SNTA1-deficient human cardiomyocytes show shorter field potential duration and slower conduction velocity

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Previous studies on SNTA1 have predominantly utilized nonhuman cardiomyocyte models. This study aims to elucidate the phenotype of SNTA1 deficiency using human cardiomyocytes. Using CRISPR/Cas9 technology, we generated SNTA1 knockout (KO) embryonic stem cell line, which were subsequently differentiated into cardiomyocytes using 2D differentiation method. Genotype analysis identified an adenine (A) insertion in the second exon of SNTA1 , resulting in a premature stop codon at the 149th amino acid position and truncation within the PDZ domain. SNTA1 -deficient cardiomyocytes exhibited a shortened action potential duration (FPD) and slower conduction velocity, as detected by micro electrode array analysis. Immunofluorescence analysis further revealed disorganized distribution of SCN5A protein in SNTA1 -deficient cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmias and plays a critical role as an essential auxiliary protein in the proper localization of SCN5A in human cardiomyocytes. Biological sciences/Stem cells/Embryonic stem cells Biological sciences/Stem cells/Stem cell differentiation Health sciences/Diseases/Cardiovascular diseases/Arrhythmias Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects Human embryonic stem cell SNTA1-defcient cardiomyocytes SCN5A Arrhythmia Action potential duration Conduction velocity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction SNTA1 is a member of the membrane-associated adaptor protein family, consisting of 505 amino acid residues in its unprocessed form, with a molecular weight of approximately 58 kDa [ 1 ]. SNTA1 contains three distinct domains: PH1, PH2, and SU. It is predominantly distributed beneath muscle membranes and at neuromuscular junctions [ 2 ]. As a peripheral cytoplasmic membrane protein, SNTA1 is primarily associated with dystrophin and dystrophin-related proteins, such as glycoproteins, utrophin, and dystrobrevin, within the dystrophin glycoprotein complex (DGC), also referred to as the dystrophin-associated protein complex (DAPC) [ 3 – 5 ]. Within the DGC, SNTA1 plays a crucial role in supporting the proper subcellular localization of associated functional proteins. G-proteins are a critical class of signal-transducing proteins located on the inner surface of the cell membrane, where they associate with transmembrane receptors. The N-terminal region of the PH1 domain in SNTA1 and the C-terminal region of the SU domain have been shown to mediate binding with the Gα subunit of heterotrimeric G-proteins, contributing to the regulation of their functions [ 6 ]. Calmodulin (CaM) transduces calcium signals by binding to Ca²⁺ ions and interacting with downstream target proteins [ 7 ]. The PH1 domain of SNTA1 interacts with CaM, playing a role in intracellular Ca²⁺ regulation [ 8 ]. SCN5A, the primary cardiac voltage-gated sodium channel alpha subunit 5, is essential for the rapid depolarization phase of the cardiac action potential and plays a key role in cardiac conduction. SNTA1 interacts with SCN5A through its PDZ domain, which binds to the internal domain of SCN5A's N-terminal [ 9 ], as well as through its association with the C-terminal of SCN5A, thereby contributing to its regulation [ 10 ]. Clinically, SNTA1 has been identified as a susceptibility locus for Long QT Syndrome 12 (LQT12), a rare arrhythmic disorder associated with an increased risk of sudden cardiac death. At present, the etiology and preventive treatment of Long QT Syndrome 12 remain inadequately understood and largely unexplored. Most current research on SNTA1 relies on animal models; however, significant differences between animal and human cardiac muscle limit their relevance for studying human cardiac arrhythmias. Induced pluripotent stem cell and gene editing technologies offer a powerful platform for creating precise human disease models to advance precision medicine [ 11 ]. Therefore, we utilized CRISPR/Cas9 technology to generate SNTA1 knockout (SNTA1-KO) human cardiomyocytes, providing a more physiologically relevant model for investigating arrhythmias resulting from SNTA1 deficiency. Our present work establishes a foundation for studying SNTA1 point mutations. Results Establishment of homozygous SNTA1 -deficient human embryonic stem cell We selected the second exon, a shared exon, as the target for SNTA1 editing. A single guide RNA (sgRNA) was designed to target this exon, with the sequence 5′-ATTGGCAGCTGACCAGACAG-3′, and the protospacer adjacent motif (PAM) sequence AGG (Fig. 1 A). The sgRNA was ligated into a linear CRISPR/Cas9 plasmid, followed by plasmid amplification. The resulting plasmid was delivered into H9 embryonic stem cells via electroporation using the LONZA Nucleofector 4D system. Post-electroporation, the cells were selected using 0.3 µg/mL puromycin. Resistant clones were picked, expanded, and subjected to genomic DNA extraction for genotyping analysis. One clone was identified with an adenine nucleotide insertion upstream of the PAM region (Fig. 1 B). This insertion introduced a premature stop codon (TGA) at amino acid position 149, truncating the SNTA1 protein in the PDZ domain which is located in the PH1 domain. This modified cell line was designated as H9SNTA1KO [ 12 ]. The H9SNTA1KO cells were cultured and compared to the parental H9 embryonic stem cells under light microscopy, revealing no discernible morphological differences (Fig. 1 C). Immunofluorescence staining confirmed positive expression of the pluripotency markers SSEA4 and OCT4 in H9SNTA1KO cells (Fig. 1 D). Furthermore, qRT-PCR analysis demonstrated that the expression levels of the pluripotency markers DPPA4, SOX2, OCT4, and NANOG in H9SNTA1KO cells were comparable to those in H9 cells, with no significant differences observed (Fig. 1 E). The results confirmed the successful establishment of H9SNTA1KO cells, which retained normal pluripotency markers. H9SNTA1KO differentiation to cardiomyocytes To obtain cardiomyocytes, we employed a 2D differentiation method as described in previous studies [ 13 – 14 ]. The process of differentiation required approximately 12 days (Fig. 2 A). Cardiomyocytes were collected at day 30 of differentiation (Fig. 2 B). Transmission electron microscopy (TEM) analysis revealed the presence of myofibrils in the cytoplasm (Fig. 2 C, yellow pentagrams), although the cells were immature, lacking T-tubules and well-organized layered myofilaments. Immunofluorescence staining was performed to detect the cardiomyocyte-specific markers TNNT2 and α-actinin, both of which were positively expressed in the H9SNTA1KO derived cardiomyocytes (KO cardiomyocytes) (Fig. 2 D). The collected cells were identified as cardiomyocytes, which exhibited spontaneous contractions. The ventricular muscle-specific marker MYL2 was assessed in both WT cardiomyocytes (H9 derived cardiomyocytes) and KO cardiomyocytes after purified medium selection using flow cytometry (Fig. 2 E). The results demonstrated that H9SNTA1KO cells differentiated into cardiomyocyte subtypes comparable to WT cells, confirming normal differentiation capacity (Fig. 2 F). All results confirmed the successful generation of H9SNTA1KO cardiomyocytes, which closely resemble those of WT. H9SNTA1KO derived cardiomyocytes showed shorter field potential duration and slower conduction velocity SNTA1, known as the LQT12 gene, has been associated with long QT syndrome (LQTS) [ 15 – 16 ]. LQTS, a hereditary condition characterized by QT interval prolongation, increases the risk of life-threatening arrhythmias. To investigate the effects of SNTA1 deficiency in cardiomyocytes, we employed the MAESTRO™ MEA SYSTEMS to assess their electrical activity (Fig. 3 A). Cardiomyocytes were seeded onto MEA (micro electrode array) plates for electrical activity analysis under normal