 mM L-glutamine, 55 mM β-mercaptoethanol, and 10 ng/mL human recombinant bFGF.\u003c/p\u003e \u003cp\u003eCM differentiation of iPSCs was performed as described by Lian et al., with some modifications. Directed CM differentiation from human pluripotent stem cells by modulating Wnt/b-catenin signaling under fully defined conditions; Nature protocols, 2013, 162, VOL.8 NO.1). hiPSC colonies were detached using dispase (17105-041; Thermo Fisher Scientific) and dissociated into single cells. Then, 1.5 × 10\u003csup\u003e6\u003c/sup\u003e hiPSCs were seeded on matrigel (354277; Corning, NY, USA)-coated 35-mm dishes, grown on mTeSR1 (#85851; STEMCELL Technologies) until reaching 100% confluency, and subjected to cardiac differentiation. The following chemicals were sequentially administered: 6 µM CHIR99021 (252917-06-9; Cayman, MI, USA) for the first two days, followed by 10 µM recombinant human/mouse/rat activin A (338-AC; R\u0026amp;D Systems) and 20 µM recombinant human bFGF (13256029; Thermo Fisher Scientific) the next day, and 5 µM IWR1 (I0161; Sigma-Aldrich, St. Louis, USA) three days after that. The medium was replaced with the Roswell Park Memorial Institute-1640 medium (11875-085; Thermo Fisher Scientific) supplemented with B27 supplement (minus insulin) once every two days until the CMs contracted. Subsequently, 7-AI (5 and 100 nM) was administered one day before cell differentiation initiation. MCU inhibitor RuR (5 µM) was also administered at the same time as 7-AI.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003emESC-CM and hiPSC-CM differentiation was assessed via flow cytometry using the cTnT marker. Cultured mESC-CMs were detached from the dish using trypsin and washed with phosphate-buffered saline (PBS) via centrifugation at 1800 rpm for 5 min. hiPSC-CMs were dissociated into single cells using accutase and washed with PBS via centrifugation at 1200 rpm for 5 min. These cells were resuspended in 1 mL of fluorescence-activated cell sorting buffer before staining. After permeabilization with the permeabilization buffer (Gibco) at 4°C for 10 min, the cells were incubated with primary antibodies at room temperature for 1 h, washed with the fluorescence-activated cell sorting buffer, incubated again with the Alexa-488-conjugated secondary antibodies at room temperature for 1 h, and subjected to flow cytometry.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence assay\u003c/h3\u003e\n\u003cp\u003eTo assess the differentiation efficiency of mESC-CMs and hiPSC-CMs after 7-AI treatment, immunostaining was performed using the α-SA Tom20 antibody to visualize the mitochondria. The cells were incubated overnight with primary antibodies at 4°C, followed by incubation with the secondary Alexa-488- and Alexa-555-conjugated antibodies at room temperature for 1 h. Images were acquired using a confocal microscope (Leica). Cell size was calculated using the mean cell area with the ImageJ software, and cell length was measured in pixels and converted to actual length (µm) for quantitative analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eReverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eNext, mRNA expression levels were quantified via real-time RT-PCR using the 7500 real-time PCR system, according to the manufacturer’s protocol. PCR was performed using a 96-well plate with three replicates at a final volume of 20 µL, and expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase RNA levels. mRNA expression analysis was performed according to the TOYOBO protocol. Quantitative real-time PCR for binding analysis was performed using the SYBR Green protocol.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eThree experimental groups (vehicle, 5 nM 7-AI-treated, and 100 nM 7-AI-treated hiPSC CMs) were lysed using the radioimmunoprecipitation assay buffer containing protease/phosphatase inhibitors and centrifuged at 15,000 rpm for 30 min at 4°C. The isolated proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk at room temperature for 30 min, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with the respective secondary antibodies. Immunoblot signals were detected using Amersham 680 and quantified using the ImageJ software.