tbuckley/cabot-search huggingface dataset:
Abstract Mo098: LMNA R190W Mutation Associated With Dilated Cardiomyopathy Causes Hypercontractility in iPSC-derived Cardiomyocytes and Engineered Heart Muscles
Authors: Melissa Hector-Greene, Jacob Kapustin, Lina Greenberg, Michael Greenberg, Kory Lavine
Journal: Circulation Research (2024)

Familial dilated cardiomyopathy (DCM) caused by mutations in sarcomeric proteins have been extensively studied in vitro , however less is known about DCM caused by alterations in non-sarcomeric genes like Lamin A (LMNA). LMNA is a ubiquitously expressed nuclear intermediate filament protein that regulates nuclear structure, chromatin access and gene expression. LMNA mutations account for 6% of DCM, and affected patients develop drug-therapy resistant severe heart failure and lethal arrythmias that are not amenable to currently available therapies. Most identified LMNA-cardiomyopathy mutations have been associated with severely depressed cardiac systolic function in patients and cardiomyocyte hypocontractility in vitro . However, the underlying mechanisms are incompletely understood. This project examines the mechanisms by which a patient-derived pathogenic mutation, LMNA R190W, leads to cardiac dysfunction at the cellular and tissue levels. We hypothesize that LMNA R190W mutation causes dysregulated expression of calcium handling genes, eventually leading to altered contractility. First, we investigated early disease pathogenesis using iPSC-derived cardiomyocytes. Intriguingly, single cardiomyocyte traction force microscopy studies showed cardiomyocyte hypercontractility, and calcium flux assays revealed prolonged calcium transients. Second, in human engineered heart muscle tissues, LMNA CMs were also consistently hypercontractile. Through gene expression studies, we identified upregulation of a cardiac-specific sarcomeric calcium regulator, calsequestrin 2 (CASQ2), which modulates intracellular calcium availability and localization, and thereby indirectly impacts calcium-dependent contraction and electrophysiology. siRNA-mediated knockdown of CASQ2 in iPSC-CMs decreased cellular calcium content, suggesting Casq2 is necessary for increased calcium and contractility in LMNA R190W cells. These findings suggest that in the initial stages of LMNA R190W-associated cardiomyopathy, cardiomyocyte contractility is not diminished, but actually augmented, and this may be related to aberrant calcium handling. We posit that chronic inappropriate calcium overload may eventually lead to cell death, and predispose to cardiomyopathy. Understanding the initial processes driving the development of LMNA cardiomyopathy will inform more accurate mechanism-based subclassification of mutations, and the rational development of precision therapies.
Abstract We140: Age-Dependent miR-34a and AGTRAP Cross-Modulation: a Novel Mechanism of Vascular Aging
Authors: Cristina Florio, Sara Sileno, Liqun Jiang, Laura Monteonofrio, Sunayana Begum Syed, Mirko Baranzini, CHIARA PRAMPOLINI, Federica Macri, Manuel Casaburo, Nadia Fanotti, Stefania Castiglione, Robert Monticone, Bruce Ziman, Richard telljohann, Mingyi Wang, Christopher Morrell, Angela Raucci, Edward Lakatta, Alessandra Magenta, Maurizio Colognesi Capogrossi
Journal: Circulation Research (2024)