culture conditions (Fig. 3 B). Data from the MAESTRO™ MEA SYSTEMS revealed that the beat period of KO cardiomyocytes was shorter than that of WT cardiomyocytes (Fig. 3 C’-C’’). The interval between depolarization and repolarization, referred to as the field potential duration (FPD), was extracted from field potential signals (Fig. 3 D’-D’’). Statistical analysis showed that the FPD of KO cardiomyocytes was significantly shorter than that of WT cardiomyocytes (Fig. 3 D’’’). FPD parameters can simulate the QT interval, the shortened FPD parameter predicted the shortened QT interval in KO cardiomyocytes. and abnormal QT variations indicate an increased susceptibility to arrhythmias in KO cardiomyocytes [ 16 ]. The stability of beat propagation patterns and conduction velocity was also assessed to evaluate the functional properties of the cardiomyocytes (Fig. 3 E). Statistical analysis demonstrated that the conduction velocity of KO cardiomyocytes was slower than that of WT cardiomyocytes (Fig. 3 F). Reduced conduction velocity may contribute to an increased risk of arrhythmias [ 16 ]. All the results showed there is a potential risk of arrhythmia in KO cardiomyocytes. H9SNTA1KO derived cardiomyocytes showed the disorganization of SCN5A SNTA1 interacts with both the C-terminal and N-terminal domains of SCN5A [ 9 ], facilitating the proper membrane localization of SCN5A [ 10 ]. To investigate this further, we conducted additional experiments. mRNA was extracted from 30-day-old cardiomyocytes, and SCN5A expression was analyzed. The results indicated no significant difference in SCN5A expression between the KO and WT cardiomyocytes at 30 days (Fig. 4 A). Similarly, mRNA extracted from 45-day-old cardiomyocytes showed no difference in SCN5A expression between the KO cardiomyocytes and WT cardiomyocytes (Fig. 4 B). These findings suggested that SNTA1 deficiency does not influence SCN5A transcription. The cellular localization of SCN5A was further examined using immunofluorescence staining (Fig. 4 C). The imaging results revealed a disorganized distribution of SCN5A in KO cardiomyocytes compared to the WT cardiomyocytes. Statistical analysis confirmed that the proportion of cells with disorganized SCN5A localization was significantly higher in the KO cardiomyocytes than in the WT cardiomyocytes (Fig. 4 D). Discussion As an adapter protein, SNTA1 is localized beneath the cell membrane and contains three functional domains: PH1, PH2, and SU. These domains are involved in the subcellular localization of various intracellular functional proteins, including the Gα subunit, CaM, and SCN5A [ 7 – 10 ]. Clinical reports have indicated that mutations in SNTA1 may be associated with Long QT Syndrome (LQTS) [ 15 ] and other cardiovascular phenotypes. Long QT syndrome (LQTS) encompasses a group of heritable conditions characterized by cardiac repolarization dysfunction [ 16 ]. To investigate the effects of SNTA1 mutations, we employed a human cell model. Using gene editing system, we targeted the second exon of SNTA1 in H9 embryonic stem cells. Following gene editing, we established a SNTA1 -deficient human embryonic stem cell line. Genotyping results revealed an adenine nucleotide insertion in the second exon of SNTA1, resulting in a premature termination of the protein. Thus, the H9SNTA1KO embryonic stem cell line was successfully generated [ 12 ]. The morphology of the H9SNTA1KO cell line was similar to that of the parental H9 cells, and both cell lines exhibited normal expression of pluripotency markers. We then used a chemically defined 2D differentiation method to induce cardiomyocyte differentiation [ 13 – 14 ]. No significant differences were observed between H9SNTA1KO-derived cardiomyocytes and those derived from H9 embryonic stem cells in terms of differentiation efficiency. To assess the electrical activity of cardiomyocytes, we employed the MAESTRO™ MEA SYSTEMS. The cardiomyocytes were seeded onto the MEA plate, and the data showed that the KO cardiomyocytes exhibited a shorter beat period compared to the WT cardiomyocytes. Additionally, the KO cardiomyocytes had a shorter field potential duration (FPD) and slower conduction velocity. FPD refers to the time interval between depolarization and repolarization, and it corresponds to the QT interval in an ECG. The shorter FPD observed in the KO cardiomyocytes indicates an abnormality in the depolarization and repolarization process. Clinically, alterations in the QT interval can lead to arrhythmias. Furthermore, the KO cardiomyocytes exhibited a slower conduction velocity. The measured conduction velocity reflects the collective effects of various factors, including cell culture health and pacemaker stability. Slower conduction propagation can potentially contribute to arrhythmias [ 18 ]. These results provide evidence that the electrical activity of SNTA1-deficient cardiomyocytes is unstable, potentially increasing the risk of cardiac arrhythmias. To further explore the underlying cause of the shorter FPD and slower conduction propagation in SNTA1-deficient cardiomyocytes, we focused on SCN5A. SCN5A is a voltage-gated sodium ion channel alpha subunit 5 located on the cardiac membrane, playing a crucial role in the depolarization process of cardiomyocyte action potentials [ 19 ]. While the expression of SCN5A showed no significant difference between KO and WT cardiomyocytes, SNTA1 acts as an adaptor protein that helps target functional proteins to the cell membrane. To investigate the localization of SCN5A in cardiomyocytes, immunofluorescence staining was performed. The results revealed that SCN5A localization was more disorganized in KO cardiomyocytes compared to WT cardiomyocytes. These findings suggest that the proper localization of SCN5A in cardiomyocytes requires SNTA1. Based on these observations, we speculate that SNTA1 deficiency may impair SCN5A localization, potentially increasing the likelihood of arrhythmia in the KO cardiomyocytes. However, the specific mechanisms underlying this effect require further investigation. Conclusion In this study, a human SNTA1-knockout cell model was established using the CRISPR/Cas9 system. This cell model provides a valuable tool for studying arrhythmias induced by SNTA1 deficiency in vitro. The findings underscore the critical role of SNTA1 as an essential auxiliary protein involved in the proper localization of SCN5A, a key cardiac ion channel in cardiomyocytes. SNTA1 is identified as a susceptibility locus for arrhythmias, highlighting its potential as a target for further research in cardiac electrophysiology. Methods Embryonic stem cell culture H9 embryonic stem cells were cultured in E8 medium and passaged using 0.5 mM EDTA upon reaching 80% confluence. Cells were typically passaged at a ratio of 1:6. Establishment the SNTA1KO embryonic stem cell line Using the Zhang Lab's resources, we designed an sgRNA targeting the second exon of SNTA1. The sgRNA sequence was 5′-attggcaggacag-3′. This sequence was ligated into a CRISPR/Cas9 plasmid, which was subsequently amplified by transforming E. coli cells (Top10 Competent Cells, CWBIO, China) and purified using an EndoFree Mini Plasmid Kit (TianGen, China). The plasmid was then delivered into H9 embryonic stem cells via electroporation (LONZA Nucleofector 4D). Following puromycin selection, individual clones were isolated and subjected to genotypic identification. An adenine insertion was introduced into the second exon, resulting in a premature stop codon at the 149th amino acid position of SNTA1. This successfully established the H9SNTA1KO cell line [ 11 ]. Cardiac differentiation Embryonic stem cells (ESCs) were cultured in E8 medium. When the cells reached approximately 70–80% confluence, they were passaged at a 1:6 ratio using E8 medium supplemented with 10 µM Y-27632 (ROCK inhibitor, MCE, USA). Once the cells reached 80–90% confluence, cardiac differentiation was used 2D differentiation