tbuckley/cabot-search huggingface dataset:
Abstract We034: Hjurp Promotes Cardiomyocyte Proliferation and Heart Regeneration by Mediating CenpA Assembly
Authors: Haotong Li, Yu Nie, Shengshou Hu
Journal: Circulation Research (2024)

Background: Loss of cardiomyocytes without replenishment is one of the main cellular mechanisms leading to heart failure. Promoting cardiomyocyte proliferation and heart regeneration has become a therapeutic strategy to rescue heart failure. Therefore, we need to understand the differences between neonatal cardiomyocytes and adult cardiomyocytes, summarize the mechanism of cardiomyocyte exit from the cell cycle, and find potential intervention targets. Method: Combined analysis of bulk RNA-seq, ATAC-seq, and histone CUT&amp;Tag of 1-day-old mouse and 7-day-old mouse cardiomyocytes to screen for Hjurp . In vitro , siRNA was used to knock down Hjurp in neonatal rat cardiomyocytes, and adenovirus was used to overexpress Hjurp . RT-qPCR and immunofluorescence were used to detect cardiomyocyte proliferation and cardiomyocyte number. In vivo , adenovirus was injected into the mouse heart in situ to knock down Hjurp . Immunofluorescence was used to detect cardiomyocyte proliferation. Masson staining was used to detect the proportion of myocardial fibrosis and echocardiography was used to detect cardiac function to reflect cardiac regeneration and repair. Results: HJURP was identified by RNA-seq, ATAC-seq, and histone CUT&amp;Tag as a key regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. The expression levels of Hjurp and Cenp-A decrease with the weakening of cardiomyocyte proliferation ability. In vitro , Hjurp overexpression promoted whereas Hjurp knockdown attenuated cardiomyocyte proliferation. In vivo , cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when Hjurp was knockdown in cardiomyocytes. Conclusion: Our study confirms that HJURP is necessary for cardiomyocyte proliferation and cardiac regeneration after cardiac injury. As an assembly element of centromeres during the cell cycle, overexpression of HJURP is sufficient to promote cardiomyocyte proliferation, which may be accomplished by promoting CENP-A assembly. Thus, HJURP might serve as a potential novel target in heart regeneration after myocardial infarction.
Abstract We022: Depletion of Tip60 After Myocardial Infarction Induces Histone H2A.Z Deacetylation Followed by Cardiomyocyte Dedifferentiation/Cell-Cycle Activation
Authors: Xinrui Wang, Katherine Kulik, Tina Wan, John Lough, John Auchampach
Journal: Circulation Research (2024)

Myocardial infarction (MI) leads to cardiomyocyte (CM) loss, resulting in cardiac dysfunction and heart failure. Remuscularization of injured myocardium requires proliferation of surviving CMs. However, approaches aimed at inducing CM regeneration following MI have been inadequate. To this end, we are targeting the acetyltransferase Tip60 (Tat-interactive protein 60 kD), a pleiotropic tumor suppressor encoded by the Kat5 gene. Using a murine genetic model, we previously reported that disruption of Kat5 promotes CM cell-cycle re-entry, and protects against the damaging effects of MI. It is becoming increasingly recognized that pre-existing CMs must undergo dedifferentiation accompanied by reversion to glycolytic metabolism in order to resume proliferation. The site-specific acetylation of the histone variant H2A.Z at lysine K4/K7 (H2A.Zac K4/K7 ) has been shown to maintain the differentiated state in hematopoietic stem cells (HSCs), neurons, and skeletal myocytes. Here, we report that Tip60 depletion post-MI results in the near-complete absence of H2A.Zac K4/K7 in CMs. This is associated with the enrichment of differentially expressed genes (DEGs) in epithelial to mesenchymal transitioning (EMT), cytoskeletal disassembly, extracellular matrix (ECM) softening, and metabolic reprogramming. To assess therapeutic relevance, we have begun to evaluate the potential cardioprotective effects of the anti-parasitic drug pentamidine, a known Tip60 inhibitor. Systemic administration of pentamidine on days 3-16 post-MI preserved cardiac function, accompanied by CM cell-cycle activation. These data suggest that pentamidine treatment enhances remuscularization, thereby promoting the retention of post-MI function, supporting the translational promise of targeting Tip60 as an innovative therapy for ischemic heart disease.
Abstract Mo095: Variant Effect Mapping by Combining Base-editing Mutagenesis and Multiplexed Functional Assays Using Human Cardiomyocytes
Authors: Yuta Yamamoto, Victoria Parikh, Euan Ashley
Journal: Circulation Research (2024)