Background: Renin-Angiotensin Aldosterone System (RAAS) activation plays a central role in vascular aging, leading to structural and functional alterations including vascular smooth muscle cell (VSMC) proliferation, migration and secretion of extracellular matrix and inflammatory molecules. The age-associated increase in Angiotensin II (Ang II) expression enhances AT1-receptor activity and determinates a pro-fibrotic and pro-inflammatory phenotype. Further, miR-34a expression is modulated by aging and increases in different tissues, including the mouse aorta, but no direct miR-34a target has been identified in the RAAS pathway that sustains age-dependent increase of pro-inflammatory signaling. Aims: The aim of our study is to evaluate the potential interaction between miR-34a and RAAS signaling in VSMCs. Methods and Results: miR-34a expression exhibited an age-dependent increase in Rhesus Monkey (8- to 26-year-old) and rat central arteries (8- and 30-month-old). In silico analysis indicated that AT1R-associated protein (AGTRAP), a negative modulator of AT1R signaling, is a potential miR-34a target suggesting that the age-dependent increase in miR-34a in the arterial vascular wall may decrease AGTRAP expression in arterial VSMC. Ang II enhanced miR-34a expression in 30-month-old rat and in human aorta SMCs (HASMCs), whereas AGTRAP and Sirtuin-1 (SIRT1) mRNA and protein level decreased. Immunohistochemistry analysis showed a decrease of AGTRAP in 18-month-old miR-34a knock-out mice compared to 2-month-old mice. By luciferase assay, we demonstrated that AGTRAP is a direct miR-34a target. Moreover, AGTRAP and SIRT1 decreased in HASMCs infected with miR-34a-encoding lentivirus and this effect was partially rescued in AGTRAP/miR-34a co-infected cells. Finally, pro-inflammatory genes IL-6, COX-2, MCP-1 and MFG-E8 expression was upregulated in Ang II-treated and miR-34a overexpressing HASMCs, and this effect was abolished in AGTRAP/miR-34a co-transfected cells. Conclusions: Our results show cross-modulation between miR-34a and AGTRAP. MiR-34a targeting AGTRAP increases the expression of pro-inflammatory molecules in VSMCs and represents a novel mechanism that modulates RAAS signaling in vascular aging. Statement: This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Abstract We029: Common and Divergent Cellular Etiologies Underlying Hypoplastic Left Heart Syndrome and Hypoplastic Right Heart Syndrome
Authors: Yang Yu, Cankun Wang, Shiqiao Ye, Hannah Qin, Megan McNutt, Matthew Alonzo, Karen Texter, Vidu Garg, Mingtao Zhao
Journal: Circulation Research (2024)

Single ventricle heart defects are the most severe forms of congenital heart defects and are classified based on the affected ventricles: hypoplastic left heart syndrome (HLHS) and hypoplastic right heart syndrome (HRHS). Cellular etiologies underlying differential left and right ventricular hypoplasia are unknown and remain intractable without an experimental model to probe common and divergent cellular etiologies between HLHS and HRHS. In this study, we leveraged patient-specific induced pluripotent stem cells (iPSCs) and single-cell transcriptomics to interrogate distinct etiologies of HLHS and HRHS. Both HLHS and HRHS iPSC-derived cardiomyocytes (iPSC-CMs) exhibit a reduced proliferation capacity compared to sex-matched family controls under both static and cyclic stretch conditions. Biological pathways related to cell cycle progression, DNA replication, and cell proliferation are downregulated in both HLHS and HRHS iPSC-CMs, suggesting an intrinsic cardiac proliferation deficiency. Single-cell transcriptomics indicate that differentiation of cardiac mesoderm towards second heart field (SHF) progenitors are compromised in both HLHS and HRHS. However, differentiation of epicardial progenitors is enhanced at the expense of SHF progenitors in HLHS, whereas first heart field (FHF) progenitors are prevalent in HRHS. Trajectory inference analysis uncovers distinct cell lineage determination pathways in FHF and SHF progenitors between HLHS and HRHS. Moreover, HLHS iPSC-CMs exhibit reduced mitochondrial activities whereas HRHS iPSC-CMs show enhanced mitochondrial respiration and ATP production compared to controls. In sum, these common and divergent cellular etiologies of HLHS and HRHS may underlie ventricular hypoplasia in the left side and right side of the heart.