method. The CardioEasy® mediums for differentiation were provided by the Cellapybio Inc (China). Flow Cytometry Cardiomyocytes were digested using CardioEasy® I and CardioEasy® II digestive solutions (Cellapybio, China) to prepare single-cell suspensions. The cells were then fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT), followed by two washes with PBS. Permeabilization was performed using 0.2% Triton X-100 for 5 minutes at RT. Next, the cells were incubated with the antibody for 30 minutes in the dark at RT, followed by two additional PBS washes to remove unbound antibodies. The samples were analyzed using a flow cytometer (Beckman, EPICS XL), and the results were processed with FlowJo VX software. Immunofluorescent staining Immunofluorescence staining was performed to visualize the localization of intracellular antigens. Cells were seeded on coverslips and cultured until reaching approximately 50% confluence. The medium was aspirated, and the cells were washed three times with PBS. The coverslip-adherent cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature (RT), followed by three washes with PBS. Permeabilization was performed using 0.3% Triton X-100 for 10 minutes at RT. The cells were then blocked with 3% BSA for 30 minutes at RT. After blocking, the cells were incubated with the primary antibody at 4°C for 24 hours. Following three PBS washes, they were incubated with the secondary antibody and DAPI (100 nM) for 1 hour at RT. The cells were subsequently washed three more times with PBS and imaged using a confocal microscope (Leica, TCS SP5). Both primary and secondary antibodies were used for immunofluorescence staining. Both primary and secondary antibodies are provided in Supplementary Table S2. Quantitative Real‑time PCR (qRT‑PCR) To compare gene expression at the transcriptional level between the KO and WT groups, real-time PCR was performed. Cells were seeded in a 6-well plate at a density sufficient to reach approximately 90% confluence for RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and treated with DNase I (Beyotime, China) at 37°C for 30 minutes to remove any contaminating DNA. Reverse transcription was performed using the PrimeScript™ reverse transcription system (TaKaRa, Japan). Relative gene expression levels were analyzed by quantitative real-time PCR (qRT-PCR) on an iCycler iQ5 system (Bio-Rad, USA) using TB Green™ Premix Ex Taq™ II (TaKaRa, Japan). Relative quantification of gene expression was determined using the ∆CT method. Primer sequences used for qRT-PCR are provided in Supplementary Table S1 . Electrical activity of cardiomyocytes detection To assess the electrical activity of cardiomyocytes under normal culture conditions, we utilized the MAESTRO™ MEA SYSTEMS. Matrigel working solution was prepared by diluting it 1:200 and used to coat the wells of a 24-well MEA plate overnight. A total of 20,000–30,000 cardiomyocytes were seeded onto one matrigel-coated well of the 24-well MEA plate and cultured in cardiac maintenance medium (Cellapybio, China) supplemented with 10 µM Y-27632. Once the cardiomyocytes spread and began beating regularly, their electrical activity was measured and analyzed using the MAESTRO™ MEA SYSTEMS(Axion BioSystems, Inc, US). Transmission Electron Microscope (TEM) Transmission electron microscopy was employed to examine the ultrastructure of cardiomyocytes. The medium was aspirated from the cardiomyocytes, and without rinsing, they were immediately fixed in 2.5% glutaral solution. The cells were then gently scraped off using a cell scraper and collected into a centrifuge tube (cells avoid being digested by enzymes). After centrifugation, a visible cell pellet should be obtained. The cells were fixed at RT for 2 hours with fresh electron microscope fixative. The samples were subsequently sent to Wuhan GoodBio Technology Company for further processing. Statistical methods The data are presented as mean ± standard deviation. Differences between two groups were analyzed using a one-tailed or two-tailed t-test, while rates were compared using Fisher's Exact test. For comparisons involving three or more groups, one-way or two-way analysis of variance (ANOVA) was applied, followed by Tukey's multiple comparison test. A 95% confidence interval was used, and statistical significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, representing four levels of significance. Limitation The cardiomyocytes we obtained lacked T-tubule structures, which is characteristic of immature cardiomyocytes rather than mature ones. Additionally, our study was conducted using a two-dimensional (2D) in vitro cell culture model. Abbreviations Symbol Full Name SNTA1 α-1-syntrophin hESCs human embryonic stem cells H9SNTA1KO SNTA1 -deficient H9 embryonic stem cells WT H9 embryonic stem cells WT cardiomyocytes cardiomyocytes derived from H9 embryonic stem cells KO SNTA1 -deficientH9 embryonic stem cells or H9SNTA1KO KO cardiomyocytes SNTA1 -deficient cardiomyocytes PAM protospacer adjacent motif CRISPR/Cas9 clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 TNNT2 troponin T2, cardiac type SSEA4 stage-specific embryonic antigen-4 NANOG Nanog homeobox SOX2 SRY-box transcription factor 2 DPPA4 developmental pluripotency associated 4 OCT-4 POU class 5 homeobox 1 MYL2 myosin light chain 2 MEA micro electrode array Declarations Funding Basic scientific research expenses of colleges and universities in Heilongjiang Province （2022-KYYWF-0808） Competing interests The authors declare that they have no conflict of interest. Ethics approval Title of the approved project, “Research on Differential Gene Expression in Human Cardiomyocytes with SNTA1 Deficiency”. Name of institutional approval committee or unit, “Ethics Committee of Qiqihar Medical University”. The approval number, (#77/2022). Date of approval. (12/12/2022). Consent to participate Not applicable Consent for publication Not applicable Data availability statement The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Code availability Not applicable Author contributions DT conceived the idea and designed the experiments; DT and ZY performed the data analysis. DT, ZM, and LWY performed the manuscript preparation. JHF, LT and LDY are responsible for the collection and assembly of data. ZKS, WYJ, and LL contributed to the molecular experiments. LJ, YHB, and ZHY contributed to the TEM results. SL and YLL contributed to the function analysis. LY and YLL have been helping with revisions. All authors read and approved the final manuscript. Acknowledgement We thank Feng Lan Professor from National Center for Cardiovascular Diseases to provide hESCs and the method of obtaining cardiomyocytes from hESCs. We thank Yong-Ming Wang Professor from the School of Life Sciences, Fudan University to provide the CRISPR/Cas9 plasmid. We are grateful to Hong-Feng Jiang Professor from Beijing Laboratory for Cardiovascular Precision Medicine for editing the article. The authors declare that they have not use AI-generated work in this manuscript. References Bhat, H. F., Adams, M. E. & Khanday, F. A. Syntrophin proteins as Santa Claus: role(s) in cell signal transduction. Cell. Mol. Life Sci. 70 (14), 2533–2554. 10.1007/s00018-012-1233-9 (2013). Epub 2012 Dec 21. PMID: 23263165; PMCID: PMC11113789. Sheng, M. & Sala, C. PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci. ;24:1–29. 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Supplementary Files supplementary19ok.docx Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Mar, 2025 Reviews received at journal 23 Mar, 2025 Reviews received at journal 21 Mar, 2025 Reviewers agreed at journal 13 Mar, 2025 Reviewers agreed at journal 13 Mar, 2025 Reviewers invited by journal 13 Mar, 2025 Editor assigned by journal 13 Mar, 2025 Editor invited by journal 07 Mar, 2025 Submission checks completed at journal 07 Mar, 2025 First submitted to journal 27 Feb, 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. 