tbuckley/cabot-search huggingface dataset:
Abstract Mo091: Analysis of GLA Gene Mutation Using a Female Fabry Disease Patient-derived Induced Pluripotent Stem Cells.
Authors: Yukihiro Saito, Kazufumi Nakamura, Toshihiro Iida, Duy Quan Vo, Shinsuke Yuasa
Journal: Circulation Research (2024)

Introduction: Fabry disease is an inborn error of metabolism caused by mutations in the alpha-galactosidase gene ( GLA ) located on the X chromosome, resulting in intracellular accumulation of globotriaosylceramide and systemic tissue injury. In addition to enzyme replacement therapy, chaperone therapy is effective for many missense mutations. One female patient had c.547G&gt;A mutation, which has been reported as p.G183S, and we checked the indication for migalastat, a pharmacological chaperone. However, migalastat reactivity testing using HEK293 cells transfected with mutant cDNA had not been performed for this mutation. Aims: We analyzed the c.547G&gt;A mutation and examined the efficacy of migalastat for this mutation. Methods: Peripheral blood mononuclear cells from patients were transfected with reprogramming factors using episomal vectors to generate induced pluripotent stem (iPS) cells. Cardiomyocytes were induced from the iPS cells. The mutation site was located at the 3' end of exon 3, and splicing mutation was suspected. Thus, PCR amplification was performed between exon 2 and exon 5 of the cDNA, and the PCR product was sequenced to confirm the mRNA sequence generated in the patient's cells. Real-time PCR was performed using Taqman probe to amplify sites unaffected by the mutation, and expression levels of normal and mutant GLA were compared. iPS cells were treated with migalastat and the effect on α-galactosidase activity was examined. Results: Since the patient was female, five iPS cell lines expressing only normal GLA and four iPS cell lines expressing only mutant GLA were established due to X-chromosome inactivation. Mutant cDNA was found to have a deletion of 36 bases, part of exon 3, resulting in a deletion of 12 amino acids. Real-time PCR result showed that the GLA expression level of mutant GLA-expressing cells was about half that of normal GLA-expressing cells, which was comparable to the GLA expression level of the cells from a healthy subject. Mutant GLA-expressing iPS cells had no α-galactosidase activity, which was not increased by treatment with migalastat. Conclusions: In patient-derived iPS cells and iPS cell-derived cardiomyocytes, c.547G&gt;A produced p.C172_G183del, not p.G183S, and migalastat was ineffective. Further therapeutic development is needed for patients with this mutation.
Abstract We033: Heart-Specific Histone Methyltransferase SMYD1 Promotes Cardiac Maturation in the Direct Conversion of Human Fibroblasts into Cardiomyocytes.
Authors: Anteneh Getachew Woldemariam, Hui Li, Meimei Wang, William Peng, Justin Bridges, Rui Lu, Yang Zhou
Journal: Circulation Research (2024)

Introduction: Direct cardiac reprogramming emerges as a promising strategy for in situ conversion of fibroblasts into induced cardiomyocyte-like cells (iCMs). While the conversion of mouse fibroblasts into functional, beating iCMs has been achieved efficiently, the reprogramming of human iCMs remains challenging, particularly their maturation with existing transcription factor combinations. Given the pivotal role of epigenetic regulation in this process, our study sought to leverage heart-specific epigenetic factors alongside cardiac-enriched transcription factors to enhance cardiac maturation, thereby yielding more reliable human iCMs. Result: Through analyzing the proteomic data across various human tissues, we found that while many epigenetic factors are broadly expressed, SMYD1 is uniquely heart-specific. To determine its involvement in reprogramming, we overexpressed SMYD1 and evaluated the reprogramming efficiency and iCM quality via flow cytometry, immunofluorescence staining, and transcriptome analysis. Notably, we observed an increase in iCMs expressing sarcomere markers, such as α-myosin heavy chain and α-actinin, as well as the ventricular specific myosin-light-chain 2v in SMYD1-treated group. Gene set enrichment analysis of RNA-sequencing data further indicated that SMYD1-induced iCMs exhibit ventricular gene signatures. Additionally, we discovered moderate activation of SMYD1 in mature iCMs generated with MEF2C, GATA4, TBX5, and TBX20. TBX20 is the key factor that leads to the co-binding of MEF2C, TBX5, and TBX20 on the SMYD1 promoter region and increases active histone marks, H3K27ac and H3K4me1. Rescue assay and transcriptomic comparison confirmed SMYD1 as the primary downstream target of TBX20 in promoting iCM functional maturation. Mechanistically, SMYD1 directly interacts with MEF2C and GATA4 to modify histone marks on cardiac regulatory regions. Conclusion: Our study provides comprehensive phenotypic and molecular evidence of SMYD1’s role as a cardiac-specific regulator during direct cardiac reprogramming. By interacting with key reprogramming factors and rewriting histone marks, SMYD1 significantly advances the maturation of human iCMs towards a ventricular subtype identity.
Abstract Mo094: Cardiomyocytes Differentiated from Human Induced Pluripotent Stem Cells Show Robust Phenotypes to Test Gene Therapy Strategies for Duchenne Muscular Dystrophy
Authors: Abiageal Keegan, Adriana Fernanda Gonzalez, Sofia Torres-Bigio, Leahlyn Mamuyac, Hung Doan, Rongruo Zhang, Fiona Lau, Asuka Eguchi
Journal: Circulation Research (2024)