Abstract We031: Optimizing Immunosuppressive Strategies for Enhanced Survival of Allogeneic Pluripotent Stem Cell-Derived Cardiomyocytes in Non-Human Primate Transplantation Model
Authors: Shuji Chino, Hajime Ichimura, Shugo Tohyama, Keiichi Fukuda, Takashi Shiina, James Chong, TAKAFUMI NAITO, Maki Ohya, MIkiko Kobayashi, Takuro Tomita, Mitsuhiko Yamada, Hideki Kobayashi, Naoko Shiba, Tatsuichiro Seto, Shin Kadota, Yuji Shiba
Journal: Circulation Research (2024)

Introduction: Accumulating evidence suggests that the transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) regenerates injured hearts in animal studies. Currently, several clinical studies are underway, transplanting allogeneic human PSC-CMs. In these clinical studies, patients have been treated with the same combination of immunosuppressants used after heart transplantation, including steroids, calcineurin inhibitors (CNIs), and mycophenolate mofetil (MMF). However, the optimal treatment following PSC-CM transplantation has yet to be established. The purpose of this study is to determine the optimal treatment for grafted PSC-CM survival by regulating the immune response using an allogeneic non-human primate transplantation model. Methods and Results: We prepared 9 male cynomolgus monkeys as recipient animals. Donor cardiomyocytes were generated from induced pluripotent stem (iPS) cells derived from another monkey, with MHC class I and class II types differing from those in the recipients. The recipient animals underwent ischemia/reperfusion injury of the left anterior descending artery two weeks before iPSC-CMs transplantation. Initially, animals were treated with the same combination of immunosuppressants (steroid/CNIs/MMF: N=3), and grafted PSC-CMs survived without apparent immune rejection. Subsequently, we conducted experiments aiming to reduce immunosuppressants. The animals were treated with the same combination, but steroids were terminated at 4 weeks post-transplantation (N=4). Histological analysis at 8 or 12 weeks post-transplantation revealed that grafted cardiomyocytes were rejected, with a significant infiltration of CD3-positive lymphocytes. When recipients were treated with another combination of immunosuppressants (steroid/abatacept/MMF), and steroids were terminated at 4 weeks post-transplantation, grafted cardiomyocytes survived without apparent infiltration of CD3-positive lymphocytes at 8 weeks post-transplantation (N=2). Conclusion: A different combination of immunosuppressants from heart transplantation allows transplanted allogeneic PSC-CMs to survive without the long-term use of steroids.
Abstract We035: PTMA-MBD3 axis is a core heart regenerative driver in mammals
Authors: Ning Liu, Pei Jianqiu, Yifan Xie, Yu Nie
Journal: Circulation Research (2024)

Introduction: The adult mammalian heart has an extremely limited ability for regeneration after injury, whereas the fetal heart can steadily regenerate. Uncovering the molecular underpinnings of proliferative cardiomyocytes and manipulating the core genes activate cardiomyocyte proliferation may provide new insight into adult heart regrowth and functional repair post-injury. Methods and Results: We performed single-cell RNA-sequencing of mouse embryonic hearts from various developmental stages (E8.5-E17.5), and identified a cardiomyocyte population in proliferative phase with 120 highly expressed feature genes. Combining with our bulk RNA-seq data on isolated cardiomyocytes from different heart developmental periods (E14.5, P1-P56), we screened and demonstrated that Ptma is the core conservative factor in promoting mouse, rat and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. Cardiac specifically knocking out Ptma blocked cardiomyocyte proliferation and neonatal mouse heart regeneration after apical resection injury. AAV9-delivered Ptma overexpression extended neonatal heart regenerative time window and showed therapeutic benefit in driving adult cardiomyocyte proliferation and cardiac repair. As PTMA is a polypeptide, we further validated that PTMA derived conservative peptide Tα1 displayed more effective in treating adult cardiac injury compared to Decapeptide. PTMA is an unstructured, intrinsically disordered nuclear protein that functions by binding to target proteins. Using Co-IP, IP and Native PAGE, we found PTMA promoted cardiomycyte proliferaton by binding to MBD3, which decreases the formation of MBD3-HDAC1 NuRD complex . We further decreased MBD3-HDAC1 NuRD complex formation by inhibiting MBD3, boosting both rat and mouse cardiomyocyte proliferation. Conclusions: We identified the PTMA-MBD3 axis as an effective target for promoting mammalian heart regeneration and provided a new insight into cardiomyocyte proliferation through blocking MBD3-HDAC1 NuRD complex.