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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-6120127","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":426298047,"identity":"16a7cedb-02cf-4b52-9cae-edb8148ef6c8","order_by":0,"name":"Tao 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Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhang","suffix":""},{"id":426298052,"identity":"bbae87bf-f7e4-4b55-bf76-f31136293c70","order_by":3,"name":"Wei-Ya Lang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Ya","middleName":"","lastName":"Lang","suffix":""},{"id":426298053,"identity":"6dd48782-31f4-41df-9f31-e5348648a160","order_by":4,"name":"Dan-Yang Liu","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dan-Yang","middleName":"","lastName":"Liu","suffix":""},{"id":426298054,"identity":"e2252b2f-ca0c-4f17-be10-931b72ce05ae","order_by":5,"name":"Ke-Shuang Zhang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke-Shuang","middleName":"","lastName":"Zhang","suffix":""},{"id":426298055,"identity":"ca6f96f8-c301-4320-9ae5-b39b17ef1097","order_by":6,"name":"Yue-Jing Wang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue-Jing","middleName":"","lastName":"Wang","suffix":""},{"id":426298056,"identity":"8bec51cb-329b-4fc5-a4c8-53fffde94763","order_by":7,"name":"Lin Li","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":426298057,"identity":"13581a8f-c957-445e-a8cc-209e02334b83","order_by":8,"name":"Jie Lian","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Lian","suffix":""},{"id":426298058,"identity":"1bf7dbf0-3533-4aaf-89b2-2e8637fc8e6d","order_by":9,"name":"Hong-Bo Yao","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong-Bo","middleName":"","lastName":"Yao","suffix":""},{"id":426298059,"identity":"d186a324-7ef7-4eb1-ac23-213aca098a5f","order_by":10,"name":"Hai-Yan Zhang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hai-Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":426298060,"identity":"e13f637d-75f8-40f9-a578-b65d503fefc4","order_by":11,"name":"Hai-Feng Jin","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hai-Feng","middleName":"","lastName":"Jin","suffix":""},{"id":426298061,"identity":"2d08585e-c5ce-4f40-9aba-cdd694b6cef4","order_by":12,"name":"Tong Lu","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Lu","suffix":""},{"id":426298062,"identity":"5ae44ca5-6581-458b-bf60-a4fe820b83cd","order_by":13,"name":"Lei Shen","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shen","suffix":""},{"id":426298063,"identity":"2f517bf9-e770-4c5e-a006-9847b57eaf8f","order_by":14,"name":"Li-Ling Yue","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li-Ling","middleName":"","lastName":"Yue","suffix":""},{"id":426298064,"identity":"a098cf42-6cb0-4ca0-bd28-bf69a18ee5aa","order_by":15,"name":"Yan Lin","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2025-02-27 10:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6120127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6120127/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16406-6","type":"published","date":"2025-08-20T16:29:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78226757,"identity":"6507c9b1-e452-4df5-b6fa-74463d7cf111","added_by":"auto","created_at":"2025-03-11 07:02:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1420907,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of homozygous \u003cem\u003eSNTA1\u003c/em\u003e-deficient human embryonic stem cell\u003c/p\u003e\n\u003cp\u003eA. Schematic of the sgRNA designed to the PH1 domain in \u003cem\u003eSNTA1\u003c/em\u003e.There is one adenine nucleotide inserted into \u003cem\u003eSNTA1\u003c/em\u003e before PAM sequence.\u003c/p\u003e\n\u003cp\u003eB. The sanger sequence of H9 and H9SNTA1KO genomic DNA. There is one adenine nucleotide inserted into the second exon of \u003cem\u003eSNTA1 \u003c/em\u003ein H9SNTA1KO.\u003c/p\u003e\n\u003cp\u003eC. The light microscope images of H9 and H9SNTA1KO. Scale bar: 100 μm.\u003c/p\u003e\n\u003cp\u003eD. Immunofluorescence staining for pluripotency was performed. Both SSEA4 and OCT-4 were positive in H9SNTA1KO. \u003cem\u003eSNTA1\u003c/em\u003e-knockout did not influence the pluripotency of hESCs. Scale bar: 25 μm.\u003c/p\u003e\n\u003cp\u003eE. The stem cell pluripotency qPCR results of DPPA-4, SOX-2, OCT-4, and NANOG. There is no significance between H9 and H9SNTA1KO. (N= 3). ns; not significant, unpaired two-sided Student’s t test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/0d41e0e08e616ed7b7abeb87.png"},{"id":78226779,"identity":"612e1195-b277-4d23-9187-fe9627755543","added_by":"auto","created_at":"2025-03-11 07:02:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1159877,"visible":true,"origin":"","legend":"\u003cp\u003eH9SNTA1KOdifferentiation to cardiomyocytes\u003c/p\u003e\n\u003cp\u003eA. Schematic of embryonic stem cell induction into cardiomyocytes using small molecular inhibitors two-dimensionaldifferentiation method.\u003c/p\u003e\n\u003cp\u003eB. The light microscope image of beating KO cardiomyocytes at day 10 of differentiation. Scale bar: 100 μm.\u003c/p\u003e\n\u003cp\u003eC. The transmission electron microscope image of KO cardiomyocyte at 30days. The yellow pentagram shows the myofilaments in cardiomyocyte. Scale bar: 2 μm.\u003c/p\u003e\n\u003cp\u003eD. Immunofluorescence staining of TNNT2 (green) and α-actinin (red) in KO cardiomyocytes. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003eE. Flow cytometry was used to detect a specific ventricular muscle marker, MYL2. The results demonstrated that the yield of WT and KOventricular muscle was similar when purified by metabolic selection.\u003c/p\u003e\n\u003cp\u003eF. Quantification of MYL2 of the flow cytometry (N = 3). ns; not significant, unpaired two-sided Student’s t test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/e29ebb696cbf93fa668d34a4.png"},{"id":78226765,"identity":"11097d43-6f0c-4166-ac48-1cdcb8381dbe","added_by":"auto","created_at":"2025-03-11 07:02:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":884470,"visible":true,"origin":"","legend":"\u003cp\u003eH9SNTA1KO derived cardiomyocytes showed shorter field potential duration and slower conduction velocity\u003c/p\u003e\n\u003cp\u003eA. Schematic of MAESTRO™ MEA SYSTEMS work flow.\u003c/p\u003e\n\u003cp\u003eB. The image of cardiomyocytes seeded on the Micro-electrode plate. Scale bar: 10 μm.\u003c/p\u003e\n\u003cp\u003eC’. The image of Continuous Waveform Plots for WT cardiomyocytes and KO cardiomyocytes.\u003c/p\u003e\n\u003cp\u003eC’’. The statistical of beat period of the WT cardiomyocytes and KO cardiomyocytes. There is a statistical difference between them. N = 9, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. The beat period of KO cardiomyocytes was shorter than that of WT.\u003c/p\u003e\n\u003cp\u003eD’. The Schematic of beat Metrics.\u003c/p\u003e\n\u003cp\u003eD’’. The image of cardiac voltage waveforms for WT cardiomyocytes and KO cardiomyocytes.\u003c/p\u003e\n\u003cp\u003eD’’’. The statistical FPD diagram of WT cardiomyocytes and KO cardiomyocytes. The MEA results indicates that the FPD of KO cardiomyocytes is shorter than that of WT. There is a statistical difference. N = 9, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003eE. The propagation map of WT cardiomyocytes and KO cardiomyocytes.\u003c/p\u003e\n\u003cp\u003eF. The MEA results showing the conduction velocity of KO cardiomyocytes is slower than the WT cardiomyocytes. There is a statistical difference. N = 3, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/66e3eb8adc7a3dfb6596c00d.png"},{"id":78228240,"identity":"a846268a-57a9-40c3-a888-f2b970d20b70","added_by":"auto","created_at":"2025-03-11 07:10:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":457310,"visible":true,"origin":"","legend":"\u003cp\u003eH9SNTA1KO derived cardiomyocytes showed the disorganization of SCN5A\u003c/p\u003e\n\u003cp\u003eA. The expression of SCN5A in 30-day WT and KO cardiomyocytes was analyzed using RT-qPCR. The results showed there is no significant. ns; not significant.