\u003c/p\u003e\n\u003ch3\u003eFLUO-4 assay\u003c/h3\u003e\n\u003cp\u003ehiPSC-CMs cultured in 35-mm dishes were stained with 1 µM FLUO-4 (F14201; Thermo Fisher Scientific, Invitrogen) and 2 mM probenecid for 15 min in a 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After FLUO-4 staining, the medium was replaced with PBS containing 1% fetal bovine serum (FBS), and the cells were incubated at room temperature for 20 min. Subsequently, the medium was switched to Tyrode’s solution containing glucose and calcium to measure the calcium changes. To determine the calcium levels in CMs, 1 mM caffeine was applied and calcium signal changes were observed using the A1 confocal laser microscope (Nikon, Melville, NY) via time-lapse confocal imaging. Images were captured every 2 s for 10 min. Fluorescence intensity data for each cell was acquired using the NIS-Elements C software (Nikon), and ΔF/F was calculated from this data. ΔF/F of calcium signals were graphed, and maximum intensity of calcium signals, signal rise rate, and time to reach 90% of the maximum calcium signal were quantified.\u003c/p\u003e \u003cp\u003eTo assess the intracellular calcium signaling changes during CM differentiation, iPSCs were seeded in a 35-mm µ-Dish (ibidi) and cultured until they reached the desired confluency. After incubating with 1 µM FLUO-4 and 2 mM probenecid in a 5% CO₂ incubator at 37°C for 15 min, the cells were incubated again at room temperature for 15 min. Then, the cells were washed with PBS, and the medium was replaced with PBS containing 1% FBS. To assess the intracellular calcium flux, 6 µM CHIR99021, a compound used in the early stage of CM differentiation, was applied to stimulate calcium movement in cells.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEP study\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSpontaneous action potentials (APs) were recorded from single hiPSC-CMs using current-clamp mode at 37°C. Cells exhibiting stable spontaneous beating activity were selected for analysis. Once AP waveforms stabilized, the average of five consecutive AP traces was analyzed under each test condition. AP recordings were conducted in an extracellular solution containing (mM) 145 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 5 glucose, and 1.8 CaCl2 (pH 7.4). The internal pipette solution consisted of (mM) 120 K-Asp, 20 KCl, 5 NaCl, 2 CaCl2, 10 HEPES, 5 EGTA, and 5 Mg-ATP (pH 7.25). Cells were monitored to ensure stable electrophysiological properties before data acquisition. AP subtypes in hiPSC-CMs were classified based on action potential duration at 90% repolarization (APD90), the time required for the membrane potential to return to 90% of its resting level following depolarization. Nodal-type APs were defined as APD90 \u0026lt; 100 ms, atrial-type APs as 100 ms ≤ APD90 \u0026lt; 250 ms, and ventricular-type APs as APD90 ≥ 250 ms. Further classification between atrial- and nodal-type APs was based on differences in amplitude (TA) and upstroke velocity (dV/dtmax)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eNuclear and cytoplasmic protein extraction\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eVehicle and hiPSC-CM groups treated with 7-AI (5 and 100 nM) from day 1 of differentiation were harvested on day 6. To extract the nuclear and cytoplasmic proteins from differentiated CMs, the cells were washed with PBS, transferred to a 15-mL tube, and centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was aspirated, and the cells were resuspended in 1 mL of cold PBS in a 1.5-mL tube, followed by centrifugation at 3500 rpm for 5 min. The pellet was resuspended in 200 µL (per 100 mm dish) of cold buffer A and vortexed. After allowing the lysate to swell on ice for 20 min, it was passed through a 1-mL syringe fitted with an 18-21-gauge needle 3–4 times, and cell lysis was confirmed via trypan blue staining. The lysate was further centrifuged at 9000 rpm for 15 min, and the resulting supernatant containing the cytosolic proteins was carefully collected. The nuclear pellet was recovered, resuspended in 20 µL of cold buffer B, and incubated on ice for 20 min. The lysate was centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatant containing the nuclear proteins was transferred to a new tube. Western blotting was performed to analyze the proteins, and concentrations of the nuclear/cytosolic proteins were determined using the BCA protein assay kit. Buffer A contained 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl, 10 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol (freshly added), and 0.2 mM phenylmethylsulfonyl fluoride (freshly added). Buffer B contained 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl₂, 25% glycerol, 0.5 mM dithiothreitol (freshly added), and 0.2 mM phenylmethylsulfonyl fluoride (freshly added).