Introduction: MYBPC3 is the most prevalent gene in hypertrophic cardiomyopathy (HCM). However, the pathogenicity of most MYBPC3 variants remains unknown, and it is still challenging to interpret variants identified in patients. Although multiplexed functional assays using cancer cell lines are powerful approaches to test large number of variants in a single experiment and facilitate the interpretation of variants, there is a limitation of the assessment of genes expressing in specific cell types, such as sarcomeric genes including MYBPC3. Hypothesis: The approach integrating base-editing mutagenesis and multiplexed functional assays using iPSC-derived cardiomyocytes (iPSC-CMs) will enable us to prospectively evaluate MYBPC3 variants in a high-throughput manner and illuminate the pathophysiological mechanism of HCM. Methods: We focused on the intron-exon boundary including intron 11 and exon 12 in MYBPC3 and introduced variants into iPSCs by CRISPR-X, which can target multiple genomic locations and introduce diverse editing, and adenine base editor. After cardiac differentiation, we performed multiplexed functional assays. iPSC-CMs were immunostained for cMyBP-C and BNP expressions and sorted based on those expressions using fluorescence-activated cell sorting. The ubiquitin-proteasome activity associated with cellular proteostasis was analyzed by ubiquitin fusion degradation reporter system. The variant scores were calculated by comparing the frequency of each variant in each population sorted based on phenotype using next-generation sequencing. Results: About 200 variants located at the mutagenesis target region were evaluated via in situ mutagenesis and each functional assay. The pathogenic/likely pathogenic variants from ClinVar exhibited higher variant scores compared to benign/likely benign variants in each assay. Even in variants that have not yet been reported clinically, nonsense variants and several missense and intronic variants including splice site variants showed higher variant scores compared to synonymous variants. Conclusion: This study demonstrates that our novel approach can prospectively evaluate MYBPC3 variant effect at scale and provide clues to elucidate pathophysiological mechanism of HCM.
Abstract We037: GPC3-mediated metabolic rewiring of diabetic mesenchymal stromal enhances their cardioprotective functions via pyruvate kinase M2 activation
Authors: Darukeshwara Joladarashi, Charan Thej Gurrala, Ajit Magadum, Vandana Mallaredy, Maria Cimini, Carolina Gonzalez, May Truongcao, Andrew Gibb, Cindy Benedict, Walter Koch, Raj Kishore
Journal: Circulation Research (2024)

Background: Mesenchymal stromal cells (MSC) represent promising stem cell therapy for treating cardiovascular diseases. Diabetes affects the functional capability of MSC and impedes cell-based therapy. However, the impact of diabetes on MSC myocardial reparative activity, metabolic fingerprint, and the mechanism of dysfunction is still poorly understood. Therefore, our study aimed to understand the molecular basis of diabetes-induced MSC functions and their metabolic mechanism, and potential metabolic reprogramming of diabetic MSC to reverse these dysfunctions may represent a strategy to enhance cell-based therapeutics for myocardial repair in diabetic patients. Hypothesis: Our central hypothesis is that increased Glypican-3 (GPC3) mediates diabetic MSC dysfunction and diminishes energy metabolism; metabolic reprogramming of diabetic MSC with GPC3 knockdown restored diabetic MSC reparative and therapeutic activities in post-MI cardiac repair. Methods: MSCs were isolated from the bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) to compare the MSC metabolic profiles and consequent reparative function. We investigated the effect of GPC3 knockdown on db/db-MSC energy metabolism, immunosuppression, angiogenic potential in vitro, and therapeutic potential for ischemic cardiac tissue repair. Results: Intra-myocardial transplantation of db/db-MSC into the ischemic myocardium of mice does not confer cardiac benefit post-MI. The seahorse metabolic flux assays and 13 C glucose tracing studies identified defective energy metabolism in db/db-MSC. Furthermore, we found that GPC3, a heparan sulfate proteoglycan, is highly upregulated in db/db-MSC. GPC3 knockdown-db/db-MSC restored their energy metabolism, immunomodulation, and angiogenesis. Intramyocardial injection of GPC3 metabolically reprogrammed-db/db-MSC confers cardiac benefit post-MI. Conclusion: Our findings indicate that metabolic rewiring using GPC3 may be used to restore the metabolic state and functions of db/db-MSC for cell therapy applications.
Abstract We025: Human Engineered Heart Tissues Demonstrate Clinically-Relevant Disease indicators of Duchenne Muscular Dystrophy
Authors: Shawn Luttrell, Ayesha Arefin, Jesse Macadangdang, Kevin Gray, Samir Kharoufeh, Christal Worthen, Greg Luerman, Nicholas Geisse
Journal: Circulation Research (2024)