\u003c/p\u003e\n\u003cp\u003eB. The expression of SCN5A in 45-day WT and KO cardiomyocytes was analyzed using RT-qPCR. The results showed there is no significant. ns; not significant.\u003c/p\u003e\n\u003cp\u003eC. Immunostaining of SCN5A in 45-day WT and KO cardiomyocytes. Scale bar: 7 μm.\u003c/p\u003e\n\u003cp\u003eD. Statistical analysis of SCN5A disorganization cell counting in 45-day WT and KO cardiomyocytes. The results showed that KO cardiomyocytes exhibit a higher number of disorganized cells compared to WT cardiomyocytes. ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/b945b96f9183d51aa9d79252.png"},{"id":89847245,"identity":"5eda7eec-656c-4229-a544-48cab4a2a843","added_by":"auto","created_at":"2025-08-25 16:42:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4228474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/a06c2ce2-17b4-4184-8c54-b50882ff1f18.pdf"},{"id":78228239,"identity":"419d0ac8-f8f7-4f10-9c4b-c48b69321414","added_by":"auto","created_at":"2025-03-11 07:10:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20690,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary19ok.docx","url":"https://assets-eu.researchsquare.com/files/rs-6120127/v1/81dc8d4351a089057f92244f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SNTA1-deficient human cardiomyocytes show shorter field potential duration and slower conduction velocity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSNTA1 is a member of the membrane-associated adaptor protein family, consisting of 505 amino acid residues in its unprocessed form, with a molecular weight of approximately 58 kDa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. SNTA1 contains three distinct domains: PH1, PH2, and SU. It is predominantly distributed beneath muscle membranes and at neuromuscular junctions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a peripheral cytoplasmic membrane protein, SNTA1 is primarily associated with dystrophin and dystrophin-related proteins, such as glycoproteins, utrophin, and dystrobrevin, within the dystrophin glycoprotein complex (DGC), also referred to as the dystrophin-associated protein complex (DAPC) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Within the DGC, SNTA1 plays a crucial role in supporting the proper subcellular localization of associated functional proteins.\u003c/p\u003e \u003cp\u003eG-proteins are a critical class of signal-transducing proteins located on the inner surface of the cell membrane, where they associate with transmembrane receptors. The N-terminal region of the PH1 domain in SNTA1 and the C-terminal region of the SU domain have been shown to mediate binding with the Gα subunit of heterotrimeric G-proteins, contributing to the regulation of their functions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Calmodulin (CaM) transduces calcium signals by binding to Ca\u0026sup2;⁺ ions and interacting with downstream target proteins [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The PH1 domain of SNTA1 interacts with CaM, playing a role in intracellular Ca\u0026sup2;⁺ regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. SCN5A, the primary cardiac voltage-gated sodium channel alpha subunit 5, is essential for the rapid depolarization phase of the cardiac action potential and plays a key role in cardiac conduction. SNTA1 interacts with SCN5A through its PDZ domain, which binds to the internal domain of SCN5A's N-terminal [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], as well as through its association with the C-terminal of SCN5A, thereby contributing to its regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Clinically, SNTA1 has been identified as a susceptibility locus for Long QT Syndrome 12 (LQT12), a rare arrhythmic disorder associated with an increased risk of sudden cardiac death. At present, the etiology and preventive treatment of Long QT Syndrome 12 remain inadequately understood and largely unexplored.\u003c/p\u003e \u003cp\u003eMost current research on SNTA1 relies on animal models; however, significant differences between animal and human cardiac muscle limit their relevance for studying human cardiac arrhythmias. Induced pluripotent stem cell and gene editing technologies offer a powerful platform for creating precise human disease models to advance precision medicine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, we utilized CRISPR/Cas9 technology to generate SNTA1 knockout (SNTA1-KO) human cardiomyocytes, providing a more physiologically relevant model for investigating arrhythmias resulting from SNTA1 deficiency. Our present work establishes a foundation for studying SNTA1 point mutations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEstablishment of homozygous\u003c/b\u003e \u003cb\u003eSNTA1\u003c/b\u003e\u003cb\u003e-deficient human embryonic stem cell\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe selected the second exon, a shared exon, as the target for \u003cem\u003eSNTA1\u003c/em\u003e editing. A single guide RNA (sgRNA) was designed to target this exon, with the sequence 5\u0026prime;-ATTGGCAGCTGACCAGACAG-3\u0026prime;, and the protospacer adjacent motif (PAM) sequence AGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The sgRNA was ligated into a linear CRISPR/Cas9 plasmid, followed by plasmid amplification. The resulting plasmid was delivered into H9 embryonic stem cells via electroporation using the LONZA Nucleofector 4D system. Post-electroporation, the cells were selected using 0.3 \u0026micro;g/mL puromycin. Resistant clones were picked, expanded, and subjected to genomic DNA extraction for genotyping analysis. One clone was identified with an adenine nucleotide insertion upstream of the PAM region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This insertion introduced a premature stop codon (TGA) at amino acid position 149, truncating the SNTA1 protein in the PDZ domain which is located in the PH1 domain. This modified cell line was designated as H9SNTA1KO [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The H9SNTA1KO cells were cultured and compared to the parental H9 embryonic stem cells under light microscopy, revealing no discernible morphological differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Immunofluorescence staining confirmed positive expression of the pluripotency markers SSEA4 and OCT4 in H9SNTA1KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, qRT-PCR analysis demonstrated that the expression levels of the pluripotency markers DPPA4, SOX2, OCT4, and NANOG in H9SNTA1KO cells were comparable to those in H9 cells, with no significant differences observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The results confirmed the successful establishment of H9SNTA1KO cells, which retained normal pluripotency markers.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eH9SNTA1KO differentiation to cardiomyocytes\u003c/h2\u003e \u003cp\u003eTo obtain cardiomyocytes, we employed a 2D differentiation method as described in previous studies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The process of differentiation required approximately 12 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cardiomyocytes were collected at day 30 of differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Transmission electron microscopy (TEM) analysis revealed the presence of myofibrils in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, yellow pentagrams), although the cells were immature, lacking T-tubules and well-organized layered myofilaments. Immunofluorescence staining was performed to detect the cardiomyocyte-specific markers TNNT2 and α-actinin, both of which were positively expressed in the H9SNTA1KO derived cardiomyocytes (KO cardiomyocytes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The collected cells were identified as cardiomyocytes, which exhibited spontaneous contractions. The ventricular muscle-specific marker MYL2 was assessed in both WT cardiomyocytes (H9 derived cardiomyocytes) and KO cardiomyocytes after purified medium selection using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The results demonstrated that H9SNTA1KO cells differentiated into cardiomyocyte subtypes comparable to WT cells, confirming normal differentiation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). All results confirmed the successful generation of H9SNTA1KO cardiomyocytes, which closely resemble those of WT.