MYBPC3 (c.194CT) mutation-mediated RyR2 dysfunction contributes to pathogenic phenotypes of DCM revealed by hiPSC modeling

MYBPC3 (c.194CT) mutation-mediated RyR2 dysfunction contributes to pathogenic phenotypes of DCM revealed by hiPSC modeling | 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 MYBPC3 (c.194CT) mutation-mediated RyR2 dysfunction contributes to pathogenic phenotypes of DCM revealed by hiPSC modeling Manting Xie, Bingbing Xie, Liang Huang, Ying Chen, Xingqiang Lai, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7455786/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Dilated cardiomyopathy (DCM) is a leading cause of heart failure and the primary indication for heart transplantation. The intricate and poorly elucidated pathogenesis of genetic DCM, coupled with the paucity of effective therapeutic options, imposes a substantial burden on both patients and their families. In this study, we identified a novel MYBPC3 mutation (c.194C > T) in a patient diagnosed with DCM and established a patient-specific human induced pluripotent stem cell (hiPSC) model. Cardiomyocytes derived from these patient-specific hiPSCs (hiPSC-CMs) exhibited hallmark features of DCM, including hypertrophic cell size, aberrant distribution of sarcomeric α-actinin, and dysregulated calcium ion homeostasis, as compared to control hiPSC-CMs derived from a healthy individual. RNA sequencing analysis revealed a significant upregulation of CASQ2 , which encodes calsequestrin, a protein that binds to Ryanodine receptor 2 (RyR2). Notably, treatment with the RyR2 inhibitor ryanodine effectively restored the abnormal calcium transients observed in DCM-hiPSC-CMs. In summary, our findings provide compelling evidence that the c.194C > T mutation of MYBPC3 plays a definitive pathogenic role in DCM, and that modulation of the RyR2 receptor may alleviate calcium dysregulation in affected cardiomyocytes. These insights enhance our understanding of the molecular mechanisms underlying DCM and offer a promising therapeutic strategy for patients with calcium ion dysregulation associated with this condition. dilated cardiomyopathy human induced pluripotent stem cells human induced pluripotent stem cell-derived cardiomyocyte MYBPC3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Cardiomyocytes differentiated from patient-specific induced pluripotent stem cells (hiPSCs) reproduces morphology of cardiac hypertrophy and sarcomeric disorders. A novel c.194C>T mutation in MYBPC3 results in abnormal calcium transients in hiPSC-derived cardiomyocytes. c.194C>T mutation of MYBPC3 leads to a significant increase in the expression of calsequestrin that binds to the ryanodine receptor 2 (RyR2). Treatment with RyR2 inhibitor markedly improves the ability of calcium handling in DCM-hiPSC-cardiomyocytes. Introduction Dilated cardiomyopathy (DCM) is a severe cardiovascular disorder characterized by left ventricular dilation and systolic dysfunction, often accompanied by arrhythmias, heart failure, thromboembolic events, and an increased risk of sudden death [ 1 ] . The global prevalence of DCM is estimated to range between 1 in 250 and 1 in 400 individuals [ 2 ] . Although advancements in pharmacological therapy, device-based interventions, mechanical circulatory support, and heart transplantation have improved survival rates, the development of comorbidities remains a significant concern, with a five-year survival rate remaining below 50% [ 3 , 4 ] . Approximately 30–50% of hereditary DCM cases can be attributed to known genetic mutations, including those affecting sarcomere proteins, cytoskeletal components, ion channels, and other relevant molecular pathways [ 5 – 7 ] . MYBPC3 encodes the cardiac isoform of myosin-binding protein C (cMyBP-C), which is a crucial regulatory protein for cardiac contraction. It is located in the cross-bridge-bearing region between myosin and actin [ 8 , 9 ] . Mutations in the MYBPC3 gene elevate the risk of developing various forms of cardiomyopathy, including DCM [ 10 – 12 ] . Mutations at distinct sites may impair the interaction between cMyBP-C and other proteins, leading to a reduction in functionality [ 13 – 15 ] . Due to ethical constraints, acquiring human heart tissues or primary cardiomyocytes (CMs) from DCM patients presents significant challenges [ 16 , 17 ] . Moreover, there are substantial differences in genetic composition, cardiac anatomy, and electrophysiology between humans and animals [ 18 – 20 ] . Human induced pluripotent stem cells (hiPSCs), which possess the ability to unlimited self-renew and the capability to differentiate into all three germ layers similar to human embryonic stem cells (hESCs), have emerged as a novel tool for advancing DCM research [ 21 , 22 ] . Patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) provide a theoretical foundation for investigating the pathophysiology of hereditary DCM and developing cardiovascular therapies by accurately modeling human DCM in vitro [ 18 , 23 ] . In this study, we examined a patient with DCM who harbors a novel MYBPC3 (c.194C > T) point mutation and generated hiPSC-CMs. These hiPSC-CMs exhibited hypertrophic morphology, disorganized sarcomeres, aberrant calcium handling, and upregulated expression of genes associated with hypertrophy. Notably, we observed a significant increase in the expression of CASQ2, which encodes calsequestrin, a protein that interacts with the Ryanodine receptor 2 (RyR2). Our findings suggest that RyR2 may represent a promising therapeutic target for DCM patients exhibiting calcium dysregulation. Materials and Methods Patient recruitment. Patients were recruited in the study following the protocols with informed consent approved by Medical Ethics Committee of Sun Yat-Sen Memorial Hospital, the ethical approval number is SYSKY-2022-010-01. Echocardiogram and electrocardiogram were performed on all patients. Peripheral blood was extracted from patients, and the entire exome sequencing process was carried out at the Guangzhou DaAn Clinical Laboratory Center. Human iPSCs reprogramming and culture. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of the patient and reprogrammed to hiPSCs using a CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) on the basis of user guide. PBMCs were plated at 5×10 5 cells/mL to a 24-well plate in complete StemPro™-34 medium (Gibco) supplemented with SCF (100 ng/mL), FLT-3 (100 ng/mL), IL-3 (20 ng/mL), and IL-6 (20 ng/mL). After four days, the cells were transduced by the reprogramming vectors at the appropriate MOI and incubated overnight. Twenty-four hours later, the cells were removed into fresh complete PBMC medium and cultured for two days. The transduced cells were then plated in MEF feeder-cells culture wells of a 24-well plate in complete StemPro™-34 medium. The cells were gradually transitioned into mTeSR™ 1 medium (Stem Cell Technologies) by replacing half of the StemPro™-34 medium with mTeSR™ 1 medium. Nearly two weeks later stem cell-like colonies were picked and transferred on Matrigel-coated plates (Corning) in mTeSR™ 1 medium under conditions at 37 ℃ with 5% CO2. Cells were dissociated with ReLeSR (StemCell Technologies) with mTeSR™ 1 medium supplemented with 10 µM Y-27632 (MCE). Human iPSCs characteristics. AP staining was performed using a Alkaline Phosphatase Assay Kit (Applygen) according to manufacturer's instructions. Karyotyping was analyzed by Guangzhou DaAn Clinical Laboratory Center. Sanger sequencing was performed by Guangzhou IGE Biotechnology Co., Ltd.. Genomic DNA from hiPSCs was extracted using a Kit (TIANGEN) according to the manufacturer’s instructions. Polymerase chain reaction was performed using EasyTaq® DNA polymerase. Quantitative RT-PCR. Total RNA was extracted using RNAzol (MOLECULAR RESEARCH) according to the manufacturer’s instructions. RNA was reverse transcribed in a 20-µL reaction system using a Reverse Transcription kit (Novoprotein). Resulting cDNA was used in qPCR using SYBR qPCR Master Mix (Vazyme) and a LightCycler480 Detection System (Roche). The relative expression of target genes was calculated by the comparative quantification method (2 −ΔΔCT ) and was normalized to the average expression of GAPDH. Immunofluorescence staining. The hiPSCs were seeded in Matrigel-coated 24-well plate and cultured for two or three days. The cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After permeabilizing with 0.3% Triton-X (BIOFROXX) at room temperature for an additional 15 minutes, the cells were incubated with 10% donkey serum (LOUNBIOTECH) and followed with primary antibodies (Table 1 ) at 4 ℃ overnight. After washing, cells were incubated with corresponding secondary antibodies Alex Fluor 488/555/647 (Invitrogen) in the dark for 1h at room temperature. Cells were counterstained with DAPI (Beyotime) for 5 minutes. Images were taken on Inverted Microscopes (Leica, DMi8) or confocal laser scanning microscope (Zeiss, LSM710/780) and analyzed with ImageJ. Table 1 Whole exome high-throughput sequencing in DCM patients. Gender Age Range Gene Location of Genome Variant information mode of inheritance 1 male 36–40 MYBPC3 chr11: 4737288835 NM_000256.3: c.194C > T p.Thr65Met AD 2 male 75–80 TNNT2 chr1: 201332518 NM_001276345.2: c.506G > A p.Arg169Gln AD 3 male 30–35 TTN chr2: 179486248 NM_001267550.2: c.45303C > A p.Asn15101Lys AD 4 female 50–55 DSP chr6: 7574888 NM_004415.4: c.2298-2A > T AD 5 male 30–35 TTN chr2: 179605357 NM_001267550.2: c.12602dup AD AD: Autosomal Dominant Inheritance Cardiomyocyte differentiation. hiPSCs were differentiated to cardiomyocytes using a small molecule Wnt-activation/inhibition protocol with heparin previously described. Briefly, hiPSCs were first treated with CHIR99021 (5 µM, MCE) in E8 basal medium for 24 hours, and then with IWP2 (3 µM, MCE) for another 72 hours on day 2 post-differentiation. Heparin (3µg/mL, Selleck) was added into the medium on day 1 and removed on day 7. The media was then replaced with E8 basal medium with insulin (20 µg/mL, Sigma) and refreshed every 2 days. Spontaneously beating clusters was normally observed on day 6 to 7 post-differentiation. On day 11 post-differentiation, hiPSC-CMs were metabolically selected in DMEM without glucose (Gibco) supplemented with lactate (20 mM, Sigma) for 96 hours. All experiments were conducted by using hiPSC-CMs between 30 and 35 days. Flow cytometry. HiPSC-CMs on day 10 post-differentiation in monolayer were dissociated with 1xTrypLE (Gibco) for 5 minutes. The cells were fixed by 4% PFA for 15 minutes and permeabilized by 0.3% Triton X-100 for 30 minutes at room temperature. Then, the cells were incubated with primary antibodies for 1 hour. After washing, the cells were incubated with second antibodies Alex Fluor 488 (1:1,500 diluted in PBS, Invitrogen) for 30 minutes. The cells were washed three times and suspended in PBS for the flow cytometry assay. The data was analyzed with the CytExpert software (BD Biosciences). Wheat germ agglutinin staining. Wheat germ agglutinin staining was performed using iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate (AAT Bioquest) according to the manufacturer’s instructions. The cells were incubated at 37 ℃ for 20 minutes. Images were taken on Inverted Microscopes (Leica, DMi8) or confocal laserscanning microscope (Zeiss, LSM710/780) and analyzed with ImageJ. Transmission Electron Microscopy. Human iPSC-CMs were fixed in 2.5% glutaraldehyde fixed solution at room temperature for 5 minutes in the dark. Then, the cells were scraped off, washed and centrifuged. The cells were fixed for an additional 30 minutes and sent to Wuhan Servicebio Technology Co., Ltd. for embedding and staining. Calcium transient analysis. hiPSC-CMs were treated with 5 µM Fluo-4 AM (Thermo Scientific) in the Tyrode’s solution (140.0 µM NaCl, 5.0 µM KCl, 2 µM MgCl 2 , 10 µM HEPES, 1.8 µM CaCl 2 , 10 µM glucose, pH 7.4) for 30 minutes at 37 ℃. The cells were then washed for three times with the Tyrode’s solution and incubated at room temperature for 20 min before use. Calcium traces were captured in the line-scan model using confocal laserscanning microscope (Zeiss, LSM710/780) with a 63× objective. The calcium transient analysis were performed with ImageJ. Western blot. Total protein was extracted by RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail for 30 minutes on ice. Protein concentration was analyzed using a BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s protocol. An equal amount of protein (10–20 µg) was loaded