Abstract We136: HnRNPA1 Modulates Expression of Myogenic Differentiation Factors by Maintenance of <i>Ppp1r1b</i> - <i>lncRNA</i> /PRC2 Complex Integrity
Authors: Xuedong Kang, Yan Zhao, Marlin Touma
Journal: Circulation Research (2024)

Objective: HnRNPA1 is an important member of the HNRNP family, serves multiple functions. The long noncoding RNA Ppp1r1b is a key regulator of myogenesis in cardiac and skeletal muscle development. This study aimed to investigate the role of hnRNPA1 in Ppp1r1b-lncRNA mediated myogenesis and identify the underlying molecular mechanisms. Methods: Ppp1r1b-lncRNA : protein complex was isolated by RNA pulldown assay. The corresponding proteins were identified by mass spectrometry. The binding sites of hnRNPA1 and EZH2 on Ppp1r1b-lncRNA were further evaluated by RNA pulldown assay and western blotting. After downregulation of hnRNPA1, expression of muscle development related genes was measured by qRT-PCR and western blot analysis. Furthermore, EZH2 RIP (RNA Immunoprecipitation), H3K27me3 CHIP (Chromatin Immunoprecipitation) and Ppp1r1b-lncRNA CHIRP (Chromatin Isolation by RNA Purification) were applied. Results: HnRNPA1 and EZH2 both bind to Ppp1r1b-lncRNA and their bindng sites are located at different positions. EZH2 RIP assay and H3K27me3 CHIP show decreased Ppp1r1b-lncRNA /EZH2 interaction and corresponded decreased H3K27me3 modification after downregulation of hnRNPA1. The expression of myogenic diffrentiation related genes was significantly suppressed by hnRNPA1-RNAi while CHIRP assay indicated elevated interaction of Ppp1r1b-lncRNA with Myod1 and Myogenin promoter. Conclusions: HnRNPA1 facilitates the integrity of Ppp1r1b-lncRNA /PRC2 complex, which preserves correct regulation of myogenesis gene expression. After hnRNPA1 suppression, the elevated promoter occupancy of Ppp1r1b- lncRNA corresponds with transcriptional repression. The hnRNPA1/ Ppp1r1b lncRNA /PRC2 complex regulatory axis is a potential therapeutic target during heart and skeletal muscle development.
Abstract Mo097: Inhibiting the Sarcomere Improves Diastolic Dysfunction in Patient-Derived hiPSC-CM Models of Pediatric Restrictive Cardiomyopathy
Authors: David Staudt, Ricardo Serrano, Dries Feyen, Mark Mercola
Journal: Circulation Research (2024)

Introduction: Pediatric patients with Restrictive Cardiomyopathy (RCM) develop heart failure due to stiffening of the wall of the cardiac ventricle leading to diastolic dysfunction but preserved cardiac contractility. Treatment options are extremely limited, and the evaluation of new therapeutic strategies is hindered due to a lack of models that recapitulate this ventricular stiffening. Here, we evaluate two novel patient-derived human induced pluripotent stem cell (hiPSC) models of restrictive cardiomyopathy due to mutations in the thin filament, and evaluate separate sarcomere-targeted therapeutic strategies to improve diastolic function. Hypothesis: Directly decreasing the thin filament’s response to calcium will improve diastolic function in RCM models with less effect on systolic function than myosin inhibition. Methods: hiPSC-cardiomyocytes (hiPSC-CMs) were differentiated from hiPSC lines generated from two patients with pediatric RCM, as well as their isogenic controls. After replating on polyacrylamide gels and staining with fluorescent calcium indicators, diastolic tension, generated tension, and calcium sensitivity were measured at baseline and upon treatment with either a troponin calcium desensitizer (W7) or a myosin inhibitor (mavacamten). Results: hiPSC-CMs from both patients showed increased diastolic tension, generated tension, and calcium sensitivity compared to their isogenic controls. Treatment with Mavacamten improved diastolic function but severely compromised systolic function, while treatment with W7 improved diastolic function with less effect on systolic function. Conclusions: Directly inhibiting the sarcomere in models of RCM improved measures of diastolic dysfunction, but directly targeting the thin filament in these lines showed less effect on systolic function than myosin inhibition, suggesting cardiac troponin calcium desensitizers may be more beneficial in this severe diastolic disease.