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eH9SNTA1KO derived cardiomyocytes showed shorter field potential duration and slower conduction velocity\u003c/h3\u003e\n\u003cp\u003eSNTA1, known as the LQT12 gene, has been associated with long QT syndrome (LQTS) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. LQTS, a hereditary condition characterized by QT interval prolongation, increases the risk of life-threatening arrhythmias. To investigate the effects of SNTA1 deficiency in cardiomyocytes, we employed the MAESTRO\u0026trade; MEA SYSTEMS to assess their electrical activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Cardiomyocytes were seeded onto MEA (micro electrode array) plates for electrical activity analysis under normal culture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Data from the MAESTRO\u0026trade; MEA SYSTEMS revealed that the beat period of KO cardiomyocytes was shorter than that of WT cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026rsquo;-C\u0026rsquo;\u0026rsquo;). The interval between depolarization and repolarization, referred to as the field potential duration (FPD), was extracted from field potential signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026rsquo;-D\u0026rsquo;\u0026rsquo;). Statistical analysis showed that the FPD of KO cardiomyocytes was significantly shorter than that of WT cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026rsquo;\u0026rsquo;\u0026rsquo;). FPD parameters can simulate the QT interval, the shortened FPD parameter predicted the shortened QT interval in KO cardiomyocytes. and abnormal QT variations indicate an increased susceptibility to arrhythmias in KO cardiomyocytes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The stability of beat propagation patterns and conduction velocity was also assessed to evaluate the functional properties of the cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Statistical analysis demonstrated that the conduction velocity of KO cardiomyocytes was slower than that of WT cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Reduced conduction velocity may contribute to an increased risk of arrhythmias [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. All the results showed there is a potential risk of arrhythmia in KO cardiomyocytes.\u003c/p\u003e\n\u003ch3\u003eH9SNTA1KO derived cardiomyocytes showed the disorganization of SCN5A\u003c/h3\u003e\n\u003cp\u003eSNTA1 interacts with both the C-terminal and N-terminal domains of SCN5A [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], facilitating the proper membrane localization of SCN5A [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To investigate this further, we conducted additional experiments. mRNA was extracted from 30-day-old cardiomyocytes, and SCN5A expression was analyzed. The results indicated no significant difference in SCN5A expression between the KO and WT cardiomyocytes at 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, mRNA extracted from 45-day-old cardiomyocytes showed no difference in SCN5A expression between the KO cardiomyocytes and WT cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings suggested that SNTA1 deficiency does not influence SCN5A transcription. The cellular localization of SCN5A was further examined using immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The imaging results revealed a disorganized distribution of SCN5A in KO cardiomyocytes compared to the WT cardiomyocytes. Statistical analysis confirmed that the proportion of cells with disorganized SCN5A localization was significantly higher in the KO cardiomyocytes than in the WT cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs an adapter protein, SNTA1 is localized beneath the cell membrane and contains three functional domains: PH1, PH2, and SU. These domains are involved in the subcellular localization of various intracellular functional proteins, including the Gα subunit, CaM, and SCN5A [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Clinical reports have indicated that mutations in SNTA1 may be associated with Long QT Syndrome (LQTS) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and other cardiovascular phenotypes. Long QT syndrome (LQTS) encompasses a group of heritable conditions characterized by cardiac repolarization dysfunction [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To investigate the effects of SNTA1 mutations, we employed a human cell model. Using gene editing system, we targeted the second exon of SNTA1 in H9 embryonic stem cells. Following gene editing, we established a \u003cem\u003eSNTA1\u003c/em\u003e-deficient human embryonic stem cell line. Genotyping results revealed an adenine nucleotide insertion in the second exon of SNTA1, resulting in a premature termination of the protein. Thus, the H9SNTA1KO embryonic stem cell line was successfully generated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The morphology of the H9SNTA1KO cell line was similar to that of the parental H9 cells, and both cell lines exhibited normal expression of pluripotency markers. We then used a chemically defined 2D differentiation method to induce cardiomyocyte differentiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. No significant differences were observed between H9SNTA1KO-derived cardiomyocytes and those derived from H9 embryonic stem cells in terms of differentiation efficiency.\u003c/p\u003e \u003cp\u003eTo assess the electrical activity of cardiomyocytes, we employed the MAESTRO\u0026trade; MEA SYSTEMS. The cardiomyocytes were seeded onto the MEA plate, and the data showed that the KO cardiomyocytes exhibited a shorter beat period compared to the WT cardiomyocytes. Additionally, the KO cardiomyocytes had a shorter field potential duration (FPD) and slower conduction velocity. FPD refers to the time interval between depolarization and repolarization, and it corresponds to the QT interval in an ECG. The shorter FPD observed in the KO cardiomyocytes indicates an abnormality in the depolarization and repolarization process. Clinically, alterations in the QT interval can lead to arrhythmias. Furthermore, the KO cardiomyocytes exhibited a slower conduction velocity. The measured conduction velocity reflects the collective effects of various factors, including cell culture health and pacemaker stability. Slower conduction propagation can potentially contribute to arrhythmias [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These results provide evidence that the electrical activity of SNTA1-deficient cardiomyocytes is unstable, potentially increasing the risk of cardiac arrhythmias.