onto a 10–12% TGX Stain-Free FastCast Acrylamide gel (Bio-Rad) at 200V for 30 minutes. The proteins were transferred to a PVDF membrane using the Trans-blot Turbo system (Bio-Rad). Membranes were then blocked in 5% BSA diluted in TBST at room temperature for 1 hour. The membranes were incubated with primary antibodies at 4℃ overnight. After incubated with secondary antibody at room temperature for 1 hour, the bands were visualized with chemiluminescence imaging system (Bio-Rad). Statistical analysis. Statistical analysis was performed with GraphPad Prism (version 9) using Student’s t-test or one-way ANOVA. All data were presented as mean ± standard error of the mean from at least three independent experiments. P -value less than 0.05 was considered statistically significant. Results Patient with DCM carries a pathogenic missense mutation (c.194C > T) in the MYBPC3 gene We obtained peripheral blood samples from nine patients diagnosed with DCM at Sun Yat-sen Memorial Hospital with informed consent. Whole exome high-throughput sequencing identified genetic mutations associated with DCM in five of these patients (Table 1 ). Notably, we focused on a male patient who was found to carry a novel pathogenic missense mutation in the MYBPC3 gene (chr11:4737288835, NM_000256.3: c.194C > T, p.T65M). This mutation results in an amino acid substitution at position 65 of the cMyBP-C, changing threonine (T) to methionine (M) (Fig. 1 A). Clinically, the patient exhibited classic symptoms of DCM, such as nocturnal dyspnea, exertional dyspnea, chest tightness, and decreased exercise tolerance. Echocardiography revealed diffuse hypokinesis of the ventricular walls and biventricular dilation (Fig. 1 B). An electrocardiogram (ECG) indicated 13 premature ventricular contractions over a 24-hour period (Fig. 1 C). Generating DCM patient-specific human induced pluripotent stem cells (DCM-hiPSCs) carrying MYBPC3 (c.194C > T) mutation To generate DCM-hiPSCs retaining all genetic information, peripheral blood mononuclear cells were isolated via density gradient centrifugation. These cells were subsequently reprogrammed into hiPSCs using a Sendai virus kit and maintained through approximately 15 passages with stable growth. Colonies exhibiting positive alkaline phosphatase staining and characteristic hESC-like morphology, including large nuclei, high nucleocytoplasmic ratios, and tightly packed cell arrangements, were selected, expanded, and established as hiPSC lines (Fig. 2 A). After extended passaging, karyotype analysis confirmed that the cells retained normal morphology and chromosomal counts (Fig. 2 B). DNA sequencing validated the presence of the specific MYBPC3 (c.194C > T) mutation in the DCM-hiPSC line (Fig. 2 C). The pluripotency of the DCM-hiPSC line was further confirmed by PCR analysis, which demonstrated robust expression of pluripotency markers NANOG , OCT4 , and SOX2 , as well as the absence of Sendai virus vectors (Fig. 2 D). Similar mRNA expression levels of NANOG , OCT4 , and SOX2 genes were observed between DCM-hiPSCs and control hiPSCs (Fig. 2 E). Immunostaining revealed high expression levels of pluripotency markers OCT4, SOX2, and SSEA4 in DCM-hiPSCs (Fig. 2 F). Additionally, DCM-hiPSCs maintained the capacity to differentiate into cells representing all three germ layers in vitro (Fig. 2 G). The characterization of the control group is detailed in Figure S1 . Collectively, these findings demonstrate the successful generation of a DCM-hiPSC line suitable for further research into disease mechanisms and potential therapeutic targets. Differentiation of DCM-hiPSCs into spontaneous contracting CMs Subsequently, we generated DCM patient-specific CMs (DCM-hiPSC-CMs) from hiPSCs utilizing a previously established small molecule-based protocol [ 25 ] (Fig. 3 A). We observed progressive changes in cell morphology and spontaneous contractions as early as day 7 post-differentiation (Fig. 3 B). The hiPSCs were successfully differentiated into hiPSC-CMs, which exhibited characteristic cardiomyocyte markers (Figs. 3 C and S2A). Flow cytometric analysis indicated that up to 90% of the cells were successfully differentiated into cardiomyocytes (Figure S2B). We also carried out differentiation on another clone and obtained the same result (Figure S3). The spontaneously contracting hiPSC-CMs, after 30 to 35 days of differentiation, were utilized for further analysis. Additionally, we predicted the mRNA secondary structure of MYBPC3 using RNAfold software (Fig. 4 A). The mutation site disrupted the stem-loop structure of the mRNA, potentially contributing to the downregulation of endogenous MYBPC3 expression in DCM-hiPSC-CMs (Fig. 4 B). To further elucidate this effect, we conducted Western blot analysis to assess the abundance of cMyBP-C protein; however, no significant differences were observed (Fig. 4 C). Furthermore, immunofluorescence imaging (Fig. 4 D) demonstrated that cMyBP-C localized to the sarcomere and exhibited a punctate distribution under high magnification. Characterization of the DCM-hiPSC-CMs carrying MYBPC3 (c.194C > T) mutation The morphology of control hiPSC-CMs (n = 143) was predominantly round or oval and exhibited a relatively uniform appearance, whereas DCM-hiPSC-CMs (n = 128) displayed more diverse morphologies with significantly larger area compared to the control group (Fig. 5 A). The relative mRNA expression levels of hypertrophic markers NPPA and NPPB were markedly elevated in DCM-hiPSC-CMs, indicating that DCM can induce cardiomyocyte hypertrophy (Fig. 5 B). Transmission electron microscopy and immunostaining were employed to evaluate myofibril organization in hiPSC-CMs. DCM-hiPSC-CMs demonstrated increased variability in sarcomeric organization, characterized by a higher number of poorly aligned Z lines (Fig. 5 C) and a punctate distribution of sarcomeric α-actinin (Fig. 5 D). To examine the functional characteristics of the beating DCM-hiPSC-CMs, we employed video imaging to track individual beating clusters at 10, 20, and 30 days post-differentiation. The beating rate of DCM-hiPSC-CMs exhibited a significant decline from 61.5 ± 2.3 bpm at day 10 to 10.7 ± 0.9 bpm at day 20, and further decreased to 6.7 ± 0.8 bpm at day 30, in comparison with the control group (Fig. 5 E). Subsequently, we utilized fluorescent Ca²⁺ imaging to evaluate the Ca²⁺ handling properties during excitation-contraction coupling. Aberrant calcium transients were observed in DCM-hiPSC-CMs, suggesting impaired function of Ca²⁺-related channels and components in both the plasma membrane and sarcoplasmic reticulum (Figs. 5 F and 5 G). Furthermore, DCM-hiPSC-CMs exhibited markedly larger intracellular Ca²⁺ transient amplitudes relative to control cells (Fig. 5 H). Additionally, the DCM-hiPSC-CMs showed prolonged time to peak and delayed decay of the calcium transient, which further underscores the impaired calcium handling observed in these cells (Fig. 5 H). MYBPC3 (c.194C > T) mutation upregulated the expression of CASQ2 and disrupted calcium homeostasis. To gain deeper insights into the impact of the MYBPC3 (c.194C > T) mutation on DCM, we conducted RNA sequencing (RNA-seq) analysis on hiPSC-CMs from both control and patient-specific DCM-hiPSCs. We identified differentially expressed genes (DEGs), with 180 up-regulated and 117 down-regulated genes common to both groups. These DEGs were subjected to comprehensive pathway and functional enrichment analyses (Fig. 6 A). The results indicated that the DEGs were significantly enriched in calcium ion binding functions at the molecular level (Fig. 6 B), as shown by the expression heatmap (Fig. 6 C). Key genes involved in calcium ion binding, including CASQ2 , DUOX2 , PCDHGA3 , SMOC2 , ACAN , and FBLN5 , were validated using real-time quantitative PCR (Fig. 6 D). Notably, CASQ2 which encodes calsequestrin exhibited the most significant upregulation in DCM-hiPSC-CMs and is associated with myocardial contraction, further implicating its role in the observed calcium dysregulation. We further examined its protein level and found that CASQ2 was significantly up-regulated in the DCM-hiPSC-CMs group. Levels of other Ca 2+ -regulating proteins, including p-RyR2, RyR2, sarcoplasmic or endoplasmic reticulum Ca 2+ ATPase 2 (SERCA2) and phospholamban (PLB), were evaluated (Fig. 6 E-I). Among these proteins, p-RyR2 and PLB showed levels that were significantly elevated, while SERCA2 showed levels that were significantly decreased. All these results suggest that the MYBPC3 (c.194C > T) mutation causes a change in intracellular calcium homeostasis. Ryanodine receptor 2 (RyR2) inhibitor restored abnormal calcium transients in DCM-hiPSC-CMs. Calsequestrin, a calcium-binding protein localized in the sarcoplasmic reticulum of cardiomyocytes, interacts directly or indirectly with RyR2. To elucidate the mechanisms underlying CASQ2 upregulation in DCM-hiPSC-CMs, we evaluated the functional responses of both control and DCM-hiPSC-CMs to various inhibitors. Specifically, we examined the effects of the RyR2 inhibitor ryanodine (1 µM) (Fig. 7 A), the IP3R inhibitor 2-Aminoethyl diphenylborinate (2-APB, 2.5 µM) (Fig. 7 C), the voltage-dependent Ca²⁺ channel inhibitor verapamil (1 µM) (Fig. 7 E), and the calmodulin-dependent kinase II (CaMKII) inhibitor KN93 (1 µM) (Fig. 7 G). In DCM-hiPSC-CMs, all treatments significantly attenuated the peak of Ca²⁺ transients, resulting in a reduction in transient amplitude (Figs. 7 B, 7 D, 7 F, and 7 H). Notably, only ryanodine treatment was capable of restoring the frequency of Ca²⁺ transients in DCM-hiPSC-CMs (Fig. 7 A). Collectively, these findings indicate that RyR2 may represent a promising therapeutic target for patients with DCM, especially those exhibiting impaired calcium handling. Discussion In this study, we utilized hiPSC-derived cardiomyocytes from a DCM patient as an in vitro disease model. Our findings reveal for the first time that MYBPC3 (c.194C > T) heterozygous mutant cardiomyocytes recapitulate several key characteristics of DCM, including increased cell areas, increased sarcomere disorders, and abnormal calcium handling. Additionally, we demonstrate that this novel mutation in MYBPC3 leads to a significant upregulation of CASQ2 gene and protein expression in DCM-hiPSC-CMs. CASQ2 encodes calsequestrin, which binds to RyR2. Treatment with a RyR2 inhibitor markedly improves calcium handling in DCM-hiPSC-CMs. These results provide a robust foundation for understanding the underlying mechanisms of DCM and exploring potential therapeutic interventions. We identified a patient harboring a heterozygous mutation in MYBPC3 (c.194C > T), which results in the substitution of threonine with methionine at codon 65 of the cMyBP-C protein (p.Thr65Met). This missense variant has been reported in individuals diagnosed with DCM; however, its pathogenicity remains undetermined. Furthermore, the lack of effective disease models, particularly human-based models, has hindered our understanding of the pathogenesis associated with MYBPC3 (c.194C > T) mutations, thereby impeding the development of effective treatments and preventive strategies for this condition. Additionally, there is a significant correlation between genotype and phenotype in DCM patients. Compared to those without genetic mutations, genetically affected patients tend to exhibit an earlier age of onset and more severe symptoms, underscoring the critical need for investigating hereditary DCM [ 26 , 27 ] . We generated hiPSCs from peripheral mononuclear cells of DCM patient harboring the MYBPC3 (c.194C > T) mutation using reprogramming technology. These hiPSCs were subsequently differentiated into cardiomyocytes in vitro to establish a cellular disease model. Our findings revealed that cardiomyocytes carrying this specific mutation exhibited phenotypes characteristic of DCM [ 28 , 29 ] . Calcium handling plays a key critical role in regulation of excitation-contraction in CMs [ 30 ] . We observed that patient-derived hiPSC-CMs exhibited abnormal Ca 2+ handling from the initial stage of cardiomyocyte differentiation until maturation, especially after 35 days, exhibiting higher amplitude of Ca 2+ transient, prolonged decay time and increased cytoplasmic Ca 2+ level. To investigate the mechanisms underlying the aberrant calcium processing induced by gene mutations, RNA-seq analysis revealed that differentially expressed genes were primarily enriched in calcium ion binding functions. Notably, among several Ca 2+ binding-related genes