Abstract We146: CHD8 is Essential for Postnatal Heart Function through Preserving Mitochondrial Integrity
Authors: Nong Chen, Masahide Sakabe, Mei Xin
Journal: Circulation Research (2024)

Objective: Epigenetic regulation plays a crucial role in heart development and disease, although its mechanisms remain elusive. Known mutations in epigenetic regulators have been linked to cardiac disease. For instance, the mutation in the chromodomain helicase DNA-binding (CHD) protein 7 gene, which leads to CHARGE syndrome, is known to results in heart defects. However, the role of CHD8, an interacting partner of CHD7, in the heart has not been investigated. This study aims to explore the function of CHD8 in the heart. Methods and Results: We generated a cardiac-specific Chd8 conditional knockout mouse line (Chd8 cKO) by breeding Chd8 fl/fl mice with aMHC Cre mice. Over 60% of Chd8 cKO mice died between 7 and 9 months of age, with increased heart weight to bodyweight ratio and reduced cardiac function, increased fibrosis, and signs of dilated cardiomyopathy. Electron microscopy analysis revealed disordered sarcomeres and escalated mitophagy in CHD8-deficient hearts. Using Genome-wide transcriptome analysis though RNA sequencing, we discovered that genes involved in mitochondrial oxidative phosphorylation and muscle contraction were down-regulated in Chd8 cKO hearts. Proteins in the electron transport chain complexes were down regulated and mitophagy marker LC3 increased in the Chd8 cKO hearts compared with the control mice. Upregulated pathways include inflammatory response suggesting that the innate immune response may contribute to dilated cardiomyopathy. Conclusions: Our findings underscore CHD8 as a key regulator of cardiac function and regeneration by influencing chromatin accessibility of genes involved in mitochondrial oxidative phosphorylation and contractility. Overall, our study provides new insights into the epigenetic regulation of cardiac function and the CHD8’s role in heart development and disease. It implies that activating CHD8 may be a promising therapeutic strategy for treating dilated cardiomyopathy and other heart-related diseases.