\u003c/p\u003e \u003cp\u003eTo further explore the underlying cause of the shorter FPD and slower conduction propagation in SNTA1-deficient cardiomyocytes, we focused on SCN5A. SCN5A is a voltage-gated sodium ion channel alpha subunit 5 located on the cardiac membrane, playing a crucial role in the depolarization process of cardiomyocyte action potentials [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While the expression of SCN5A showed no significant difference between KO and WT cardiomyocytes, SNTA1 acts as an adaptor protein that helps target functional proteins to the cell membrane. To investigate the localization of SCN5A in cardiomyocytes, immunofluorescence staining was performed. The results revealed that SCN5A localization was more disorganized in KO cardiomyocytes compared to WT cardiomyocytes. These findings suggest that the proper localization of SCN5A in cardiomyocytes requires SNTA1. Based on these observations, we speculate that SNTA1 deficiency may impair SCN5A localization, potentially increasing the likelihood of arrhythmia in the KO cardiomyocytes. However, the specific mechanisms underlying this effect require further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a human SNTA1-knockout cell model was established using the CRISPR/Cas9 system. This cell model provides a valuable tool for studying arrhythmias induced by SNTA1 deficiency in vitro. The findings underscore the critical role of SNTA1 as an essential auxiliary protein involved in the proper localization of SCN5A, a key cardiac ion channel in cardiomyocytes. SNTA1 is identified as a susceptibility locus for arrhythmias, highlighting its potential as a target for further research in cardiac electrophysiology.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e\n\n "},{"header":"Methods","content":"\u003ch2\u003eEmbryonic stem cell culture\u003c/h2\u003e\u003cp\u003eH9 embryonic stem cells were cultured in E8 medium and passaged using 0.5 mM EDTA upon reaching 80% confluence. Cells were typically passaged at a ratio of 1:6.\u003c/p\u003e\u003ch3\u003eEstablishment the SNTA1KO embryonic stem cell line\u003c/h3\u003e\u003cp\u003eUsing the Zhang Lab's resources, we designed an sgRNA targeting the second exon of SNTA1. The sgRNA sequence was 5′-attggcaggacag-3′. This sequence was ligated into a CRISPR/Cas9 plasmid, which was subsequently amplified by transforming E. coli cells (Top10 Competent Cells, CWBIO, China) and purified using an EndoFree Mini Plasmid Kit (TianGen, China). The plasmid was then delivered into H9 embryonic stem cells via electroporation (LONZA Nucleofector 4D). Following puromycin selection, individual clones were isolated and subjected to genotypic identification. An adenine insertion was introduced into the second exon, resulting in a premature stop codon at the 149th amino acid position of SNTA1. This successfully established the H9SNTA1KO cell line [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eCardiac differentiation\u003c/h2\u003e\u003cp\u003eEmbryonic stem cells (ESCs) were cultured in E8 medium. When the cells reached approximately 70–80% confluence, they were passaged at a 1:6 ratio using E8 medium supplemented with 10 µM Y-27632 (ROCK inhibitor, MCE, USA). Once the cells reached 80–90% confluence, cardiac differentiation was used 2D differentiation method. The CardioEasy® mediums for differentiation were provided by the Cellapybio Inc (China).\u003c/p\u003e\u003ch2\u003eFlow Cytometry\u003c/h2\u003e\u003cp\u003eCardiomyocytes were digested using CardioEasy® I and CardioEasy® II digestive solutions (Cellapybio, China) to prepare single-cell suspensions. The cells were then fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT), followed by two washes with PBS. Permeabilization was performed using 0.2% Triton X-100 for 5 minutes at RT. Next, the cells were incubated with the antibody for 30 minutes in the dark at RT, followed by two additional PBS washes to remove unbound antibodies. The samples were analyzed using a flow cytometer (Beckman, EPICS XL), and the results were processed with FlowJo VX software.\u003c/p\u003e\u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was performed to visualize the localization of intracellular antigens. Cells were seeded on coverslips and cultured until reaching approximately 50% confluence. The medium was aspirated, and the cells were washed three times with PBS. The coverslip-adherent cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature (RT), followed by three washes with PBS. Permeabilization was performed using 0.3% Triton X-100 for 10 minutes at RT. The cells were then blocked with 3% BSA for 30 minutes at RT. After blocking, the cells were incubated with the primary antibody at 4°C for 24 hours. Following three PBS washes, they were incubated with the secondary antibody and DAPI (100 nM) for 1 hour at RT. The cells were subsequently washed three more times with PBS and imaged using a confocal microscope (Leica, TCS SP5). Both primary and secondary antibodies were used for immunofluorescence staining. Both primary and secondary antibodies are provided in Supplementary Table S2.\u003c/p\u003e\u003ch2\u003eQuantitative Real‑time PCR (qRT‑PCR)\u003c/h2\u003e\u003cp\u003eTo compare gene expression at the transcriptional level between the KO and WT groups, real-time PCR was performed. Cells were seeded in a 6-well plate at a density sufficient to reach approximately 90% confluence for RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and treated with DNase I (Beyotime, China) at 37°C for 30 minutes to remove any contaminating DNA. Reverse transcription was performed using the PrimeScript™ reverse transcription system (TaKaRa, Japan). Relative gene expression levels were analyzed by quantitative real-time PCR (qRT-PCR) on an iCycler iQ5 system (Bio-Rad, USA) using TB Green™ Premix Ex Taq™ II (TaKaRa, Japan). Relative quantification of gene expression was determined using the ∆CT method. Primer sequences used for qRT-PCR are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eElectrical activity of cardiomyocytes detection\u003c/h2\u003e\u003cp\u003eTo assess the electrical activity of cardiomyocytes under normal culture conditions, we utilized the MAESTRO™ MEA SYSTEMS. Matrigel working solution was prepared by diluting it 1:200 and used to coat the wells of a 24-well MEA plate overnight. A total of 20,000–30,000 cardiomyocytes were seeded onto one matrigel-coated well of the 24-well MEA plate and cultured in cardiac maintenance medium (Cellapybio, China) supplemented with 10 µM Y-27632. Once the cardiomyocytes spread and began beating regularly, their electrical activity was measured and analyzed using the MAESTRO™ MEA SYSTEMS(Axion BioSystems, Inc, US).\u003c/p\u003e\u003ch2\u003eTransmission Electron Microscope (TEM)\u003c/h2\u003e\u003cp\u003eTransmission electron microscopy was employed to examine the ultrastructure of cardiomyocytes. The medium was aspirated from the cardiomyocytes, and without rinsing, they were immediately fixed in 2.5% glutaral solution. The cells were then gently scraped off using a cell scraper and collected into a centrifuge tube (cells avoid being digested by enzymes). After centrifugation, a visible cell pellet should be obtained. The cells were fixed at RT for 2 hours with fresh electron microscope fixative. The samples were subsequently sent to Wuhan GoodBio Technology Company for further processing.\u003c/p\u003e\u003ch2\u003eStatistical methods\u003c/h2\u003e\u003cp\u003eThe data are presented as mean ± standard deviation. Differences between two groups were analyzed using a one-tailed or two-tailed t-test, while rates were compared using Fisher's Exact test. For comparisons involving three or more groups, one-way or two-way analysis of variance (ANOVA) was applied, followed by Tukey's multiple comparison test. A 95% confidence interval was used, and statistical significance was defined as follows: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, representing four levels of significance.\u003c/p\u003e"},{"header":"Limitation","content":"\u003cp\u003eThe cardiomyocytes we obtained lacked T-tubule structures, which is characteristic of immature cardiomyocytes rather than mature ones. Additionally, our study was conducted using a two-dimensional (2D) in vitro cell culture model.