examined, only CASQ2 , which encodes the calsequestrin protein, showed a significant increase in DCM-hiPSC-CMs. Calsequestrin is a calcium-binding protein responsible for calcium buffering in sarcoplasmic reticulum [ 31 ] . Functionally, calsequestrin regulates the release of Ca²⁺ from the sarcoplasmic reticulum via RyR2 during excitation-contraction coupling [ 32 ] . Consequently, we hypothesize that MYBPC3 (c.194C > T) mutation may influence RyR2-mediated calcium release by upregulating the expression of calsequestrin. In addition, studies have demonstrated a direct interaction between the RyR2 protein and cMyBP-C protein, with cMyBP-C potentially inhibiting RyR2-dependent Ca 2+ release [ 33 ] . Based on these findings, we hypothesize that the MYBPC3 (c.194C > T) mutation may weaken the inhibitory effect of cMyBP-C on RyR2, leading to sarcoplasmic reticulum Ca 2+ leakage and intracellular Ca 2+ overload. RyR2 is a large tetrameric protein that regulates Ca 2+ release from the sarcoplasmic reticulum in cardiomyocytes and plays a crucial role during myocardial contraction. RyR2, PLB and SERCA2a regulate myocardial calcium signal through the dynamic cooperation of "release and recycling". The coordination of the three functions is the core guarantee for the normal pumping function of the heart, and their imbalance is an important pathological mechanism of various heart diseases. This channel's function may be impaired in DCM [ 34 , 35 ] . PLB is an endogenous inhibitor of SERCA2a, and the upregulation of PLB protein in DCM will further enhance the inhibition of SERCA2a, resulting in decreased calcium recycling capacity and prolonged cytosolic calcium retention time, which is consistent with the phenotype of prolonged time to peak, and the increase in the level of phosphorylated ryr2 further exacerbated the calcium leakage from the sarcoplasmic reticulum. In order to correct calcium homeostasis, we utilized the RyR2 modulator ryanodine to intervene in DCM-hiPSC-CMs and found that ryanodine effectively inhibited the RyR2 receptor, thereby reducing Ca 2+ release into the cytoplasm during myocardial excitation. Consequently, it significantly diminished the amplitude of calcium transients in DCM-hiPSC-CMs, accelerated the frequency of calcium transients, and improved the abnormal calcium handling observed in these cells. To specifically exclude the contribution of other channels to the increase in intracellular Ca 2+ , DCM-hiPSC-CMs were individually treated with the IP3R inhibitor 2-APB, the L-type calcium channel inhibitor Verapamil, and the CaMKII inhibitor KN93. The results indicated that the calcium transient abnormalities in cardiomyocytes remained unaltered. These findings suggest that Ryanodine may serve as a promising therapeutic agent for DCM patients harboring the MYBPC3 (c.194C > T) mutation, and RyR2 may represent a potential therapeutic target for DCM patients. Conclusion In summary, we identified a MYBPC3 (c.194C > T) mutation in a patient diagnosed with DCM and established a patient-specific hiPSC model. Our study revealed significant impairments in myofilament regulation and calcium handling, as well as increased expression of CASQ2 , which encodes calsequestrin, a protein that binds to RyR2 in DCM-hiPSC-CMs. Treatment with a RyR2 inhibitor ameliorated the abnormal calcium transients observed in DCM-hiPSC-CMs. These findings provide a robust foundation for a deeper understanding of the underlying mechanisms of DCM and pave the way for exploring novel therapeutic interventions. Abbreviations cMyBP-C, cardiac myosin-binding protein C; DCM, dilated cardiomyopathy; hiPSC, human induced pluripotent stem cell; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; RyR2, Ryanodine receptor 2; PLB, phospholamban; SERCA2, sarcoplasmic or endoplasmic reticulum Ca 2+ ATPase 2. Declarations Acknowledgments The authors would like to thank the Experimental Instrument Sharing Platform of Sun Yat-sen University, the Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University, and Sun Yat-sen Memorial Hospital of Sun Yat-sen University for their assistance. We thank Prof. Nan Cao for kindly providing the WTB hiPSC line in this study. Thanks for all the contributors. Sources of Funding This work was supported by the National Natural Science Foundation of China (grant number 82470367 and 82272164), the Natural Science Foundation of Guangdong Province (grant number 2024A1515013135). Author information Manting Xie and Bingbing Xie contributed equally to this work. Authors and Affiliations Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, P. R. China (510080) Manting Xie, Bingbing Xie, Liang Huang, Xingqiang Lai, Jixing Gong, Nan Cao, Andy Peng Xiang & Qiuling Xiang Scientific Development and Management Office, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China(510235) Manting Xie Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China(510235) Ying Chen Maternal-Fetal Medicine Institute, Department of Obstetrics and Gynaecology, Shenzhen Baoan Women's and Children's Hospital, Shenzhen, China(518104) Xingqiang Lai Contributions Manting Xie: Writing - original draft, Investigation, Data curation. Bingbing Xie: Investigation, Writing - review & editing. Liang Huang: Methodology, Writing - review & editing. Ying Chen: Investigation, Formal analysis. Xingqiang Lai: Data curation. Jixing Gong: Methodology. Nan Cao: review & editing. Andy Peng Xiang: Supervision, review & editing. Qiuling Xiang: financial support, conceptualization, Project administration, Supervision, Writing - review & editing. Consent for publication All authors consent to the publication of the article. Competing interests None. Ethical Approval All procedures performed in this study were in accordance with the Ethical Standards Research Committee and the Helsinki declaration. This study was approved by the Medical Ethics Committee of Sun Yat-Sen Memorial Hospital(SYSKY-2022-010-01). Data availability The data sets utilized and examined in this study can be obtained from the corresponding author upon a reasonable request. References Heymans S, Lakdawala NK, Tschöpe C, Klingel K. Dilated cardiomyopathy: causes, mechanisms, and current and future treatment approaches. Lancet. 2023; 402(10406): 998-1011. Reichart D, Magnussen C, Zeller T, Blankenberg S. Dilated cardiomyopathy: from epidemiologic to genetic phenotypes: A translational review of current literature. J Intern Med 2019; 286(4): 362-372. Schultheiss HP, Fairweather D, Caforio ALP, Escher F, Hershberger RE, Lipshultz SE, Liu PP, Matsumori A, Mazzanti A, McMurray J, Priori SG. Dilated cardiomyopathy. Nat Rev Dis Primers 2019; 5(1): 32. McNally EM, Mestroni L. Dilated Cardiomyopathy: Genetic Determinants and Mechanisms. Circ Res 2017; 121(7): 731-748. Eldemire R, Mestroni L, Taylor MRG. Genetics of Dilated Cardiomyopathy. Annu Rev Med 2024; 75: 417-426. Kaski JP, Cannie D. Clinical Screening for Dilated Cardiomyopathy in At-Risk First-Degree Relatives: Who, When, and How? J Am Coll Cardiol 2023; 81(21): 2072-2074. Rosenbaum AN, Agre KE, Pereira NL. Genetics of dilated cardiomyopathy: practical implications for heart failure management. Nat Rev Cardiol 2020; 17(5): 286-297. Carrier L, Mearini G, Stathopoulou K, Cuello F. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology. Gene 2015; 573(2): 188-197. Mahdieh N, Hosseini Moghaddam M, Motavaf M, Rabbani A, Soveizi M, Maleki M, Rabbani B, Alizadeh-Asl A. Genotypic effect of a mutation of the MYBPC3 gene and two phenotypes with different patterns of inheritance. J Clin Lab Anal 2018; 32(6): e22419. Marian AJ. Molecular Genetic Basis of Hypertrophic Cardiomyopathy. Circ Res 2021; 128(10): 1533-1553. de Frutos F, Ochoa JP, Fernández AI, Gallego-Delgado M, Navarro-Peñalver M, Casas G, Basurte MT, Larrañaga-Moreira JM, Mogollón MV, Robles-Mezcua A, García-Granja PE, Climent V, Palomino-Doza J, García-Álvarez A, Brion M, Brugada R, Jiménez-Jáimez J, Bayes-Genis A, Ripoll-Vera T, Peña-Peña ML, Rodríguez-Palomares JF, Gonzalez-Carrillo J, Villacorta E, Espinosa MA, Garcia-Pavia P, Mirelis JG. Late gadolinium enhancement distribution patterns in non-ischaemic dilated cardiomyopathy: genotype-phenotype correlation. Eur Heart J Cardiovasc Imaging 2023; 25(1): 75-85. Blagova O, Pavlenko E, Sedov V, Kogan E, Polyak M, Zaklyazminskaya E, Lutokhina Y. Different Phenotypes of Sarcomeric MyBPC3-Cardiomyopathy in the Same Family: Hypertrophic, Left Ventricular Noncompaction and Restrictive Phenotypes (in Association with Sarcoidosis). Genes (Basel) 2022; 13(8). Rasicci DV, Tiwari P, Bodt SML, Desetty R, Sadler FR, Sivaramakrishnan S, Craig R, Yengo CM. Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin. Elife 2022; 11. Yang KC, Breitbart A, De Lange WJ, Hofsteen P, Futakuchi-Tsuchida A, Xu J, Schopf C, Razumova MV, Jiao A, Boucek R, Pabon L, Reinecke H, Kim DH, Ralphe JC, Regnier M, Murry CE. Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells. JACC Basic Transl Sci 2018; 3(6): 728-740. Toepfer CN, Wakimoto H, Garfinkel AC, McDonough B, Liao D, Jiang J, Tai AC, Gorham JM, Lunde IG, Lun M, Lynch TLt, McNamara JW, Sadayappan S, Redwood CS, Watkins HC, Seidman JG, Seidman CE. Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin. Sci Transl Med 2019; 11(476). Ansari AA, Wang YC, Danner DJ, Gravanis MB, Mayne A, Neckelmann N, Sell KW, Herskowitz A. Abnormal expression of histocompatibility and mitochondrial antigens by cardiac tissue from patients with myocarditis and dilated cardiomyopathy. Am J Pathol 1991; 139(2): 337-354. Katagiri T, Kitsu T, Akiyama K, Takeyama Y, Niitani H. Alterations in fine structures of myofibrils and structural proteins in patients with dilated cardiomyopathy--studies with biopsied heart tissues. Jpn Circ J 1987; 51(6): 682-688. Karakikes I, Ameen M, Termglinchan V, Wu JC. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Insights Into Molecular, Cellular, and Functional Phenotypes. Circulation Research 2015; 117(1): 80-88. Ntelios D, Meditskou S, Efthimiadis G, Pitsis A, Zegkos T, Parcharidou D, Theotokis P, Alexouda S, Karvounis H, Tzimagiorgis G. α-Myosin heavy chain (MYH6) in hypertrophic cardiomyopathy: Prominent expression in areas with vacuolar degeneration of myocardial cells. Pathol Int 2022; 72(5): 308-310. Thomas D. Allometric Relations and Scaling Laws for the Cardiovascular System of Mammals. Systems 2014; 2(2): 168-185. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126(4): 663-676. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108(3): 407-414. Smith AS, Macadangdang J, Leung W, Laflamme MA, Kim DH. Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening. Biotechnol Adv 2017; 35(1): 77-94. van Lint FHM, Mook ORF, Alders M, Bikker H, Lekanne Dit Deprez RH, Christiaans I. Large next-generation sequencing gene panels in genetic heart disease: yield of pathogenic variants and variants of unknown significance. Neth Heart J 2019; 27(6): 304-309. Lin Y, Linask KL, Mallon B, Johnson K, Klein M, Beers J, Xie W, Du Y, Liu C, Lai Y, Zou J, Haigney M, Yang H, Rao M, Chen G. Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes. Stem Cells Transl Med 2017; 6(2): 527-538. Gacita AM, Fullenkamp DE, Ohiri J, Pottinger T, Puckelwartz MJ, Nobrega MA, McNally EM. Genetic Variation in Enhancers Modifies Cardiomyopathy Gene Expression and Progression. Circulation 2021; 143(13): 1302-1316. Zhang XL, Xie J, Lan RF, Kang LN, Wang L, Xu W, Xu B. Genetic Basis and Genotype-Phenotype Correlations in Han Chinese Patients with Idiopathic Dilated Cardiomyopathy. Sci Rep 2020; 10(1): 2226. Streckfuss-Bömeke K, Tiburcy M, Fomin A, Luo X, Li W, Fischer C, Özcelik C, Perrot A, Sossalla S, Haas J, Vidal RO, Rebs S, Khadjeh S, Meder B, Bonn S, Linke WA, Zimmermann WH, Hasenfuss G, Guan K. Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes. J Mol Cell Cardiol 2017; 113: 9-21. Shah D, Virtanen L, Prajapati C, Kiamehr M, Gullmets J, West G, Kreutzer J, Pekkanen-Mattila M, Heliö T, Kallio P, Taimen P, Aalto-Setälä K. Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells. Cells 2019; 8(6). Powers JD, Malingen SA, Regnier M, Daniel TL. The Sliding Filament Theory Since Andrew Huxley: Multiscale and Multidisciplinary Muscle Research. Annu Rev Biophys, 2021, 50: 373-400. Sibbles ET, Waddell HMM, Mereacre V, Jones PP, Munro ML. The function and regulation of calsequestrin-2: implications in calcium-mediated arrhythmias. Biophys Rev. 2022 Jan 7;14(1):329-352. Wleklinski MJ, Kryshtal DO, Kim K, Parikh SS, Blackwell DJ, Marty I, Iyer VR, Knollmann BC. Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2. Circ Res. 2022 Sep 30;131(8):673-686. Stanczyk PJ, Seidel M, White J, Viero C, George CH, Zissimopoulos S, Lai FA. Association of cardiac myosin-binding protein-C with the ryanodine receptor channel - putative retrograde regulation? J Cell Sci. 2018 Aug 3;131(15):jcs210443. Steinberg C, Roston TM, van der Werf C, Sanatani S, Chen SRW, Wilde AAM, Krahn AD. RYR2-ryanodinopathies: from calcium overload to calcium deficiency. Europace. 2023 Jun 2;25(6):euad156. Wescott AP, Jafri MS, Lederer WJ, Williams GS. Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling. J Mol Cell Cardiol. 2016 Mar;92:82-92. Dulhunty AF, Wium E, Li L, Hanna AD, Mirza S, Talukder S, Ghazali NA, Beard NA. Proteins within the intracellular calcium store determine cardiac RyR channel activity and cardiac output. Clin Exp Pharmacol Physiol 2012; 39(5): 477-484. Supplementary Files image8.png 8.25SupplementalMaterial.docx Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Minor Revision 28 Oct, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 28 Aug, 2025 First submitted to journal 26 Aug, 2025 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. 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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-7455786","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511231167,"identity":"cc5d7ce9-1c48-4c8f-b0b1-2ae513fc45d3","order_by":0,"name":"Manting Xie","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Manting","middleName":"","lastName":"Xie","suffix":""},{"id":511231168,"identity":"4494a868-3571-48a2-b528-3c283400009a","order_by":1,"name":"Bingbing Xie","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Xie","suffix":""},{"id":511231169,"identity":"8ba0b2db-8b9a-4040-8d0e-f99b5086db48","order_by":2,"name":"Liang Huang","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Huang","suffix":""},{"id":511231170,"identity":"bd718284-119f-428f-8aec-cc38e80e218d","order_by":3,"name":"Ying Chen","email":"","orcid":"","institution":"Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":511231171,"identity":"f6555383-e02b-43d6-817c-e0265fcd18f9","order_by":4,"name":"Xingqiang Lai","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Xingqiang","middleName":"","lastName":"Lai","suffix":""},{"id":511231172,"identity":"39329153-36cb-497e-8327-7447dc3a176f","order_by":5,"name":"Jixing Gong","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jixing","middleName":"","lastName":"Gong","suffix":""},{"id":511231173,"identity":"8a2c4d46-2ff8-4236-be85-bb296111fbae","order_by":6,"name":"Nan Cao","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Cao","suffix":""},{"id":511231174,"identity":"ca49106c-0779-4ab8-89f5-b37b97f56091","order_by":7,"name":"Andy Peng Xiang","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Andy","middleName":"Peng","lastName":"Xiang","suffix":""},{"id":511231175,"identity":"c6779e92-1afc-474e-8a5c-8d16d8e87d77","order_by":8,"name":"Qiuling Xiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYDACZjBpIcfAwNh4ACYoQYQWCWOglgYitUDVJDYASeK0GBxnfvbwS5lE+tr2w0Bb/hy2NzjAfPA2D4NdHi4tks1s5sYy5yRyt51JbDjA2HY4ccMBtmRrHobkYlxa+JkZzKQl24BaDoC0NBxOMDjAYybNw3AA7FRsgI2Z/RtIS7rZ+Ycwh/F/w6uFn5nHTPJjm0SC2Q2gLQxshxk3HOBhw6tFspmnTJrhnIThthtAWxLb0hNnHmYztpxjkIxTi8H549skf5TZyJudT3/44MMfa3u+480Pb7ypsMOpBQSYedigrASGZmjkGuBRDwSMP9jg7Dr8SkfBKBgFo2BEAgCqv1fFFatFFAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9921-8699","institution":"Sun Yat-Sen University","correspondingAuthor":true,"prefix":"","firstName":"Qiuling","middleName":"","lastName":"Xiang","suffix":""}],"badges":[],"createdAt":"2025-08-25 16:43:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7455786/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7455786/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-026-06130-3","type":"published","date":"2026-02-18T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91194884,"identity":"51628431-4f21-46bc-9438-7a9b3b40fcf1","added_by":"auto","created_at":"2025-09-12 14:53:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":498951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical phenotype of a dilated cardiomyopathy patient with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYBPC3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(c.194C\u0026gt;T) mutation.\u003c/strong\u003e (A) Whole exome sequencing of \u003cem\u003eMYBPC3\u003c/em\u003emutant. (B) Echocardiogram for cardiac struture. LV: left ventricle; LA: left atrium; RV: right ventricle; AAO: ascending aorta. (C) Electrocardiogram (EKG) of the DCM patient. These data were measured one time.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/1d5ac410f83e45856390f13b.png"},{"id":91193598,"identity":"45fe2459-95a8-46a2-ad6f-c40fb1a093c4","added_by":"auto","created_at":"2025-09-12 14:45:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1852559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and validation of the dilated cardiomyopathy patient-specific human induced pluripotent stem cells (DCM-hiPSCs) carrying \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYBPC3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(c.194C\u0026gt;T) mutation.\u003c/strong\u003e (A) Brightfield image and alkaline phosphatase staining of the peripheral blood derived hiPSCs (scale bar: right—100 μm; left—50 μm). (B) Karyotype analysis of DCM-hiPSCs line. (C) Sanger sequencing of \u003cem\u003eMYBPC3\u003c/em\u003e mutant. (D) RT-PCR of the gene expression of pluripotency. 1: \u003cem\u003eNANOG\u003c/em\u003e; 2: \u003cem\u003eOCT4;\u003c/em\u003e 3: \u003cem\u003eSOX2\u003c/em\u003e; 4: \u003cem\u003eSev\u003c/em\u003e; 5: \u003cem\u003eKOS\u003c/em\u003e; 6: \u003cem\u003eMYC\u003c/em\u003e; 7: \u003cem\u003eKLF\u003c/em\u003e. (E) Relative mRNA expression of \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eSOX2\u003c/em\u003e. A healthy person derived hiPSCs line (Ctrl) was included as a positive control. Mean±standard error of mean, n=3. Data are represented as mean ± SEM, \u003cem\u003eP \u003c/em\u003ecompared to control using two-way ANOVA. (F) Immunofluorescent staining for OCT4, SOX2, SSEA4 and DAPI (scale bar: 45 μm). (G) Immunofluorescent staining of three germ layers (scale bar: 45 μm).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/b1b9f2960f29e1f4bf4b08a2.png"},{"id":91193602,"identity":"a890c26e-1c5d-4550-833d-c9a67151aad5","added_by":"auto","created_at":"2025-09-12 14:45:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1284720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of the dilated cardiomyopathy patient-specific induced pluripotent stem cell derived cardiomyocytes (DCM-hiPSC-CMs). \u003c/strong\u003e(A) Schematic of hiPSC-CMs induction. (B) Brightfield images of hiPSC-CMs induction from day -1 to day 10 (scale bar—100 μm). (C) Relative mRNA expression of pluripotent stem cells, mesoderm cells, cardiac progenitor cells and cardiomyocytes markers. Mean±standard error of mean, n=3.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/3359e241108aa3057e20f33e.png"},{"id":91193616,"identity":"3f95f53e-8e60-4361-a270-3066a045d030","added_by":"auto","created_at":"2025-09-12 14:45:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1439944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mRNA expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYBPC3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the protein expression of cMyBP-C.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA structure of \u003cem\u003eMYBPC3 \u003c/em\u003epredicted by RNAfold. The red arrow points to the site of the mutation. (B) Relative mRNA expression of \u003cem\u003eMYBPC3\u003c/em\u003e. Mean±standard error of mean, n=3. \u003cem\u003eP\u003c/em\u003e compared to control using unpaired, two-tailed Student’s t-test. (C) Western blot and quantification of cMyBP-C protein expression. Mean±standard error of mean, n=3. \u003cem\u003eP\u003c/em\u003e compared to control using unpaired, two-tailed Student’s t-test. (D) Immunofluorescent staining of cMyBP-C (scale bar—20 μm).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/a648af4e83899271031e72ec.png"},{"id":91193607,"identity":"65a1695a-a3cd-4b06-a567-af968097fac4","added_by":"auto","created_at":"2025-09-12 14:45:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":928345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the dilated cardiomyopathy patient-specific induced pluripotent stem cell derived cardiomyocytes (DCM-hiPSC-CMs) carrying \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYBPC3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(c.194C\u0026gt;T) mutation.\u003c/strong\u003e (A) Wheat germ agglutinin staining and quantification of cell size (scale bar—40 μm). Mean±standard error of mean, n=143 and 128, respectively. \u003cem\u003eP\u003c/em\u003ecompared to control using unpaired, two-tailed Student’s t-test. (B) Relative mRNA expression of hypertrophic genes (\u003cem\u003eNPPA\u003c/em\u003e and \u003cem\u003eNPPB\u003c/em\u003e). Mean±standard error of mean, n=3. \u003cem\u003eP\u003c/em\u003e compared to control using unpaired, two-tailed Student’s t-test. (C) Transmission electron microscope of sarcomere arrangement (scale bar—20 μm). (D) Immunofluorescent staining of sarcomeric markers (scale bar—45 μm). (E) Beating rates of hiPSC-CMs on day 10, 20 and 30. Mean±standard error of mean, n=11-30. Data are represented as mean ± SEM, \u003cem\u003eP\u003c/em\u003ecompared to control using two-way ANOVA. (F) Representative calcium transients of hiPSC-CMs. (G) Proportion of regular and abnormal calcium transients. Mean±standard error of mean, n=15 and 51, respectively. (H) Quantitation of baseline, amplitude, time to peak and time to decay in calcium transients. Mean±standard error of mean, n=4. \u003cem\u003eP\u003c/em\u003e compared to control using unpaired, two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/d0b5cb972a30ade8f646699c.png"},{"id":91193604,"identity":"d0e3e707-15ea-48e3-80ab-146ae965d227","added_by":"auto","created_at":"2025-09-12 14:45:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":658155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCASQ2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene in calcium ion binding function increases. \u003c/strong\u003e(A) The scatter diagram illustrating differentially expressed genes in Ctrl-hiPSC-CMs and DCM-hiPSC-CMs (significance cut-off of P=0.05 for fold-change≥1 up or down). (B) Gene Ontology molecular function enrichment analysis. (C) Heat map illustrating differentially expressed genes in calcium ion binding function. (D) Relative mRNA expression of \u003cem\u003eCASQ2\u003c/em\u003e, \u003cem\u003eDUOX2\u003c/em\u003e, \u003cem\u003ePCDHGA3\u003c/em\u003e, \u003cem\u003eSMOC2\u003c/em\u003e,\u003cem\u003e ACAN \u003c/em\u003eand\u003cem\u003e FBLN5\u003c/em\u003e. (E) Changes in the protein expressions of CASQ2, p-RyR2, PLB and SERCA2a. (F) Quantitative analysis of protein expression of CASQ2. (G) Quantitative analysis of protein expression of p-RyR2. (H) Quantitative analysis of protein expression of PLB. (I) Quantitative analysis of protein expression of SERCA2a. Data represent mean±standard error of mean, n=3. \u003cem\u003eP\u003c/em\u003e compared to control using unpaired, two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/04bb7434e34f3b4b51310307.png"},{"id":91193614,"identity":"32f07fa9-8609-48df-a9cf-595504786f28","added_by":"auto","created_at":"2025-09-12 14:45:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":570055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRyanodine improves abnormal calcium transients in DCM-hiPSC-CMs.\u003c/strong\u003e(A) Representative calcium transients of hiPSC-CMs treated with ryanodine. (B) Quantitation of amplitude and duration in calcium transients treated with ryanodine. (C) Representative calcium transients of hiPSC-CMs treated with 2-APB. (D) Quantitation of amplitude and duration in calcium transients treated with 2-APB. (E) Representative calcium transients of hiPSC-CMs treated with verapamil. (F) Quantitation of amplitude and duration in calcium transients treated with verapamil. (G) Representative calcium transients of hiPSC-CMs treated with KN93. (H) Quantitation of amplitude and duration in calcium transients treated with KN93. Data represent mean±standard error of mean, n=3. \u003cem\u003eP\u003c/em\u003ecompared to control using one-way ANOVA.