\u003c/p\u003e \u003cp\u003eWestern blotting analysis was performed to determine the transcription factor expression levels using the phospho-CREB (Ser133; 87G3) rabbit monoclonal (#9198; Cell Signaling Technology), CREB (48H2) rabbit monoclonal (#9197; Cell Signaling Technology), anti-SERCA2 (#2861; Abcam), anti-NFATc1 (#MA3024; Thermo Fisher), and anti-lamin A/C (#2032S; Cell Signaling technology) antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eLentiviral transduction of hiPSC-CMs\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eLentiviral transduction was performed on day 4 of hiPSC-CM differentiation. Lentivirus, prepared according to the manufacturer’s protocol, and polybrene (1 µg/mL) were used for transduction over 2–3 d. TRC \u003cem\u003eCREB1\u003c/em\u003e shRNA vector was constructed by Horizon (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTransfection of the calcium indicator vector into hiPSC-CMs\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eCalcium indicator vectors CMV-NLS-R-GECO and CMV-mito-GEM-GECO1 were kindly gifted by R. E. Campbell (plasmids #32462 and #32461; Addgene).\u003csup\u003e13\u003c/sup\u003e On day 12 of hiPSC-CM differentiation, the cells were washed with PBS, and the medium was replaced with the Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific). Plasmid transfection was performed using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer’s recommended protocol. Plasmid DNA (5 µg) was diluted in 125 µL Opti-MEM and gently mixed. In a separate tube, 12.5 µL of Lipofectamine 2000 Reagent was added to 125 µL Opti-MEM, mixed gently, and incubated at room temperature for 5 min. Then, the plasmid DNA dilution was added to the Lipofectamine 2000 dilution, mixed gently, and incubated at room temperature for 20 min to allow the formation of the plasmid DNA–Lipofectamine complex. After 20 min, the complex was applied dropwise to the cells, gently mixed by rocking the dish, and incubated in at 37°C. After 48 h, the cells were first incubated with 1 µM FLUO-4 and 2 mM probenecid in a 5% CO₂ incubator at 37°C for 15 min and then at room temperature for 15 min. After washing with PBS, the medium was replaced with PBS containing 1% FBS. The cells were treated with 2 µM thapsigargin and 100 nM 7-AI or PBS as a vehicle, and calcium signaling was examined. The cells were treated with 50 µM tBH (Sigma-Aldrich) for strong calcium stimulation. Finally, calcium signaling and fluorescence intensity analyses were conducted as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eStatistical analyses\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eData are represented as the mean ± standard error of the mean. Statistical analyses were conducted via one-way analysis of variance using the GraphPad Prism 5 software. Statistical significance was set at P \u0026lt; 0.05.\u003c/p\u003e \u003c/div\u003e\n\n"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e7-AI enhances cardiac differentiation efficiency in mouse embryonic stem cells\u003c/h2\u003e \u003cp\u003eWe investigated the effects of 7-AI on CM differentiation using mESCs, which are relatively easy to differentiate. mESCs were treated with 7-AI (5 and 100 nM) during CM differentiation, and differentiation efficiency was compared with that of the vehicle group. Notably, 7-AI-treated groups exhibited significantly higher beating rates and more than twice the contractile area than the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, on day 10 post-differentiation, CM-specific marker cTnT levels were significantly elevated in the 7-AI-treated groups, indicating that 7-AI increased the CM differentiation efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e7-AI promotes the maturation of mESC-derived cardiomyocytes\u003c/h2\u003e \u003cp\u003eNext, genetic and functional maturation of differentiated CMs treated with 7-AI was evaluated using α-sarcomeric actin (α-SA), a key sarcomeric structural protein. α-SA is specifically expressed in striated muscle tissues, such as CMs, making it a valuable marker to assess the functional and structural maturation of CMs. Immunofluorescence staining for α-SA, followed by length measurement revealed that the average length of α-SA was significantly longer in the 7-AI-treated groups than in the control group. Furthermore, CM size increased in a concentration-dependent manner in the 7-AI-treated groups relative to that in the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression levels of ion channel genes playing key roles in the electrical and mechanical functions of mature CMs were also examined. Levels of ion channel genes \u003cem\u003eKV4.3\u003c/em\u003e, \u003cem\u003eNCX1\u003c/em\u003e, and \u003cem\u003eNaV1.5\u003c/em\u003e were higher in the 