Introduction: Duchenne muscular dystrophy is a severe muscle wasting disease caused by the lack of dystrophin expression. Patients die of heart failure in their second or third decade of life. Delivery of full-length dystrophin is not practical due to the large size of the gene; however, smaller variants of dystrophin, called microdystrophins, have shown promise in restoring function to the skeletal muscle. These microdystrophins are currently being dosed to patients in clinical trials; however, therapeutic efficacy in the heart is unknown due to challenges in modeling DMD-associated heart failure in animal models. Hypothesis: We hypothesized that microdystrophin gene therapy can ameliorate disease phenotypes in DMD iPSC-cardiomyocytes. Methods: This study uses human induced pluripotent stem cells (iPSCs) to model the functional deficits of cardiomyocytes. By comparing DMD cells with isogenic controls where the dystrophin mutations are corrected, we are able to focus on disease phenotypes attributed to the disease in vitro. We performed viability assays to assess cell survival, ratiometric calcium imaging to evaluate calcium handling, and traction force microscopy to measure the force of contraction. Results: We found that iPSC-cardiomyocytes exhibit poor viability, aberrant calcium handling, and reduced force of contraction. When we delivered three microdystrophin variants to DMD iPSC-cardiomyocytes, we found the microdystrophins improved cell viability and partially rescued functional deficits. Conclusions: These findings demonstrate that this platform can be used to test the therapeutic efficacy of dystrophin variants in the molecular context of human cells.
Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation
Authors: Nivedhitha Velayutham, Jessica Garbern, Hannah Elwell, Zhu Zhuo, Laura Ruland, Farid Elcure Alvarez, Sara Frontini, Yago Rodriguez Carreras, Elisabeth Ricci-Blair, Jeanna Shaw, Aldric BOUFFARD, Sokol Morgan, Estela Mancheno Juncosa, Seth Rhoades, Daphne van den Berg, Alexander Kreymerman, Junya Aoyama, Jens Hoefflin, Herb Ryan, Shannan Ho Sui, Richard Lee
Journal: Circulation Research (2024)

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.
Abstract We021: Connexin 43 mediates macrophage induced cardiomyocyte proliferation and heart regeneration
Authors: Yandong Li, Jie Feng, Jian Zhang, Yu Nie
Journal: Circulation Research (2024)

Background: Heart regeneration is a potential strategy to recover pumping function after cardiac injury and protect against heart failure. Macrophages have been demonstrated to promote heart regeneration, while its mechanism has not been addressed. Modulating the interaction between macrophages and cardiomyocytes through connexin 43 (CX43) may provide a clinical therapy for preventing further deterioration of patients with heart injury and restoring the lost myocardium. Hypothesis: We hypothesize that macrophages can interact with cardiomyocytes via CX43, leading to promotion of cardiac proliferation and heart regeneration. Aims: Our study aims to provide the evidence of macrophages contacting cardiomyocytes through CX43 and promoting heart regeneration. Methods and Results: Through cell co-culture experiments, we found that macrophages attache to cardiomyocytes via CX43, subsequently promoting myocyte proliferation as evidenced by proliferative markers. Blocking the CX43 connection by SiRNA between two cells suspended cardiomyocyte proliferation in vitro. We generated macrophage-specific CX43 deficient mice (CX43 Mφ/- ) by crosing CD11b-Cre mice and CX43-flox mice. We performed apical resection operation on 1 day-old CX43 Mφ/- mice and harvested the hearts at 21 day-post-resection. CX43 Mφ/- mice demonstrated either the heart regeneration ability nor pumping function. Applying single-cell RNA sequencing in CX43 Mφ/- hearts, we illustrated the unique transcriptional signature of macrophage and cardiomyocyte in macrophage-specific CX43 deficient mice. Conclusion: Our study provides the evidence that macrophages attache to cardiomyocyte via CX43 after cardiac injury, promote cardiomyocyte proliferation and heart regeneration. This discovery may offer a new target for clinical treatment of myocardial infarction.
Abstract Mo089: Fontan-Associated Liver Disease On A Chip
Authors: Sarah Rezapourdamanab, Yamini Singh, Sophia Norton, Boeun Hwang, Linqi Jin, Mehdi Salar Amoli, Shweta Karnik, Reza Avazmohammadi, Sunder Neelakantan, Lakshmi Dasi, Rene Romero, Holly Bauser Heaton, Vahid Serpooshan
Journal: Circulation Research (2024)