Accurate in vitro modeling of healthy and disease conditions is crucial for assessing the safety and efficacy of novel therapeutics before clinical trials. For cardiac diseases, direct evaluation of muscle contraction is a reliable indicator of overall tissue function, however, this often necessitates expensive and low-throughput non-human in vivo models. Moreover, animal models may not accurately predict human responses to compound exposure. Here, we have developed a turnkey platform for facile fabrication of 3D engineered heart tissues (EHTs) that enables label-free, push-button measurements of contractility. The system allows for customized stimulation protocols to individual constructs, while simultaneously measuring contractility across 24 tissues in parallel with minimal user input. This approach enables stratification of muscle phenotypes and facilitates compound safety and efficacy screening. We present a 3D model of Duchenne muscular dystrophy that displays functional deficits consistent with in vivo disease indicators, including reduced contractile force and slowed relaxation kinetics, though beat rate remains unchanged. We show both acute and chronic effects of cardiotoxic drugs, as well as positive inotropic responses to external calcium and Isoproterenol. This highlights the utility of using EHTs to identify toxicity and derisk drug development and clinical testing outcomes. Spontaneously beating EHTs were also subjected to in situ stretching, leading to increased levels of cardiac troponin I (cTnI) in the tissue medium, indicating stretch induced injury. These data demonstrate a first-and-only commercial platform that integrates individual, fully-customizable, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Heart organoids were stretched under load presenting a challenge for therapeutics targeting injury prevention in the myocardium. Stimulation is coupled with automated assessment of 3D muscle contraction, providing an inclusive, medium-throughput platform for studying muscle-related disorders, advancing therapeutic discovery using a modality-agnostic biosystem.
Abstract We027: Gut Microbiota Modulation by Immunosuppression and Cardiac Cell Therapy in a Nonhuman Primate Ischemia/Reperfusion Model of Cardiac Regeneration
Authors: Patrick Hsieh
Journal: Circulation Research (2024)

End-stage ischemic heart disease necessitates heart transplantation, and emerging cell therapy presents a promising solution to address donor scarcity. Disruption of gut microbiota significantly influences various diseases and treatments, including transplantation. However, the impact of immunosuppression and cardiac cell therapy on gut microbiota remains largely unexplored. To elucidate this relationship, we investigated gut microbiota dynamics in response to immunosuppression and cell therapy in a nonhuman primate (NHP) cardiac ischemia/reperfusion (IR) model, with controlled genetic, dietary, and environmental factors. Immunosuppression enriched anaerobes (Faecalibacterium, Streptococcus, Anaerovibrio, Dialister), increasing gut microbiota diversity. These changes correlated with metabolic shifts towards amino acid metabolism and nucleosides/nucleotides biosynthesis. Combined treatment of human induced pluripotent stem cell (iPSC)-derived endothelial cells (EC) and cardiomyocytes (CM) also increased gut microbiota diversity, with specific genera alterations. The EC/CM co-treatment group displayed gut microbiota resembling the pre-injury group, with host metabolism shifting towards amino acid and fatty acid/lipid biosynthesis post-cell therapy. These observed microbiota changes and metabolic shifts could serve as biomarkers for monitoring cell therapy and immunosuppression outcomes, offering potential therapeutic targets to enhance efficacy.
Abstract We145: δ-catenin mRNA m <sup>6</sup> A Methylation Promotes Cardiac Fibrosis following Acute Myocardial Infarction
Authors: Roshan Dutta, Prabhat Ranjan, Vasundhara Pathak, Karen Colin, qinkun zhang, Min Xie, Hind Lal, martin young, Suresh Verma
Journal: Circulation Research (2024)