7-AI-treated groups than in the vehicle group, although no significant differences were observed in the expression levels of \u003cem\u003emERG\u003c/em\u003e, \u003cem\u003eKV2.2\u003c/em\u003e, and \u003cem\u003eCaV1.2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eCalcium kinetics analysis using Fluo-4 revealed that differentiated CMs treated with 100 nM 7-AI exhibited more than double the amplitude (ΔF/F) of the vehicle CMs during beating. Additionally, the treated group exhibited Vmax upstroke (ΔF/F/sec) more than three-fold higher than that of the vehicle group. Therefore, 7-AI-treated CMs exhibited high intracellular calcium fluctuations and rapid calcium release and uptake, showing enhanced contraction speed and efficiency. Furthermore, time to 90% peak(s) in the 7-AI-treated CM group was reduced by more than three-fold compared to that in the control group, indicating efficient calcium release in the 7-AI-treated CM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eCollectively, these results suggest that 7-AI treatment during CM differentiation promotes the activation of ion channels regulating calcium release and uptake in calcium storage compartments, thereby supporting CM differentiation into functionally mature cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e7-AI enhances cardiac differentiation efficiency in human induced pluripotent stem cells\u003c/h2\u003e \u003cp\u003eNext, we examined the effects of 7-AI on CM differentiation using hiPSC-CMs. During differentiation, human fibroblast-derived iPSCs were treated with 7-AI (5 and 100 nM). Notably, 7-AI-treated groups exhibited significantly stronger contractions and larger beating areas than the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, cTnT levels were elevated in the 7-AI-treated groups, indicating that 7-AI increased the CM differentiation efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e7-AI promotes the maturation of hiPSC-derived cardiomyocytes\u003c/h2\u003e \u003cp\u003eStructural maturity of hiPSC-CMs was examined via α-SA staining. Total cell size was significantly larger and sarcomere length was significantly longer in the 7-AI-treated group than in the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To evaluate genetic maturation, expression levels of the CM-specific markers, such as \u003cem\u003ecTnT\u003c/em\u003e and \u003cem\u003eMHC6\u003c/em\u003e, and genes encoding ion channels characteristic of mature CMs were analyzed. Upon treatment with 7-AI (5, 100, and 500 nM), highest increase in \u003cem\u003ecTnT\u003c/em\u003e and \u003cem\u003eMHC6\u003c/em\u003e levels was observed in the 100 nM 7-AI-treated group. Analysis of ion channel gene expression revealed that the 7-AI-treated hiPSC-CMs exhibited significantly higher levels of \u003cem\u003eKv4.3\u003c/em\u003e and \u003cem\u003eNCX1\u003c/em\u003e (similar to mESC-CMs) as well as \u003cem\u003ehERG\u003c/em\u003e, \u003cem\u003eKv2.2\u003c/em\u003e, and \u003cem\u003eCaV1.2\u003c/em\u003e (different from mESC-CMs) than the vehicle CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the calcium kinetics analysis of hiPSC-CMs, 100 nM 7-AI-treated group exhibited a higher amplitude (ΔF/F) and Vmax upstroke (ΔF/F/sec) than the vehicle group, indicating enhanced contraction speed and calcium handling efficiency. Furthermore, time to 90% peak(s) was reduced in the 7-AI-treated group, indicating rapid calcium release. Overall, 7-AI-treated groups exhibited more stable and efficient excitation–contraction coupling than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Electrophysiological (EP) studies were also conducted to classify the hiPSC-CM cell types AP subtypes in hiPSC-CMs were classified as nodal-type (APD90 \u0026lt; 100 ms), atrial-type (100 ms ≤ APD90 \u0026lt; 250 ms), and ventricular-type (APD90 ≥ 250 ms) based on AP duration. Pie charts show the distribution of AP subtypes in vehicle- and 7-AI-treated cells. Representative AP traces illustrate differences in AP morphology among the subtypes. 