Abstract We015: Modeling Cardiac Arrhythmogenicity of hiPSC-CMs and Cardiac Fibroblasts Nanopatterned Coculture and Machine Learning
Authors: Huaxiao Yang, Nicolas Rogozinski, Hung-Ta Wo, Gautham Mohanraj, Joseph Wu, Oscar Abilez
Journal: Circulation Research (2024)

Background: Sudden cardiac death is one of the most threatening heart conditions in the U.S. Most individuals have an underlying structural cardiac disease associated with cardiac fibrosis and ventricular arrhythmia. The relationship between fibrosis and arrhythmias is found to be related to cardiovascular cell couplings, especially cardiomyocytes (CMs) and cardiac fibroblasts (CFs). While animal models have been applied to model cardiac arrhythmias, the disparities between animal models and humans limit the accuracy of pro-arrhythmic predictions. The use of human induced pluripotent stem cells (hiPSCs)-derived CMs has permitted highly modular testing conditions, specifically the ratio of CFs to CMs in an engineered coculture system. Moreover, machine learning (ML) has become an emerging tool in cardiology, such as cardiac toxicity and function evaluation and classification, with its unique advantages of high accuracy and efficiency with less human bias and intuition. Hypothesis: We hypothesize that cardiac fibrosis-associated arrhythmia will be modeled by nanopatterned coculture of hiPSC-CMs and CFs at varied ratios, and the arrhythmic samples will be accurately and efficiently classified by ML algorithms. Approach: The hiPSC-CMs were cocultured and nanopatterned with CFs at ratios of 0 to 30% for 7 days. Upon the electrical pacing at 1Hz, the action potential was recorded by live-cell optical mapping with FluoVolt. The recorded membrane potential and electrical propagation were analyzed by Matlab-based Electomap software. Results: The cocultured hiPSC-CMs and CFs were aligned with aligned sarcomeres and uni-directional contraction ( Fig. 1a ). The cardiac arrhythmia was observed in 15% CFs with spiral wave ( Fig. 1b ) in comparison to the hiPSC-CMs with 0% CF. Beat-to-beat and depolarization durations were found significant with top ranks in Feature Importance to facilitate the distinction between arrhythmic and non-arrhythmic samples by the unsupervised K-means Clustering ( Figs. 1c and d ). Conclusions: We have successfully established a nanopatterned coculturing system of hiPSC-CMs and CFs for modeling cardiac fibrosis-induced arrhythmia, which was further analyzed and classified by ML algorithms. This system reveals great potential for better understanding the relationship between cardiac fibrosis-associated arrhythmia and sudden cardiac death and also drug evaluation for anti-cardiac arrhythmia.
Abstract We041: In vitro characterization of different human-induced pluripotent stem cell-derived cardiac progenitors
Authors: Uzair Ahmed, Divya Sridharan, Mahmood Khan
Journal: Circulation Research (2024)

Introduction: Myocardial Infarction (MI) is the number one cause of death in the United States. The heart undergoes severe trauma during MI, which results in the death of cardiac cells. Although the cardiac tissue cannot regenerate on its own, regenerative medicine approaches involving cell transplantation have been shown to improve the cardiac function. Among different cell types, human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSCs) have been shown to have the ability to proliferate and differentiate into cardiac lineage cells and therefore, are a promising source for myocardial repair. Here, we characterized two distinct populations of hiPSC-CPCs and assessed their differentiation potential. Methods: We compared hiPSC-CPCs differentiated using the small molecule, isoxazole-9 (ISX-9), ISX9-hiPSC-CPCs with the commercially acquired iCell hiPSC-CPCs (Fujifilm Cellular Dynamics Int.). The expression of different cardiac lineage markers were assessed via immunostaining, qPCR and flow cytometry. Furthermore, we assessed the proliferation of the cells using immunostaining and XTT assays. We also evaluated their in vitro differentiation to cardiomyocytes, via immunostaining and qPCR analysis. Results: Our studies showed similar expression of different cardiac progentior markers (Nkx2.5, GATA4, KFL-4, ISL-1, BRY-T, MESP-1, MYH-6, CTnT) in the two hiPSC-CPC populations. However, while the iCell hiPSC-CPCs predominantly expressed the cell surface marker KDR, the ISX-9-hiPSC-CPCs predominantly expressed SSEA-1. The iCell-hiPSC-CPCs showed higher proliferation rates when compared to the ISX-9-hiPSC-CPCs. However, &gt;95% of both the cell populations expressed the proliferation marker, ki67. Finally, the while both the CPC populations could differentiate into cardiomyocytes (cTNT + ), only a few of these differentiated cardiomyocytes showed spontaneous contractility in vitro. Conclusion: In our study, we have compared and characterized two distinct populations of hiPSC-CPCs to identify a promising candidate for myocardial regeneration therapies.