Background: Cardiac fibrosis is pivotal in heart failure progression, where excessive extracellular matrix (ECM) secretion by activated fibroblasts leads to adverse remodeling and dysfunction. While Wnt/β-catenin signaling influences fibroblast activation and cardiac fibrosis post-MI, its precise regulation remains unclear. Emerging evidence suggests N6-methyladenosine (m 6 A) mRNA methylation's role in disease pathology, yet its specific contribution to post-MI cardiac fibrosis is not well understood. Thus, we hypothesized that “ MI-induced METTL3 (a Key m6A mRNA methyltransferase) activation stabilizes δ-catenin mRNA, facilitating cardiac fibrosis and adverse remodeling ”. Methods: The mice underwent sham/MI surgeries for 4 weeks, after which heart tissues were collected for biochemical and histological analysis following heart function measurements. Additionally, methyl-immunoprecipitation followed by RNA sequencing (MeRIP-sequencing) was performed on the heart tissue post-surgery, and the data were analyzed to identify fibrosis-associated targets. Results: Ischemic injury significant increase in m6A mRNA methylation in heart tissues. This increase in m 6 A RNA was also observed in adult cardiac fibroblasts (AMFs) following TGFb treatment. Interestingly, METTL3 inhibition (METTL3 siRNA) resulted in a significant reduction in TGFb-induced periostin and fibronectin gene expression, while METTL3 overexpression enhanced the expression of fibrotic genes in AMFs. To identify differentially regulated m6A target genes, MeRIP-seq was performed on RNA isolated from mice hearts post-MI. The sequencing data suggested hypermethylation of fibrosis-associated genes, including δ-catenin, post-MI. Notably, TGFb-induced increased δ-catenin mRNA methylation led to δ-catenin mRNA stabilization. In contrast, METTL3 inhibition using siMETTL3 in AMFs and in METTL3 KO mice significantly reduced fibroblast activation and cardiac fibrosis. Conclusion: Our data suggests that hypermethylation of δ-catenin mRNA plays a significant role in the progression of cardiac fibrosis following AMI. Therefore, regulating METTL3 could be a potential therapeutic target for attenuating cardiac fibrosis.
Abstract We038: Metal-organic frameworks as next-generation cardiac therapeutics
Authors: Syed Baseeruddin Alvi, Muhamad Mergaye, Pankaj Singh Dholaniya, Ghazala Sameer, Xianyao Xu, Helena Islam, Mahmood Khan
Journal: Circulation Research (2024)