7-AI treatment increased the proportion of ventricular-type APs while reducing atrial-type populations, suggesting an influence on cardiomyocyte electrophysiological maturation. APD90, action potential duration at 90% repolarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e7-AI treatment upregulates cardiomyocyte-specific genes and proteins\u003c/h2\u003e \u003cp\u003eMature CMs efficiently regulate the calcium dynamics. Therefore, we analyzed the expression levels of genes and proteins involved in calcium release, uptake, and storage. Levels of CaV1.2, an L-type calcium channel responsible for calcium influx from the extracellular space into the cytoplasm, and ryanodine receptor 2 and IP3R2, receptors facilitating calcium release from the sarcoplasmic reticulum into the cytoplasm, were significantly higher in the 7-AI-treated groups than in the vehicle group. Levels of SERCA2, which promotes calcium reuptake from the cytoplasm into the sarcoplasmic reticulum, were also elevated. However, no significant difference in the expression levels of sequestrin, a key protein associated with calcium storage, was observed among the groups. Expression levels of phospholamban, which regulates SERCA2 activity and controls the calcium reuptake rate, were lower in the 7-AI-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Protein expression analysis revealed significantly increased cTnT and ryanodine receptor levels, slightly increased SERCA2 and IP3R2 levels, and no change in phospholamban levels in the 7-AI-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe examined the nuclear translocation of transcription factors responding to calcium signaling to determine the mechanisms underlying the increased expression levels of calcium-related proteins involved in intracellular calcium transport and CM differentiation. To confirm the activation of specific transcription factors during CM differentiation, nuclear and cytoplasmic proteins were separated and analyzed. cTnT transcription factors, including Nkx2.5[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and GATA4, rely on CREB phosphorylation in response to calcium signaling.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Therefore, we evaluated whether 7-AI treatment induces CREB phosphorylation. Compared to the vehicle, 7-AI increased the nuclear translocation of phosphorylated CREB, even at a low concentration of 5 nM. However, total amount of CREB was unaffected by 7-AI treatment, with most CREB remaining in the cytoplasm. In contrast, NFATc1, another calcium-responsive transcription factor, did not show any increase in nuclear translocation following 7-AI treatment. Relative amounts of nuclear and cytoplasmic proteins were quantified using the nuclear housekeeping protein, lamin A/C, and cytoplasmic housekeeping protein, β-actin, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo further assess the effect of increased nuclear translocation of phosphorylated CREB on CM differentiation of hiPSCs, we used a short hairpin RNA (shRNA) to inhibit CREB expression in the early differentiation stage and subsequently differentiated the cells with or without 7-AI treatment. Flow cytometric analysis was used to count the cTnT-positive CMs and evaluate their differentiation efficiency. In the control group (without CREB knockdown), 7-AI treatment significantly promoted CM differentiation. However, 7-AI treatment did not enhance CM differentiation in the cells treated with the shRNA, which inhibited CREB expression. Notably, vehicle cells showed no significant reduction in differentiation efficiency after CREB knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between intranuclear calcium transport and cardiomyocyte differentiation and maturation\u003c/h2\u003e \u003cp\u003eWe further investigated the effect of 7-AI treatment on calcium transport to clarify the mechanisms underlying calcium redistribution during the CM differentiation of hiPSCs. In the early differentiation stage, hiPSCs were treated with CHIR99021, a glycogen synthase kinase-3β inhibitor promoting mesoderm progenitor cell differentiation, and calcium movement was monitored using FLUO-4, a calcium-sensitive fluorescent dye. CHIR99021 treatment rapidly increased the cytoplasmic calcium levels. In the 7-AI-treated group, cytoplasmic calcium was transported into the nucleus, resulting in a three-fold increase in nuclear calcium levels compared to those in the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This increase in nuclear calcium levels was unexpectedly large, prompting us to investigate the underlying mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough 7-AI inhibits mPTP, this effect alone cannot explain the excessive increase in nuclear calcium levels. An earlier version of the 7-AI compound acted as an MCU inhibitor.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Therefore, we hypothesized that MCU inhibition by 7-AI blocks mitochondrial calcium uptake, leading to the accumulation of excess cytoplasmic calcium, which translocates to the nucleus, considerably increasing the nuclear calcium levels. To verify this hypothesis, we used genetically encoded calcium indicators (GECOs), fluorescent indicators changing their fluorescence intensity based on the intracellular calcium levels, to monitor calcium transport over time.