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/09336fb15e5a39144cb92c49.png"},{"id":103251049,"identity":"d802d196-395a-42ca-b3dc-1146c21374a8","added_by":"auto","created_at":"2026-02-23 16:02:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8378606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/939b032c-fc6d-499b-9f19-f2564b1c4de5.pdf"},{"id":91193594,"identity":"4a0a3e9b-b521-4a0f-99ae-5da78461529d","added_by":"auto","created_at":"2025-09-12 14:45:33","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":500276,"visible":true,"origin":"","legend":"","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/706554cda43cd0b257ffb015.png"},{"id":91193603,"identity":"4231b4d1-a7c3-42d3-9508-3f05e7fa519c","added_by":"auto","created_at":"2025-09-12 14:45:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3858294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.25SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7455786/v1/8dcba35ddbc3fbf73ce14805.docx"}],"financialInterests":"","formattedTitle":"MYBPC3 (c.194CT) mutation-mediated RyR2 dysfunction contributes to pathogenic phenotypes of DCM revealed by hiPSC modeling","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eCardiomyocytes differentiated from patient-specific induced pluripotent stem cells (hiPSCs) reproduces morphology of cardiac hypertrophy and sarcomeric disorders.\u003c/li\u003e\n \u003cli\u003eA novel c.194C\u0026gt;T mutation in \u003cem\u003eMYBPC3\u003c/em\u003e results in abnormal calcium transients in hiPSC-derived cardiomyocytes.\u003c/li\u003e\n \u003cli\u003ec.194C\u0026gt;T\u003cem\u003e\u0026nbsp;\u003c/em\u003emutation of \u003cem\u003eMYBPC3\u003c/em\u003e leads to a significant increase in the expression of calsequestrin that binds to the ryanodine receptor 2 (RyR2).\u003c/li\u003e\n \u003cli\u003eTreatment with RyR2 inhibitor markedly improves the ability of calcium handling in DCM-hiPSC-cardiomyocytes.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eDilated cardiomyopathy (DCM) is a severe cardiovascular disorder characterized by left ventricular dilation and systolic dysfunction, often accompanied by arrhythmias, heart failure, thromboembolic events, and an increased risk of sudden death\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The global prevalence of DCM is estimated to range between 1 in 250 and 1 in 400 individuals\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although advancements in pharmacological therapy, device-based interventions, mechanical circulatory support, and heart transplantation have improved survival rates, the development of comorbidities remains a significant concern, with a five-year survival rate remaining below 50%\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Approximately 30\u0026ndash;50% of hereditary DCM cases can be attributed to known genetic mutations, including those affecting sarcomere proteins, cytoskeletal components, ion channels, and other relevant molecular pathways\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMYBPC3\u003c/em\u003e encodes the cardiac isoform of myosin-binding protein C (cMyBP-C), which is a crucial regulatory protein for cardiac contraction. It is located in the cross-bridge-bearing region between myosin and actin\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Mutations in the \u003cem\u003eMYBPC3\u003c/em\u003e gene elevate the risk of developing various forms of cardiomyopathy, including DCM\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Mutations at distinct sites may impair the interaction between cMyBP-C and other proteins, leading to a reduction in functionality\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDue to ethical constraints, acquiring human heart tissues or primary cardiomyocytes (CMs) from DCM patients presents significant challenges\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Moreover, there are substantial differences in genetic composition, cardiac anatomy, and electrophysiology between humans and animals\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Human induced pluripotent stem cells (hiPSCs), which possess the ability to unlimited self-renew and the capability to differentiate into all three germ layers similar to human embryonic stem cells (hESCs), have emerged as a novel tool for advancing DCM research\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) provide a theoretical foundation for investigating the pathophysiology of hereditary DCM and developing cardiovascular therapies by accurately modeling human DCM \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we examined a patient with DCM who harbors a novel \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) point mutation and generated hiPSC-CMs. These hiPSC-CMs exhibited hypertrophic morphology, disorganized sarcomeres, aberrant calcium handling, and upregulated expression of genes associated with hypertrophy. Notably, we observed a significant increase in the expression of CASQ2, which encodes calsequestrin, a protein that interacts with the Ryanodine receptor 2 (RyR2). Our findings suggest that RyR2 may represent a promising therapeutic target for DCM patients exhibiting calcium dysregulation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePatient recruitment.\u003c/b\u003e Patients were recruited in the study following the protocols with informed consent approved by Medical Ethics Committee of Sun Yat-Sen Memorial Hospital, the ethical approval number is SYSKY-2022-010-01. Echocardiogram and electrocardiogram were performed on all patients. Peripheral blood was extracted from patients, and the entire exome sequencing process was carried out at the Guangzhou DaAn Clinical Laboratory Center.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman iPSCs reprogramming and culture.\u003c/b\u003e Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of the patient and reprogrammed to hiPSCs using a CytoTune\u0026trade;-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) on the basis of user guide. PBMCs were plated at 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL to a 24-well plate in complete StemPro\u0026trade;-34 medium (Gibco) supplemented with SCF (100 ng/mL), FLT-3 (100 ng/mL), IL-3 (20 ng/mL), and IL-6 (20 ng/mL). After four days, the cells were transduced by the reprogramming vectors at the appropriate MOI and incubated overnight. Twenty-four hours later, the cells were removed into fresh complete PBMC medium and cultured for two days. The transduced cells were then plated in MEF feeder-cells culture wells of a 24-well plate in complete StemPro\u0026trade;-34 medium. The cells were gradually transitioned into mTeSR\u0026trade; 1 medium (Stem Cell Technologies) by replacing half of the StemPro\u0026trade;-34 medium with mTeSR\u0026trade; 1 medium. Nearly two weeks later stem cell-like colonies were picked and transferred on Matrigel-coated plates (Corning) in mTeSR\u0026trade; 1 medium under conditions at 37 ℃ with 5% CO2. Cells were dissociated with ReLeSR (StemCell Technologies) with mTeSR\u0026trade; 1 medium supplemented with 10 \u0026micro;M Y-27632 (MCE).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman iPSCs characteristics.\u003c/b\u003e AP staining was performed using a Alkaline Phosphatase Assay Kit (Applygen) according to manufacturer's instructions. Karyotyping was analyzed by Guangzhou DaAn Clinical Laboratory Center. Sanger sequencing was performed by Guangzhou IGE Biotechnology Co., Ltd.. Genomic DNA from hiPSCs was extracted using a Kit (TIANGEN) according to the manufacturer\u0026rsquo;s instructions. Polymerase chain reaction was performed using EasyTaq\u0026reg; DNA polymerase.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative RT-PCR.\u003c/b\u003e Total RNA was extracted using RNAzol (MOLECULAR RESEARCH) according to the manufacturer\u0026rsquo;s instructions. RNA was reverse transcribed in a 20-\u0026micro;L reaction system using a Reverse Transcription kit (Novoprotein). Resulting cDNA was used in qPCR using SYBR qPCR Master Mix (Vazyme) and a LightCycler480 Detection System (Roche). The relative expression of target genes was calculated by the comparative quantification method (2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e) and was normalized to the average expression of GAPDH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence staining.\u003c/b\u003e The hiPSCs were seeded in Matrigel-coated 24-well plate and cultured for two or three days. The cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After permeabilizing with 0.3% Triton-X (BIOFROXX) at room temperature for an additional 15 minutes, the cells were incubated with 10% donkey serum (LOUNBIOTECH) and followed with primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at 4 ℃ overnight. After washing, cells were incubated with corresponding secondary antibodies Alex Fluor 488/555/647 (Invitrogen) in the dark for 1h at room temperature. Cells were counterstained with DAPI (Beyotime) for 5 minutes. Images were taken on Inverted Microscopes (Leica, DMi8) or confocal laser scanning microscope (Zeiss, LSM710/780) and analyzed with ImageJ.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eWhole exome high-throughput sequencing in DCM patients.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAge Range\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLocation of Genome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVariant information\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003emode of inheritance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36\u0026ndash;40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMYBPC3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003echr11: 4737288835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNM_000256.3:\u003c/p\u003e\u003cp\u003ec.194C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003ep.Thr65Met\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026ndash;80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTNNT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003echr1:\u003c/p\u003e\u003cp\u003e201332518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNM_001276345.2: c.506G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003ep.Arg169Gln\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u0026ndash;35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTTN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003echr2:\u003c/p\u003e\u003cp\u003e179486248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNM_001267550.2: c.45303C\u0026thinsp;\u0026gt;\u0026thinsp;A p.Asn15101Lys\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003efemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u0026ndash;55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDSP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003echr6:\u003c/p\u003e\u003cp\u003e7574888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNM_004415.4:\u003c/p\u003e\u003cp\u003ec.2298-2A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u0026ndash;35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTTN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003echr2:\u003c/p\u003e\u003cp\u003e179605357\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNM_001267550.2: c.12602dup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eAD: Autosomal Dominant Inheritance\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCardiomyocyte differentiation.\u003c/b\u003e hiPSCs were differentiated to cardiomyocytes using a small molecule Wnt-activation/inhibition protocol with heparin previously described. Briefly, hiPSCs were first treated with CHIR99021 (5 \u0026micro;M, MCE) in E8 basal medium for 24 hours, and then with IWP2 (3 \u0026micro;M, MCE) for another 72 hours on day 2 post-differentiation. Heparin (3\u0026micro;g/mL, Selleck) was added into the medium on day 1 and removed on day 7. The media was then replaced with E8 basal medium with insulin (20 \u0026micro;g/mL, Sigma) and refreshed every 2 days. Spontaneously beating clusters was normally observed on day 6 to 7 post-differentiation. On day 11 post-differentiation, hiPSC-CMs were metabolically selected in DMEM without glucose (Gibco) supplemented with lactate (20 mM, Sigma) for 96 hours. All experiments were conducted by using hiPSC-CMs between 30 and 35 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry.\u003c/b\u003e HiPSC-CMs on day 10 post-differentiation in monolayer were dissociated with 1xTrypLE (Gibco) for 5 minutes. The cells were fixed by 4% PFA for 15 minutes and permeabilized by 0.3% Triton X-100 for 30 minutes at room temperature. Then, the cells were incubated with primary antibodies for 1 hour. After washing, the cells were incubated with second antibodies Alex Fluor 488 (1:1,500 diluted in PBS, Invitrogen) for 30 minutes. The cells were washed three times and suspended in PBS for the flow cytometry assay. The data was analyzed with the CytExpert software (BD Biosciences).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWheat germ agglutinin staining.\u003c/b\u003e Wheat germ agglutinin staining was performed using iFluor\u0026reg; 488-Wheat Germ Agglutinin (WGA) Conjugate (AAT Bioquest) according to the manufacturer\u0026rsquo;s instructions. The cells were incubated at 37 ℃ for 20 minutes. Images were taken on Inverted Microscopes (Leica, DMi8) or confocal laserscanning microscope (Zeiss, LSM710/780) and analyzed with ImageJ.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission Electron Microscopy.\u003c/b\u003e Human iPSC-CMs were fixed in 2.5% glutaraldehyde fixed solution at room temperature for 5 minutes in the dark. Then, the cells were scraped off, washed and centrifuged. The cells were fixed for an additional 30 minutes and sent to Wuhan Servicebio Technology Co., Ltd. for embedding and staining.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCalcium transient analysis.\u003c/b\u003e hiPSC-CMs were treated with 5 \u0026micro;M Fluo-4 AM (Thermo Scientific) in the Tyrode\u0026rsquo;s solution (140.0 \u0026micro;M NaCl, 5.0 \u0026micro;M KCl, 2 \u0026micro;M MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M HEPES, 1.8 \u0026micro;M CaCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M glucose, pH 7.4) for 30 minutes at 37 ℃. The cells were then washed for three times with the Tyrode\u0026rsquo;s solution and incubated at room temperature for 20 min before use. Calcium traces were captured in the line-scan model using confocal laserscanning microscope (Zeiss, LSM710/780) with a 63\u0026times; objective. The calcium transient analysis were performed with ImageJ.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot.\u003c/b\u003e Total protein was extracted by RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail for 30 minutes on ice. Protein concentration was analyzed using a BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer\u0026rsquo;s protocol. An equal amount of protein (10\u0026ndash;20 \u0026micro;g) was loaded onto a 10\u0026ndash;12% TGX Stain-Free FastCast Acrylamide gel (Bio-Rad) at 200V for 30 minutes. The proteins were transferred to a PVDF membrane using the Trans-blot Turbo system (Bio-Rad). Membranes were then blocked in 5% BSA diluted in TBST at room temperature for 1 hour. The membranes were incubated with primary antibodies at 4℃ overnight. After incubated with secondary antibody at room temperature for 1 hour, the bands were visualized with chemiluminescence imaging system (Bio-Rad).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analysis was performed with GraphPad Prism (version 9) using Student\u0026rsquo;s t-test or one-way ANOVA. All data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean from at least three independent experiments. \u003cem\u003eP\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePatient with DCM carries a pathogenic missense mutation (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) in the\u003c/b\u003e \u003cb\u003eMYBPC3\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003cp\u003e We obtained peripheral blood samples from nine patients diagnosed with DCM at Sun Yat-sen Memorial Hospital with informed consent. Whole exome high-throughput sequencing identified genetic mutations associated with DCM in five of these patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, we focused on a male patient who was found to carry a novel pathogenic missense mutation in the \u003cem\u003eMYBPC3\u003c/em\u003e gene (chr11:4737288835, NM_000256.3: c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.T65M). This mutation results in an amino acid substitution at position 65 of the cMyBP-C, changing threonine (T) to methionine (M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Clinically, the patient exhibited classic symptoms of DCM, such as nocturnal dyspnea, exertional dyspnea, chest tightness, and decreased exercise tolerance. Echocardiography revealed diffuse hypokinesis of the ventricular walls and biventricular dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). An electrocardiogram (ECG) indicated 13 premature ventricular contractions over a 24-hour period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenerating DCM patient-specific human induced pluripotent stem cells (DCM-hiPSCs) carrying\u003c/b\u003e \u003cb\u003eMYBPC3\u003c/b\u003e \u003cb\u003e(c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo generate DCM-hiPSCs retaining all genetic information, peripheral blood mononuclear cells were isolated via density gradient centrifugation. These cells were subsequently reprogrammed into hiPSCs using a Sendai virus kit and maintained through approximately 15 passages with stable growth. Colonies exhibiting positive alkaline phosphatase staining and characteristic hESC-like morphology, including large nuclei, high nucleocytoplasmic ratios, and tightly packed cell arrangements, were selected, expanded, and established as hiPSC lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After extended passaging, karyotype analysis confirmed that the cells retained normal morphology and chromosomal counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). DNA sequencing validated the presence of the specific \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation in the DCM-hiPSC line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The pluripotency of the DCM-hiPSC line was further confirmed by PCR analysis, which demonstrated robust expression of pluripotency markers \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eSOX2\u003c/em\u003e, as well as the absence of Sendai virus vectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similar mRNA expression levels of \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eSOX2\u003c/em\u003e genes were observed between DCM-hiPSCs and control hiPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Immunostaining revealed high expression levels of pluripotency markers OCT4, SOX2, and SSEA4 in DCM-hiPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Additionally, DCM-hiPSCs maintained the capacity to differentiate into cells representing all three germ layers in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The characterization of the control group is detailed in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Collectively, these findings demonstrate the successful generation of a DCM-hiPSC line suitable for further research into disease mechanisms and potential therapeutic targets.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDifferentiation of DCM-hiPSCs into spontaneous contracting CMs\u003c/h3\u003e\n\u003cp\u003eSubsequently, we generated DCM patient-specific CMs (DCM-hiPSC-CMs) from hiPSCs utilizing a previously established small molecule-based protocol\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We observed progressive changes in cell morphology and spontaneous contractions as early as day 7 post-differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The hiPSCs were successfully differentiated into hiPSC-CMs, which exhibited characteristic cardiomyocyte markers (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and S2A). Flow cytometric analysis indicated that up to 90% of the cells were successfully differentiated into cardiomyocytes (Figure S2B). We also carried out differentiation on another clone and obtained the same result (Figure S3). The spontaneously contracting hiPSC-CMs, after 30 to 35 days of differentiation, were utilized for further analysis. Additionally, we predicted the mRNA secondary structure of \u003cem\u003eMYBPC3\u003c/em\u003e using RNAfold software (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The mutation site disrupted the stem-loop structure of the mRNA, potentially contributing to the downregulation of endogenous \u003cem\u003eMYBPC3\u003c/em\u003e expression in DCM-hiPSC-CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To further elucidate this effect, we conducted Western blot analysis to assess the abundance of cMyBP-C protein; however, no significant differences were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, immunofluorescence imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) demonstrated that cMyBP-C localized to the sarcomere and exhibited a punctate distribution under high magnification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of the DCM-hiPSC-CMs carrying\u003c/b\u003e \u003cb\u003eMYBPC3\u003c/b\u003e \u003cb\u003e(c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe morphology of control hiPSC-CMs (n\u0026thinsp;=\u0026thinsp;143) was predominantly round or oval and exhibited a relatively uniform appearance, whereas DCM-hiPSC-CMs (n\u0026thinsp;=\u0026thinsp;128) displayed more diverse morphologies with significantly larger area compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The relative mRNA expression levels of hypertrophic markers \u003cem\u003eNPPA\u003c/em\u003e and \u003cem\u003eNPPB\u003c/em\u003e were markedly elevated in DCM-hiPSC-CMs, indicating that DCM can induce cardiomyocyte hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Transmission electron microscopy and immunostaining were employed to evaluate myofibril organization in hiPSC-CMs. DCM-hiPSC-CMs demonstrated increased variability in sarcomeric organization, characterized by a higher number of poorly aligned Z lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and a punctate distribution of sarcomeric α-actinin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo examine the functional characteristics of the beating DCM-hiPSC-CMs, we employed video imaging to track individual beating clusters at 10, 20, and 30 days post-differentiation. The beating rate of DCM-hiPSC-CMs exhibited a significant decline from 61.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 bpm at day 10 to 10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 bpm at day 20, and further decreased to 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 bpm at day 30, in comparison with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Subsequently, we utilized fluorescent Ca\u0026sup2;⁺ imaging to evaluate the Ca\u0026sup2;⁺ handling properties during excitation-contraction coupling. Aberrant calcium transients were observed in DCM-hiPSC-CMs, suggesting impaired function of Ca\u0026sup2;⁺-related channels and components in both the plasma membrane and sarcoplasmic reticulum (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Furthermore, DCM-hiPSC-CMs exhibited markedly larger intracellular Ca\u0026sup2;⁺ transient amplitudes relative to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Additionally, the DCM-hiPSC-CMs showed prolonged time to peak and delayed decay of the calcium transient, which further underscores the impaired calcium handling observed in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMYBPC3\u003c/b\u003e \u003cb\u003e(c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation upregulated the expression of CASQ2 and disrupted calcium homeostasis.