Introduction: The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current in vitro liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for in vitro study of hepatic fibrosis in response to altered flow conditions in FALD patients. Methods: Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured in vitro for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy vs. disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses. Results: Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers. Conclusion: This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for in vitro investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.
Abstract Mo093: Rescue of Desmin Insufficiency Restores Contractile Function in Cardiomyocytes with <i>MYH7</i> E848G Dilated Cardiomyopathy Variant
Authors: Alexander Loiben, Wei-Ming Chien, Ashley Mckinstry, Dania Ahmed, Kai-chun Yang
Journal: Circulation Research (2024)

Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 ( MYH7 ) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for MYH7 DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic MYH7 E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force. MYH7 E848G/+ hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to MYH7 +/+ hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of MYH7 E848G/+ hiPSC-CMs compared to isogenic control MYH7 +/+ hiPSC-CMs found significant downregulation of desmin ( DES) (76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in MYH7 E848G/+ hiPSC-CMs was significantly attenuated relative to MYH7 +/+ hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor MEF2C , a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting MYH7 E848G/+ suppresses the MEF2C - DES signaling axis. We hypothesized restoring desmin protein levels may improve contractility in MYH7 E848G/+ hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple + MYH7 E848G/+ hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple - MYH7 E848G/+ hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple + MYH7 +/+ hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the MEF2C - DES signaling axis is a potential novel mechanism by which pathogenic MYH7 variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.
Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity
Authors: David Staudt, Peter Tran, Dongju Han, Ricardo Serrano, Mark Mercola
Journal: Circulation Research (2024)

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.
Abstract We141: Leveraging Cardiac Gene Reprogramming at Single-Cell Resolution to Understand Heart Failure
Authors: Megan Russell, Bin Zhou, Deyou Zheng
Journal: Circulation Research (2024)

Background: A defining characteristic of heart failure is cardiac gene reprogramming. Heart failure-induced pathological stressors activate transcription factors, including Gata4 , Tbx5 , and Mef2, required for cardiogenesis. These transcription factors activate fetal cardiac genes in the failing adult heart. The mechanism and role of such cardiac gene reprogramming in response to heart failure remain not fully characterized. Here, we leverage transcriptional reprogramming induced by thyroid hormone deficiency as a model system to understand adult heart failure at the single-cell level. Methods: We engineered a novel Myh6 / Myh7 reporter mouse line. Hypothyroidism was induced in mice with propylthiouracil (PTU). PTU treatment led to global cardiac gene reprogramming. To profile gene expression and chromatin accessibility during cardiac gene reprogramming, single nuclei multiomic sequencing was conducted. Echocardiographs on control and PTU-treated mice evaluated cardiac function changes associated with reprogramming. Cardiac tissues were collected to validate both known and newly identified candidates involved in cardiac gene reprogramming. Results: Characterization of hypothyroidic mice as a model for cardiac reprogramming revealed that the ventricles of PTU-treated hearts significantly switch from Myh6 to dominantly express Myh7 . Evaluation of echocardiographs showed that PTU-treated hearts are significantly atrophic, have chamber dilatation, and reduced cardiac function. Analysis of multiome datasets revealed a unique gene set that is differentially expressed and accessible during cardiac gene reprogramming. Upregulated and more accessible genes in PTU-treated cardiomyocytes (CM) are involved in muscle cell differentiation, muscle hypertrophy, and mitochondrion disassembly ( Tbx5 , Sox4 , Wnt2 , Rgs2 , Hey2 , Mfn2 ). Downregulated and less accessible genes in PTU-treated CMs are related to heart contraction, regulation of heart rate, and ion membrane transport ( Kcnd3 , Jarid2 , Epas1, Atp2a2 , Scn5a , Slc25a4 ). Conclusion: Induction of cardiac gene reprogramming by thyroid inhibition can be applied to understand adult heart failure mechanisms. Hypothyroidism effectively induces global remodeling in the adult heart. Single nuclei multiomic analysis reveals new mechanistic insights into cardiac gene reprogramming that alters the expression and accessibility of genes essential for heart development and maturation.