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] These indicators were targeted to specific cellular organelles. Using vectors obtained from Addgene, we monitored the real-time calcium fluctuations in specific subcellular compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eDifferentiated CMs were transfected with the CMV-NLS-R-GECO and CMV-mito-GEM-GECO1 vectors to measure the nuclear and mitochondrial calcium levels, respectively. CMs transfected with these GECO vectors were pretreated with thapsigargin, a SERCA inhibitor blocking calcium reuptake by ER. Next, we added tert-butylhydroquinone (tBH) to rapidly increase the intracellular calcium levels and monitored the calcium dynamics in the presence and absence of 7-AI.\u003c/p\u003e \u003cp\u003eUpon tBH treatment, vehicle cells showed calcium influx into both the nucleus and mitochondria, followed by a gradual decrease in calcium levels. In contrast, 7-AI-treated cells exhibited minimal calcium uptake by the mitochondria, and calcium remained concentrated in the nucleus at significantly high levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Consistently, quantitative analysis showed that nuclear calcium levels and time to peak intensity were higher in the 7-AI-treated group than in the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These results suggest that 7-AI inhibits mitochondrial calcium uptake, leading to the accumulation of excess cytoplasmic calcium, which translocates to the nucleus, causing nuclear calcium overload.\u003c/p\u003e \u003cp\u003eTo validate the hypothesis that MCU inhibition enhances CM differentiation by blocking mitochondrial calcium uptake and promoting nuclear calcium accumulation, we tested whether the effect of 7-AI is replicated by the MCU inhibitor, ruthenium red (RuR). hiPSCs were treated with RuR or 7-AI, followed by the induction of CM differentiation. Flow cytometric analysis of cTnT-positive cells revealed that both the RuR-treated and 7-AI-treated groups showed similar increases in the proportions of cTnT-positive cells, which were significantly higher than that in the vehicle group. These findings suggest that inhibiting mitochondrial calcium uptake increases nuclear calcium accumulation, thereby promoting the differentiation and maturation of hiPSC-CMs.\u003c/p\u003e \u003c/div\u003e\n\n "},{"header":"Discussion","content":"\u003cp\u003eThis study provides novel insights into the mechanisms by which calcium signaling modulates cardiomyocyte (CM) differentiation and maturation. Generally, 7-aminoindole (7-AI) was known to promote muscle cell maturation by inhibiting mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) accumulation. Here, we found that 7-AI enhances CM differentiation via an alternative pathway based on intracellular calcium redistribution.\u003c/p\u003e\u003cp\u003eWe demonstrated that 7-AI treatment significantly enhanced the differentiation efficiency and functional maturation of both mESC-CMs and hiPSC-CMs. Specifically, 7-AI treatment increased the contractile area, beating rate, and proportion of cardiac troponin T (cTnT)-expressing cells, as revealed by flow cytometry. Additionally, 7-AI-treated groups exhibited morphological changes, including increased cell size and sarcomere length, as well as upregulation of CM-specific markers and ion channel levels.\u003c/p\u003e\u003cp\u003eFurther investigation into the underlying mechanisms revealed that 7-AI primarily inhibited MCU, which facilitates cytosolic calcium uptake by mitochondria. This inhibition prevents excessive mitochondrial calcium accumulation and promotes calcium redistribution to the nucleus. Consequently, nuclear calcium activates the transcription factor cAMP response element-binding protein (CREB), which in turn promotes the expression of key genes involved in CM differentiation and maturation. Similar effects were observed with RuR, another MCU inhibitor, supporting our hypothesis.\u003c/p\u003e\u003cp\u003eOur findings underscore the importance of calcium dynamics in the CM development. While calcium is essential for normal CM function, its dysregulation can lead to various pathological conditions, such as hypertrophy and fibrosis. Interestingly, our study demonstrates that controlled nuclear calcium influx promotes CM differentiation and maturation in progenitor cells without inducing pathological remodeling.