\u003c/b\u003e To gain deeper insights into the impact of the \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation on DCM, we conducted RNA sequencing (RNA-seq) analysis on hiPSC-CMs from both control and patient-specific DCM-hiPSCs. We identified differentially expressed genes (DEGs), with 180 up-regulated and 117 down-regulated genes common to both groups. These DEGs were subjected to comprehensive pathway and functional enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results indicated that the DEGs were significantly enriched in calcium ion binding functions at the molecular level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), as shown by the expression heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Key genes involved in calcium ion binding, including \u003cem\u003eCASQ2\u003c/em\u003e, \u003cem\u003eDUOX2\u003c/em\u003e, \u003cem\u003ePCDHGA3\u003c/em\u003e, \u003cem\u003eSMOC2\u003c/em\u003e, \u003cem\u003eACAN\u003c/em\u003e, and \u003cem\u003eFBLN5\u003c/em\u003e, were validated using real-time quantitative PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Notably, \u003cem\u003eCASQ2\u003c/em\u003e which encodes calsequestrin exhibited the most significant upregulation in DCM-hiPSC-CMs and is associated with myocardial contraction, further implicating its role in the observed calcium dysregulation. We further examined its protein level and found that CASQ2 was significantly up-regulated in the DCM-hiPSC-CMs group. Levels of other Ca\u003csup\u003e2+\u003c/sup\u003e-regulating proteins, including p-RyR2, RyR2, sarcoplasmic or endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e ATPase 2 (SERCA2) and phospholamban (PLB), were evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-I). Among these proteins, p-RyR2 and PLB showed levels that were significantly elevated, while SERCA2 showed levels that were significantly decreased. All these results suggest that the \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation causes a change in intracellular calcium homeostasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRyanodine receptor 2 (RyR2) inhibitor restored abnormal calcium transients in DCM-hiPSC-CMs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCalsequestrin, a calcium-binding protein localized in the sarcoplasmic reticulum of cardiomyocytes, interacts directly or indirectly with RyR2. To elucidate the mechanisms underlying \u003cem\u003eCASQ2\u003c/em\u003e upregulation in DCM-hiPSC-CMs, we evaluated the functional responses of both control and DCM-hiPSC-CMs to various inhibitors. Specifically, we examined the effects of the RyR2 inhibitor ryanodine (1 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), the IP3R inhibitor 2-Aminoethyl diphenylborinate (2-APB, 2.5 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the voltage-dependent Ca\u0026sup2;⁺ channel inhibitor verapamil (1 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), and the calmodulin-dependent kinase II (CaMKII) inhibitor KN93 (1 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). In DCM-hiPSC-CMs, all treatments significantly attenuated the peak of Ca\u0026sup2;⁺ transients, resulting in a reduction in transient amplitude (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Notably, only ryanodine treatment was capable of restoring the frequency of Ca\u0026sup2;⁺ transients in DCM-hiPSC-CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Collectively, these findings indicate that RyR2 may represent a promising therapeutic target for patients with DCM, especially those exhibiting impaired calcium handling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we utilized hiPSC-derived cardiomyocytes from a DCM patient as an in vitro disease model. Our findings reveal for the first time that \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) heterozygous mutant cardiomyocytes recapitulate several key characteristics of DCM, including increased cell areas, increased sarcomere disorders, and abnormal calcium handling. Additionally, we demonstrate that this novel mutation in \u003cem\u003eMYBPC3\u003c/em\u003e leads to a significant upregulation of CASQ2 gene and protein expression in DCM-hiPSC-CMs. CASQ2 encodes calsequestrin, which binds to RyR2. Treatment with a RyR2 inhibitor markedly improves calcium handling in DCM-hiPSC-CMs. These results provide a robust foundation for understanding the underlying mechanisms of DCM and exploring potential therapeutic interventions.\u003c/p\u003e\u003cp\u003eWe identified a patient harboring a heterozygous mutation in \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T), which results in the substitution of threonine with methionine at codon 65 of the cMyBP-C protein (p.Thr65Met). This missense variant has been reported in individuals diagnosed with DCM; however, its pathogenicity remains undetermined. Furthermore, the lack of effective disease models, particularly human-based models, has hindered our understanding of the pathogenesis associated with \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutations, thereby impeding the development of effective treatments and preventive strategies for this condition. Additionally, there is a significant correlation between genotype and phenotype in DCM patients. Compared to those without genetic mutations, genetically affected patients tend to exhibit an earlier age of onset and more severe symptoms, underscoring the critical need for investigating hereditary DCM\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. We generated hiPSCs from peripheral mononuclear cells of DCM patient harboring the \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation using reprogramming technology. These hiPSCs were subsequently differentiated into cardiomyocytes \u003cem\u003ein vitro\u003c/em\u003e to establish a cellular disease model. Our findings revealed that cardiomyocytes carrying this specific mutation exhibited phenotypes characteristic of DCM\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCalcium handling plays a key critical role in regulation of excitation-contraction in CMs\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. We observed that patient-derived hiPSC-CMs exhibited abnormal Ca\u003csup\u003e2+\u003c/sup\u003e handling from the initial stage of cardiomyocyte differentiation until maturation, especially after 35 days, exhibiting higher amplitude of Ca\u003csup\u003e2+\u003c/sup\u003e transient, prolonged decay time and increased cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e level. To investigate the mechanisms underlying the aberrant calcium processing induced by gene mutations, RNA-seq analysis revealed that differentially expressed genes were primarily enriched in calcium ion binding functions. Notably, among several Ca\u003csup\u003e2+\u003c/sup\u003e binding-related genes examined, only \u003cem\u003eCASQ2\u003c/em\u003e, which encodes the calsequestrin protein, showed a significant increase in DCM-hiPSC-CMs. Calsequestrin is a calcium-binding protein responsible for calcium buffering in sarcoplasmic reticulum\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Functionally, calsequestrin regulates the release of Ca\u0026sup2;⁺ from the sarcoplasmic reticulum via RyR2 during excitation-contraction coupling\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Consequently, we hypothesize that \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation may influence RyR2-mediated calcium release by upregulating the expression of calsequestrin. In addition, studies have demonstrated a direct interaction between the RyR2 protein and cMyBP-C protein, with cMyBP-C potentially inhibiting RyR2-dependent Ca\u003csup\u003e2+\u003c/sup\u003e release\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Based on these findings, we hypothesize that the \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation may weaken the inhibitory effect of cMyBP-C on RyR2, leading to sarcoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e leakage and intracellular Ca\u003csup\u003e2+\u003c/sup\u003e overload.\u003c/p\u003e\u003cp\u003eRyR2 is a large tetrameric protein that regulates Ca\u003csup\u003e2+\u003c/sup\u003e release from the sarcoplasmic reticulum in cardiomyocytes and plays a crucial role during myocardial contraction. RyR2, PLB and SERCA2a regulate myocardial calcium signal through the dynamic cooperation of \"release and recycling\". The coordination of the three functions is the core guarantee for the normal pumping function of the heart, and their imbalance is an important pathological mechanism of various heart diseases. This channel's function may be impaired in DCM\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. PLB is an endogenous inhibitor of SERCA2a, and the upregulation of PLB protein in DCM will further enhance the inhibition of SERCA2a, resulting in decreased calcium recycling capacity and prolonged cytosolic calcium retention time, which is consistent with the phenotype of prolonged time to peak, and the increase in the level of phosphorylated ryr2 further exacerbated the calcium leakage from the sarcoplasmic reticulum. In order to correct calcium homeostasis, we utilized the RyR2 modulator ryanodine to intervene in DCM-hiPSC-CMs and found that ryanodine effectively inhibited the RyR2 receptor, thereby reducing Ca\u003csup\u003e2+\u003c/sup\u003e release into the cytoplasm during myocardial excitation. Consequently, it significantly diminished the amplitude of calcium transients in DCM-hiPSC-CMs, accelerated the frequency of calcium transients, and improved the abnormal calcium handling observed in these cells. To specifically exclude the contribution of other channels to the increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e, DCM-hiPSC-CMs were individually treated with the IP3R inhibitor 2-APB, the L-type calcium channel inhibitor Verapamil, and the CaMKII inhibitor KN93. The results indicated that the calcium transient abnormalities in cardiomyocytes remained unaltered. These findings suggest that Ryanodine may serve as a promising therapeutic agent for DCM patients harboring the \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation, and RyR2 may represent a potential therapeutic target for DCM patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we identified a \u003cem\u003eMYBPC3\u003c/em\u003e (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) mutation in a patient diagnosed with DCM and established a patient-specific hiPSC model. Our study revealed significant impairments in myofilament regulation and calcium handling, as well as increased expression of \u003cem\u003eCASQ2\u003c/em\u003e, which encodes calsequestrin, a protein that binds to RyR2 in DCM-hiPSC-CMs. Treatment with a RyR2 inhibitor ameliorated the abnormal calcium transients observed in DCM-hiPSC-CMs. These findings provide a robust foundation for a deeper understanding of the underlying mechanisms of DCM and pave the way for exploring novel therapeutic interventions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ecMyBP-C, cardiac myosin-binding protein C;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCM, dilated cardiomyopathy;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehiPSC, human induced pluripotent stem cell;\u003c/p\u003e\n\u003cp\u003ehiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte;\u003c/p\u003e\n\u003cp\u003eRyR2, Ryanodine receptor 2;\u003c/p\u003e\n\u003cp\u003ePLB, phospholamban;\u003c/p\u003e\n\u003cp\u003eSERCA2, sarcoplasmic or endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e ATPase 2.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Experimental Instrument Sharing Platform of Sun Yat-sen University, the Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University, and Sun Yat-sen Memorial Hospital of Sun Yat-sen University for their assistance. We thank Prof. Nan Cao for kindly providing the WTB hiPSC line in this study. Thanks for all the contributors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number 82470367 and 82272164), the Natural Science Foundation of Guangdong Province (grant number 2024A1515013135).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManting Xie and Bingbing Xie contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCenter for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, P. R. China (510080)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManting Xie, Bingbing Xie, Liang Huang, Xingqiang Lai, Jixing Gong, Nan Cao, Andy Peng Xiang \u0026amp; Qiuling Xiang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScientific Development and Management Office, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China(510235)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManting Xie\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China(510235)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing Chen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaternal-Fetal Medicine Institute, Department of Obstetrics and Gynaecology, Shenzhen Baoan Women\u0026apos;s and Children\u0026apos;s Hospital, Shenzhen, China(518104)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXingqiang Lai\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManting Xie: Writing - original draft, Investigation, Data curation. Bingbing Xie: Investigation, Writing - review \u0026amp; editing. Liang Huang: Methodology, Writing - review \u0026amp; editing. Ying Chen: Investigation, Formal analysis. Xingqiang Lai: Data curation. Jixing Gong: Methodology. Nan Cao: review \u0026amp; editing. Andy Peng Xiang: Supervision, review \u0026amp; editing. Qiuling Xiang: financial support, conceptualization, Project administration, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this study were in accordance with the Ethical Standards Research Committee and the Helsinki declaration. This study was approved by the Medical Ethics Committee of Sun Yat-Sen Memorial Hospital(SYSKY-2022-010-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets utilized and examined in this study can be obtained from the corresponding author upon a reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHeymans S, Lakdawala NK, Tsch\u0026ouml;pe C, Klingel K. Dilated cardiomyopathy: causes, mechanisms, and current and future treatment approaches. Lancet. 