Background: Zinc (Zn) is an essential trace element for the normal functioning of the heart. Long-term dietary supplementation of Zn has been shown to improve cardiac function and prevent myocardial damage caused by ischemia-reperfusion injury. However, the major limitation in delivering Zn to the cardiac tissue is the off-targeted absorption and the duration of treatment to achieve a therapeutic effect. Hypothesis: Cardiac-targeted delivery of trace elements like Zn may attenuate myocardial damage caused by ischemia-reperfusion injury, improve cardiac function, and exert a cardioprotective effect. Methods: Zn-based metal-organic frameworks (ZIF-NPs) were fabricated and tested on Microvascular Endothelial Cells (MVECs), and human-induced pluripotent derived cardiomyocytes (hiCMs) for cytoprotective effect against hypoxic stress using Live/dead assay and multi-electrode array (MEA) analysis respectively. The in vivo efficacy of ZIF-NPs was tested in the rodent myocardial infarction (MI) model and cardiac function was assessed via M-mode echocardiography. Results: The fabricated ZIF-NPs were biocompatible and showed significant intracellular uptake causing the induction of cytoprotective metallothioneins. ZIF-NP treatment showed enhanced cell survival in serum-starved MVECs and exhibited pro-angiogenic effects in vitro. Similarly, hiCMs treated with ZIF-NPs displayed enhanced endurance and electrophysiological function, when subjected to hypoxic stress and protected the cells against ROS-induced DNA damage. Intramyocardial delivery of ZIF-NPs in the cardiac tissue improved cardiac function and attenuated fibrosis post-MI. Conclusions: ZIF-NPs exerted a pro-survival, cytoprotective response in both in vitro and in vivo conditions. Our study highlights the significance of cardiac targeted Zn delivery by ZIF-NPs for cardioprotection as next-generation cardiac therapeutics.
Abstract Mo090: Altered Cardiac Cell Populations in Hypoplastic Left Heart Syndrome
Authors: Sarah Morton, Kemar Brown, Eric Wei, Joshua Gorham, BARBARA MCDONOUGH, Martin Beyer, Meraj Neyazi, Olivia Layton, Jonathan Seidman, Christine Seidman
Journal: Circulation Research (2024)

Background: Congenital heart disease (CHD) affects ~1% of infants. Hypoplastic left heart syndrome (HLHS), a severe form of CHD in which the left ventricle is underdeveloped, is associated by a 15% incidence of heart failure by 6 years of age. Only 6% of HLHS patients have a genetic cause identified on exome sequencing, limiting the ability of patients to receive a diagnosis and potentially benefit from targeted treatments. Hypothesis: Novel HLHS genes are differentially expressed in HLHS cardiac tissues, or in cells with differential abundance in HLHS hearts. Goals: To use single nucleus RNA sequencing of HLHS patient cardiac tissues to identify candidate HLHS genes. Methods: Single nucleus RNA sequencing (nucSeq) was performed on paired left and right ventricular tissues (LV, RV) from 9 participants with HLHS. Left ventricular cardiac tissues from children (4) and adults (12) without CHD were used for comparison. Filtering with CellBender and Solo were used to remove low-quality nuclei. Analysis was performed in R using the Seurat package. Results: HLHS LV tissues had a higher proportion of cardiomyocytes than pediatric or adult control tissues, (78.7% vs 64.1%, p=0.005; 78.7 % vs 47.0%, p=1.8E-08, respectively), and a lower proportion of mural cells (3.1% vs 9.3%, p=3.9E-05; 3.1 % vs 23.5%, p=7.3E-15, respectively). By contrast, there were similar proportions of endothelial cells in the HLHS, pediatric and adult tissues (7.1%, 8.8% and 7.9%, respectively; p=0.56). Within HLHS tissues, the LV had a higher proportion of cardiomyocytes than the RV (78.7% vs 51.2%, p=4.8E-04) while the proportion of endothelial cells were similar in the LV and RV (7.3% and 9.1%, respectively; p=0.33). LV tissues from HLHS participants 2-18 years of age had fewer capillary endothelial cells than pediatric controls (31.2% vs 57.8%, p=0.001), and gene markers of HLHS endothelial cells included PBX1 , which is required for Hox D-3 mediated angiogenesis, as well as ARHGAP26 and RORA, inhibitors of VEGF signaling. Conclusion: HLHS cardiac tissues have a higher proportion of cardiomyocytes in the LV despite a smaller chamber size. Differences in gene expression that accompany the differences in cell proportion could identify novel HLHS genes as well as potential therapeutic targets.
Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an <i>In Vitro</i> Model to Evaluate the Efficacy of a Gene Replacement Therapy for <i>Plakophilin-2-</i> Associated Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Isaac Perea Gil, J. Alex Aycinena, Rishikesan Chandrakumar, Olga Cisne-Thompson, Jonathan Tsui, Bernardo Zepeda, Anley Tefera, Reva Shenwai, Farshad Farshidfar, Chayse Jones, Zhihong Jane Yang, Timothy Hoey, Gretchen Argast
Journal: Circulation Research (2024)

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G&gt;C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.