\u003c/p\u003e\u003cp\u003eOur findings provide valuable insights for improving the cardiac differentiation protocols. By targeting the MCU and modulating intracellular calcium distribution during the critical cardiac lineage commitment phase, 7-AI enhanced the differentiation efficiency and functional maturation of CMs. These effects should be considered when developing and optimizing functional CM development protocols for applications in regenerative medicine and disease modeling.\u003c/p\u003e\u003cp\u003eNevertheless, a notable limitation of our study was the lack of in vivo validation experiments. Future studies are essential to confirm the therapeutic potential and safety profile of 7-AI in animal models, thereby bridging the gap between our in vitro findings and the clinical applications in myocardial regeneration.\u003c/p\u003e\u003cp\u003eFuture studies should optimize the dosage and timing of 7-AI treatment to maximize its beneficial effects and delineate its impact on other calcium-dependent transcription factors such as NFATc1. Moreover, investigating the effects of 7-AI on the differentiation of other cell types to expand its application as a versatile regulator of calcium dynamics in developmental biology.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study revealed new roles of 7-AI in modulating intracellular calcium distribution to significantly enhance the differentiation efficiency and functional maturation of CMs, highlighting a new avenue for refining cardiac differentiation protocols and advancing myocardial regeneration strategies.\u003c/p\u003e \u003cp\u003eImportantly, this study introduces 7-AI as a promising small molecule modulator of nuclear calcium signaling, offering a novel strategy to fine-tune cardiomyocyte development. Given the simplicity of chemical modulation, these findings have great translational potential for scalable stem cell-based cardiac therapy applications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eRESOURCE AVAILABILITY\u003c/p\u003e\n\u003cp\u003eLead contact\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact,\u0026nbsp;Hyun-Jai Cho([email protected])\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Availability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ethics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe human induced pluripotent stem cells (hiPSCs) used in this study were generated from Nuff (newborn foreskin fibroblast) cells (cat. no. AMS.GSC-3006G, AMS Biotechnology), a commercially available cell line. According to the supplier, the Nuff cells were obtained with informed consent from donors and in compliance with ethical guidelines. The reprogramming factors OCT4, SOX2, KLF4, and cMYC were introduced using lentiviruses(Takahashi et al.,2007). No additional ethics approval was required for this study, as no new human samples were collected.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Bio \u0026amp; Medical Technology Development Program of the National Research Foundation (NRF)\u0026amp; funded by the Korean government (MSIT) (No. RS-2022-NR067329).\u003c/p\u003e\n\u003cp\u003eNational Research Foundation of Korea (NRF) grant (2019R1F1A1063542) funded by the Korea Government (MSIT).\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used AI-generated work in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;AUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eConceptualization, JY Kim, HM Yang; Experiments, JY Kim, HJ Seo, JE Lee, SB Bang, MK Jeon ; visualization, JY Kim, HJ Seo; AP study, HA Lee; supervision, HJ Cho. .; writing\u0026nbsp;\u0026ndash; original draft, JY Kim, HJ Seo; writing\u0026nbsp;\u0026ndash; review\u0026nbsp;\u0026amp; editing, JY Kim, YJ Shin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDECLARATION OF INTERESTS\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFujiwara M, Yan P, Otsuji TG, Narazaki G, Uosaki H, Fukushima H, Kuwahara K, Harada M, Matsuda H, Matsuoka S\u003cem\u003e et al\u003c/em\u003e: Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A. \u003cem\u003ePLoS One \u003c/em\u003e2011, 6(2):e16734.\u003c/li\u003e\n\u003cli\u003eBirket MJ, Casini S, Kosmidis G, Elliott DA, Gerencser AA, Baartscheer A, Schumacher C, Mastroberardino PG, Elefanty AG, Stanley EG\u003cem\u003e et al\u003c/em\u003e: PGC-1alpha and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function. \u003cem\u003eStem Cell Reports \u003c/em\u003e2013, 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Emery JF, Li B, Hoedt E, Zhang G, Neubert TA, Tsien RW: gammaCaMKII shuttles Ca(2)(+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression. \u003cem\u003eCell \u003c/em\u003e2014, 159(2):281-294.