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSymbol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFull Name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSNTA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eα-1-syntrophin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehESCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehuman embryonic stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH9SNTA1KO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSNTA1\u003c/em\u003e-deficient H9 embryonic stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH9 embryonic stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWT cardiomyocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecardiomyocytes\u0026nbsp;derived from H9 embryonic stem cells\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSNTA1\u003c/em\u003e-deficientH9 embryonic stem cells or H9SNTA1KO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKO cardiomyocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSNTA1\u003c/em\u003e-deficient cardiomyocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eprotospacer adjacent motif\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCRISPR/Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eclustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNNT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etroponin T2, cardiac type\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSSEA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003estage-specific embryonic antigen-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNANOG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNanog homeobox\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSOX2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSRY-box transcription factor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDPPA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edevelopmental pluripotency associated 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOCT-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePOU class 5 homeobox 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMYL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emyosin light chain 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emicro electrode array\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBasic scientific research expenses of colleges and universities in Heilongjiang Province\u0026nbsp;（2022-KYYWF-0808）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTitle of the approved project, “Research on Differential Gene Expression in Human Cardiomyocytes with SNTA1 Deficiency”. Name of institutional approval committee or unit, “Ethics Committee of Qiqihar Medical University”. The approval number, (#77/2022). Date of approval. (12/12/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDT conceived the idea and designed the experiments; DT and ZY performed the data analysis. DT, ZM, and LWY performed the manuscript preparation.\u0026nbsp;JHF, LT and LDY are responsible for the collection and assembly of data.\u0026nbsp;ZKS, WYJ, and LL contributed to the molecular experiments.\u0026nbsp;LJ, YHB, and ZHY contributed to the TEM results. SL and YLL contributed to the function analysis. LY and YLL have been helping with revisions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Feng Lan Professor from National Center for Cardiovascular Diseases to provide hESCs and the method of obtaining cardiomyocytes from hESCs. We thank Yong-Ming Wang Professor from the School of Life Sciences, Fudan University to provide the CRISPR/Cas9 plasmid. We are grateful to Hong-Feng Jiang Professor from Beijing Laboratory for Cardiovascular Precision Medicine for editing the article. The authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhat, H. F., Adams, M. E. \u0026amp; Khanday, F. A. Syntrophin proteins as Santa Claus: role(s) in cell signal transduction. \u003cem\u003eCell. Mol. Life Sci.\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e (14), 2533\u0026ndash;2554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00018-012-1233-9\u003c/span\u003e\u003cspan address=\"10.1007/s00018-012-1233-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013). Epub 2012 Dec 21. PMID: 23263165; PMCID: PMC11113789.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheng, M. \u0026amp; Sala, C. PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci. ;24:1\u0026ndash;29. 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PMID: 34575961; PMCID: PMC8472079.\u003c/span\u003e\u003c/li\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Human embryonic stem cell, SNTA1-defcient cardiomyocytes, SCN5A, Arrhythmia, Action potential duration, Conduction velocity","lastPublishedDoi":"10.21203/rs.3.rs-6120127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6120127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn clinical settings, patients with SNTA1 point mutations are often associated with rare arrhythmias, including Long QT syndrome, Brugada syndrome, and sudden infant death syndrome. Previous studies on SNTA1 have predominantly utilized nonhuman cardiomyocyte models. This study aims to elucidate the phenotype of SNTA1 deficiency using human cardiomyocytes. Using CRISPR/Cas9 technology, we generated SNTA1 knockout (KO) embryonic stem cell line, which were subsequently differentiated into cardiomyocytes using 2D differentiation method. Genotype analysis identified an adenine (A) insertion in the second exon of \u003cem\u003eSNTA1\u003c/em\u003e, resulting in a premature stop codon at the 149th amino acid position and truncation within the PDZ domain. \u003cem\u003eSNTA1\u003c/em\u003e-deficient cardiomyocytes exhibited a shortened action potential duration (FPD) and slower conduction velocity, as detected by micro electrode array analysis. Immunofluorescence analysis further revealed disorganized distribution of SCN5A protein in \u003cem\u003eSNTA1\u003c/em\u003e-deficient cardiomyocytes. \u003cem\u003eSNTA1\u003c/em\u003e is a susceptibility locus for arrhythmias and plays a critical role as an essential auxiliary protein in the proper localization of SCN5A in human cardiomyocytes.\u003c/p\u003e","manuscriptTitle":"SNTA1-deficient human cardiomyocytes show shorter field potential duration and slower conduction velocity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 07:02:37","doi":"10.21203/rs.3.rs-6120127/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-24T05:17:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-23T13:28:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-21T16:13:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339910535225086805469845506159147032075","date":"2025-03-13T07:42:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253053397297571513116610819749517466827","date":"2025-03-13T07:05:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-13T06:50:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-13T06:35:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-07T23:15:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-07T08:23:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-27T10:27:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4dff573d-3635-4116-90f1-a307d5cffd5a","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45424389,"name":"Biological sciences/Stem cells/Embryonic stem cells"},{"id":45424390,"name":"Biological sciences/Stem cells/Stem cell differentiation"},{"id":45424391,"name":"Health sciences/Diseases/Cardiovascular diseases/Arrhythmias"},{"id":45424392,"name":"Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects"}],"tags":[],"updatedAt":"2025-08-25T16:34:40+00:00","versionOfRecord":{"articleIdentity":"rs-6120127","link":"https://doi.org/10.1038/s41598-025-16406-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-20 16:29:22","publishedOnDateReadable":"August 20th, 2025"},"versionCreatedAt":"2025-03-11 07:02:37","video":"","vorDoi":"10.1038/s41598-025-16406-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16406-6","workflowStages":[]},"version":"v1","identity":"rs-6120127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6120127","identity":"rs-6120127","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}