2023; 402(10406): 998-1011.\u003c/li\u003e\n\u003cli\u003eReichart D, Magnussen C, Zeller T, Blankenberg S. Dilated cardiomyopathy: from epidemiologic to genetic phenotypes: A translational review of current literature. J Intern Med 2019; 286(4): 362-372.\u003c/li\u003e\n\u003cli\u003eSchultheiss HP, Fairweather D, Caforio ALP, Escher F, Hershberger RE, Lipshultz SE, Liu PP, Matsumori A, Mazzanti A, McMurray J, Priori SG. Dilated cardiomyopathy. Nat Rev Dis Primers 2019; 5(1): 32.\u003c/li\u003e\n\u003cli\u003eMcNally EM, Mestroni L. Dilated Cardiomyopathy: Genetic Determinants and Mechanisms. Circ Res 2017; 121(7): 731-748.\u003c/li\u003e\n\u003cli\u003eEldemire R, Mestroni L, Taylor MRG. Genetics of Dilated Cardiomyopathy. Annu Rev Med 2024; 75: 417-426.\u003c/li\u003e\n\u003cli\u003eKaski JP, Cannie D. Clinical Screening for Dilated Cardiomyopathy in At-Risk First-Degree Relatives: Who, When, and How? J Am Coll Cardiol 2023; 81(21): 2072-2074.\u003c/li\u003e\n\u003cli\u003eRosenbaum AN, Agre KE, Pereira NL. Genetics of dilated cardiomyopathy: practical implications for heart failure management. Nat Rev Cardiol 2020; 17(5): 286-297.\u003c/li\u003e\n\u003cli\u003eCarrier L, Mearini G, Stathopoulou K, Cuello F. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology. Gene 2015; 573(2): 188-197.\u003c/li\u003e\n\u003cli\u003eMahdieh N, Hosseini Moghaddam M, Motavaf M, Rabbani A, Soveizi M, Maleki M, Rabbani B, Alizadeh-Asl A. Genotypic effect of a mutation of the MYBPC3 gene and two phenotypes with different patterns of inheritance. J Clin Lab Anal 2018; 32(6): e22419.\u003c/li\u003e\n\u003cli\u003eMarian AJ. Molecular Genetic Basis of Hypertrophic Cardiomyopathy. Circ Res 2021; 128(10): 1533-1553.\u003c/li\u003e\n\u003cli\u003ede Frutos F, Ochoa JP, Fern\u0026aacute;ndez AI, Gallego-Delgado M, Navarro-Pe\u0026ntilde;alver M, Casas G, Basurte MT, Larra\u0026ntilde;aga-Moreira JM, Mogoll\u0026oacute;n MV, Robles-Mezcua A, Garc\u0026iacute;a-Granja PE, Climent V, Palomino-Doza J, Garc\u0026iacute;a-\u0026Aacute;lvarez A, Brion M, Brugada R, Jim\u0026eacute;nez-J\u0026aacute;imez J, Bayes-Genis A, Ripoll-Vera T, Pe\u0026ntilde;a-Pe\u0026ntilde;a ML, Rodr\u0026iacute;guez-Palomares JF, Gonzalez-Carrillo J, Villacorta E, Espinosa MA, Garcia-Pavia P, Mirelis JG. Late gadolinium enhancement distribution patterns in non-ischaemic dilated cardiomyopathy: genotype-phenotype correlation. Eur Heart J Cardiovasc Imaging 2023; 25(1): 75-85.\u003c/li\u003e\n\u003cli\u003eBlagova O, Pavlenko E, Sedov V, Kogan E, Polyak M, Zaklyazminskaya E, Lutokhina Y. Different Phenotypes of Sarcomeric MyBPC3-Cardiomyopathy in the Same Family: Hypertrophic, Left Ventricular Noncompaction and Restrictive Phenotypes (in Association with Sarcoidosis). Genes (Basel) 2022; 13(8).\u003c/li\u003e\n\u003cli\u003eRasicci DV, Tiwari P, Bodt SML, Desetty R, Sadler FR, Sivaramakrishnan S, Craig R, Yengo CM. Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin. Elife 2022; 11.\u003c/li\u003e\n\u003cli\u003eYang KC, Breitbart A, De Lange WJ, Hofsteen P, Futakuchi-Tsuchida A, Xu J, Schopf C, Razumova MV, Jiao A, Boucek R, Pabon L, Reinecke H, Kim DH, Ralphe JC, Regnier M, Murry CE. Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells. JACC Basic Transl Sci 2018; 3(6): 728-740.\u003c/li\u003e\n\u003cli\u003eToepfer CN, Wakimoto H, Garfinkel AC, McDonough B, Liao D, Jiang J, Tai AC, Gorham JM, Lunde IG, Lun M, Lynch TLt, McNamara JW, Sadayappan S, Redwood CS, Watkins HC, Seidman JG, Seidman CE. Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin. Sci Transl Med 2019; 11(476).\u003c/li\u003e\n\u003cli\u003eAnsari AA, Wang YC, Danner DJ, Gravanis MB, Mayne A, Neckelmann N, Sell KW, Herskowitz A. Abnormal expression of histocompatibility and mitochondrial antigens by cardiac tissue from patients with myocarditis and dilated cardiomyopathy. Am J Pathol 1991; 139(2): 337-354.\u003c/li\u003e\n\u003cli\u003eKatagiri T, Kitsu T, Akiyama K, Takeyama Y, Niitani H. Alterations in fine structures of myofibrils and structural proteins in patients with dilated cardiomyopathy--studies with biopsied heart tissues. Jpn Circ J 1987; 51(6): 682-688.\u003c/li\u003e\n\u003cli\u003eKarakikes I, Ameen M, Termglinchan V, Wu JC. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Insights Into Molecular, Cellular, and Functional Phenotypes. Circulation Research 2015; 117(1): 80-88.\u003c/li\u003e\n\u003cli\u003eNtelios D, Meditskou S, Efthimiadis G, Pitsis A, Zegkos T, Parcharidou D, Theotokis P, Alexouda S, Karvounis H, Tzimagiorgis G. \u0026alpha;-Myosin heavy chain (MYH6) in hypertrophic cardiomyopathy: Prominent expression in areas with vacuolar degeneration of myocardial cells. Pathol Int 2022; 72(5): 308-310.\u003c/li\u003e\n\u003cli\u003eThomas D. Allometric Relations and Scaling Laws for the Cardiovascular System of Mammals. Systems 2014; 2(2): 168-185.\u003c/li\u003e\n\u003cli\u003eTakahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126(4): 663-676.\u003c/li\u003e\n\u003cli\u003eKehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108(3): 407-414.\u003c/li\u003e\n\u003cli\u003eSmith AS, Macadangdang J, Leung W, Laflamme MA, Kim DH. Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening. Biotechnol Adv 2017; 35(1): 77-94.\u003c/li\u003e\n\u003cli\u003evan Lint FHM, Mook ORF, Alders M, Bikker H, Lekanne Dit Deprez RH, Christiaans I. Large next-generation sequencing gene panels in genetic heart disease: yield of pathogenic variants and variants of unknown significance. Neth Heart J 2019; 27(6): 304-309.\u003c/li\u003e\n\u003cli\u003eLin Y, Linask KL, Mallon B, Johnson K, Klein M, Beers J, Xie W, Du Y, Liu C, Lai Y, Zou J, Haigney M, Yang H, Rao M, Chen G. Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes. Stem Cells Transl Med 2017; 6(2): 527-538.\u003c/li\u003e\n\u003cli\u003eGacita AM, Fullenkamp DE, Ohiri J, Pottinger T, Puckelwartz MJ, Nobrega MA, McNally EM. Genetic Variation in Enhancers Modifies Cardiomyopathy Gene Expression and Progression. Circulation 2021; 143(13): 1302-1316.\u003c/li\u003e\n\u003cli\u003eZhang XL, Xie J, Lan RF, Kang LN, Wang L, Xu W, Xu B. Genetic Basis and Genotype-Phenotype Correlations in Han Chinese Patients with Idiopathic Dilated Cardiomyopathy. Sci Rep 2020; 10(1): 2226.\u003c/li\u003e\n\u003cli\u003eStreckfuss-B\u0026ouml;meke K, Tiburcy M, Fomin A, Luo X, Li W, Fischer C, \u0026Ouml;zcelik C, Perrot A, Sossalla S, Haas J, Vidal RO, Rebs S, Khadjeh S, Meder B, Bonn S, Linke WA, Zimmermann WH, Hasenfuss G, Guan K. Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes. J Mol Cell Cardiol 2017; 113: 9-21.\u003c/li\u003e\n\u003cli\u003eShah D, Virtanen L, Prajapati C, Kiamehr M, Gullmets J, West G, Kreutzer J, Pekkanen-Mattila M, Heli\u0026ouml; T, Kallio P, Taimen P, Aalto-Set\u0026auml;l\u0026auml; K. Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells. Cells 2019; 8(6).\u003c/li\u003e\n\u003cli\u003ePowers JD, Malingen SA, Regnier M, Daniel TL. The Sliding Filament Theory Since Andrew Huxley: Multiscale and Multidisciplinary Muscle Research. Annu Rev Biophys, 2021, 50: 373-400.\u003c/li\u003e\n\u003cli\u003eSibbles ET, Waddell HMM, Mereacre V, Jones PP, Munro ML. The function and regulation of calsequestrin-2: implications in calcium-mediated arrhythmias. Biophys Rev. 2022 Jan 7;14(1):329-352.\u003c/li\u003e\n\u003cli\u003eWleklinski MJ, Kryshtal DO, Kim K, Parikh SS, Blackwell DJ, Marty I, Iyer VR, Knollmann BC. Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2. Circ Res. 2022 Sep 30;131(8):673-686.\u003c/li\u003e\n\u003cli\u003eStanczyk PJ, Seidel M, White J, Viero C, George CH, Zissimopoulos S, Lai FA. Association of cardiac myosin-binding protein-C with the ryanodine receptor channel - putative retrograde regulation? J Cell Sci. 2018 Aug 3;131(15):jcs210443. \u003c/li\u003e\n\u003cli\u003eSteinberg C, Roston TM, van der Werf C, Sanatani S, Chen SRW, Wilde AAM, Krahn AD. RYR2-ryanodinopathies: from calcium overload to calcium deficiency. Europace. 2023 Jun 2;25(6):euad156. \u003c/li\u003e\n\u003cli\u003eWescott AP, Jafri MS, Lederer WJ, Williams GS. Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling. J Mol Cell Cardiol. 2016 Mar;92:82-92.\u003c/li\u003e\n\u003cli\u003eDulhunty AF, Wium E, Li L, Hanna AD, Mirza S, Talukder S, Ghazali NA, Beard NA. Proteins within the intracellular calcium store determine cardiac RyR channel activity and cardiac output. Clin Exp Pharmacol Physiol 2012; 39(5): 477-484.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"dilated cardiomyopathy, human induced pluripotent stem cells, human induced pluripotent stem cell-derived cardiomyocyte, MYBPC3","lastPublishedDoi":"10.21203/rs.3.rs-7455786/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7455786/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDilated cardiomyopathy (DCM) is a leading cause of heart failure and the primary indication for heart transplantation. The intricate and poorly elucidated pathogenesis of genetic DCM, coupled with the paucity of effective therapeutic options, imposes a substantial burden on both patients and their families. In this study, we identified a novel \u003cem\u003eMYBPC3\u003c/em\u003e mutation (c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T) in a patient diagnosed with DCM and established a patient-specific human induced pluripotent stem cell (hiPSC) model. Cardiomyocytes derived from these patient-specific hiPSCs (hiPSC-CMs) exhibited hallmark features of DCM, including hypertrophic cell size, aberrant distribution of sarcomeric α-actinin, and dysregulated calcium ion homeostasis, as compared to control hiPSC-CMs derived from a healthy individual. RNA sequencing analysis revealed a significant upregulation of \u003cem\u003eCASQ2\u003c/em\u003e, which encodes calsequestrin, a protein that binds to Ryanodine receptor 2 (RyR2). Notably, treatment with the RyR2 inhibitor ryanodine effectively restored the abnormal calcium transients observed in DCM-hiPSC-CMs. In summary, our findings provide compelling evidence that the c.194C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation of \u003cem\u003eMYBPC3\u003c/em\u003e plays a definitive pathogenic role in DCM, and that modulation of the RyR2 receptor may alleviate calcium dysregulation in affected cardiomyocytes. These insights enhance our understanding of the molecular mechanisms underlying DCM and offer a promising therapeutic strategy for patients with calcium ion dysregulation associated with this condition.\u003c/p\u003e","manuscriptTitle":"MYBPC3 (c.194CT) mutation-mediated RyR2 dysfunction contributes to pathogenic phenotypes of DCM revealed by hiPSC modeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:45:28","doi":"10.21203/rs.3.rs-7455786/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2025-10-28T09:41:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-13T09:56:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T17:04:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T09:35:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-08-27T03:49:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d66b2fdb-6733-4987-b30f-e7d3b8f21a23","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:00:43+00:00","versionOfRecord":{"articleIdentity":"rs-7455786","link":"https://doi.org/10.1007/s00018-026-06130-3","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2026-02-18 15:57:23","publishedOnDateReadable":"February 18th, 2026"},"versionCreatedAt":"2025-09-12 14:45:28","video":"","vorDoi":"10.1007/s00018-026-06130-3","vorDoiUrl":"https://doi.org/10.1007/s00018-026-06130-3","workflowStages":[]},"version":"v1","identity":"rs-7455786","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7455786","identity":"rs-7455786","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}