\u003c/li\u003e\n\u003cli\u003eThu VT, Kim HK, Long le T, Lee SR, Hanh TM, Ko TH, Heo HJ, Kim N, Kim SH, Ko KS\u003cem\u003e et al\u003c/em\u003e: NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter. \u003cem\u003eCardiovasc Res \u003c/em\u003e2012, 94(2):342-350.\u003c/li\u003e\n\u003cli\u003eWu J, Prole DL, Shen Y, Lin Z, Gnanasekaran A, Liu Y, Chen L, Zhou H, Chen SR, Usachev YM\u003cem\u003e et al\u003c/em\u003e: Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum. \u003cem\u003eBiochem J \u003c/em\u003e2014, 464(1):13-22.\u003c/li\u003e\n\u003cli\u003eZhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T\u003cem\u003e et al\u003c/em\u003e: An expanded palette of genetically encoded Ca(2)(+) indicators. \u003cem\u003eScience \u003c/em\u003e2011, 333(6051):1888-1891.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"7-aminoindole, mitochondrial calcium uniporter (MCU), CaMK–CREB signaling, maturation, human induced pluripotent stem cell derived cardiomyocyte","lastPublishedDoi":"10.21203/rs.3.rs-6392558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6392558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eABSTRCT\u003c/span\u003e \u003c/p\u003e \u003cp\u003eBackground: Cardiovascular diseases remain the leading cause of death worldwide, and the limited efficiency of human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation hampers its potential in disease modeling and regenerative therapy. Calcium signaling plays a central role in cardiac maturation, and proper regulation of intracellular calcium dynamics is essential for activating transcriptional programs that drive cardiomyocyte differentiation. Recent studies have suggested that closure of the mitochondrial permeability transition pore (mPTP) enhances cardiomyocyte differentiation by modulating calcium homeostasis and reducing reactive oxygen species (ROS). Building on this concept, we investigated the effects of 7-aminoindole (7-AI), a novel compound that inhibits mitochondrial calcium influx via the mitochondrial calcium uniporter (MCU), on cardiomyocyte differentiation.\u003c/p\u003e \u003cp\u003eMethods: Using both mouse embryonic stem cells and hiPSCs, we treated cells undergoing differentiation with 7-AI at the cardiac progenitor stage (day 4) to inhibit MCU activity. We assessed differentiation efficiency by measuring nuclear and cytosolic calcium levels, activation of Ca\u0026sup2;⁺/calmodulin-dependent protein kinase (CaMK), and phosphorylation status of the transcription factor cAMP response element-binding protein (CREB). Cardiac-specific gene expression was evaluated by quantifying cardiac cTnT, α-SA, and MYH6/7. Structural and functional maturation of the derived cardiomyocytes was determined using immunostaining and contractility assays.\u003c/p\u003e \u003cp\u003eResults: Treatment with 7-AI significantly increased nuclear calcium levels and activated both CaMK and CREB, leading to the enhanced expression of cardiac-specific genes. Both mouse embryonic and hiPSC-derived cardiomyocytes displayed improved structural organization and contractile properties after 7-AI treatment. Comparative analysis between wild-type and CREB-deficient cells confirmed that CREB is essential for proper cardiomyocyte maturation because CREB deficiency leads to reduced cardiac marker expression and impaired myofibrillar organization.\u003c/p\u003e \u003cp\u003eConclusions: Our study demonstrated that 7-AI enhanced cardiomyocyte differentiation by inhibiting MCU, thereby redistributing calcium from the mitochondria to the nucleus. This redistribution activates CaMK and CREB, which in turn upregulate cardiac-specific gene expression, ultimately promoting the structural and functional maturation of cardiomyocytes. Targeting calcium dynamics during the cardiac progenitor stage represents a novel strategy for improving the efficiency of cardiac differentiation. These findings provide valuable insights into the molecular mechanisms governing cardiac maturation and offer a promising approach to generate functional cardiomyocytes for therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 10:18:45","doi":"10.21203/rs.3.rs-6392558/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e371a61c-03a1-43bb-86ae-6f71447c63f2","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T09:50:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-15 10:18:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6392558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6392558","identity":"rs-6392558","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}