bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract We001: Doxorubicin-Induced hiPSC Hormetic Model
Authors: Patel Jahanvi , Gonzalez Vargas Hazel , Lopez Perez Iris , Toussaint Tania , Joshi-Mukherjee Rosy
Published: October 2024

Doxorubicin (Dox), an anticancer anthracycline drug, is an important tool to understand how cells develop resistance to anthracyclines. Hormesis is a biological phenomenon where low doses of drugs can have beneficial effects on cell proliferation and differentiation. hiPSCs are becoming the model of choice for Dox studies yet the low dose hormetic effect of Dox on hiPSC proliferation and differentiation is not well understood. We assess the hypothesis that low-dose Dox preconditioning of hiPSCs enhances pluripotency gene expression while maintaining pluripotency state. To determine the low-dose effect (50 nM), we treated the JHU001 hiPSC line with Dox (hiPSC-D50) for two hours and a vehicle control without Dox (hiPSC-D0). Both hiPSC-D50 and hiPSC-D0 were maintained in E8 flex medium culture until reaching 70% confluency and were passaged five times and frozen for experimental studies. hiPSC-D50 and -D0 were thawed at passage 5 and cultured on coverslips for pluripotent gene expression (Oct-4, Nanog, and SSEA-4) analysis by immunocytochemistry. The cells were imaged using epifluorescence Nikon Eclipse Ti2 microscopy and the images were analyzed using NIS-Elements BR software. Phase-contrast microscopy shows significant cell death in hiPSC-D50 24 hours post-treatment compared to hiPSC-D0. However, 72 hours post-Dox-treatment, several colonies with pluripotent morphology were observed in hiPSC-D50 that were similar to hiPSC-D0. While some differentiated cell morphologies were observed in hiPSC-D50, these were significantly reduced after five passages. Immunofluorescent staining of Oct-4 and Nanog showed nuclear localization whereas SSEA-4 was localized on the cell surface, in both hiPSC-D0 and hiPSC-D50 cells at passage five. However, immunocytochemistry analysis of pluripotent markers revealed significantly higher expression of Oct-4, Nanog and SSEA-4 in hiPSC-D50 when compared to hiPSC-D0 (1.3-fold,1.9-fold and 2.9-fold increase with p-values: 0.00057, 0.018, and 0.0000026, respectively; n= 5) suggesting a more robust pluripotent state and self-renewal capacity. Interestingly, higher SSEA-4 expression might also be associated with resistance to Dox. Our data suggest that low-dose Dox preconditioning enhances pluripotency state. This Dox-induced hormesis model of hiPSCs is a unique platform to help understand how Dox upregulate pluripotency markers and the potential link between SSEA-4 and drug resistance.
Abstract We003: Identification of CircRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathways Related to Cardiomyocyte Proliferation in Neonatal Mice
Authors: Tang Ling , Nyarige Verah , Li Pengsheng , Zhu Wuqiang
Published: October 2024

Introduction:
Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis.

Methods:
Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining.

Results:
Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle.

Conclusion:
Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.
Abstract We007: High Throughput 3D-Printed Human Engineered Heart Tissues for Cardiac Disease Modeling
Authors: Juarros Miranda , Dhand Abhishek , Martin Thomas , Valle-Ayala Henry , Hunt Dakota , Burdick Jason , Leinwand Leslie
Published: October 2024

While cell models have been used to study cardiac diseases, there is currently no standardization or fully mature
in vitro
system. Obtaining a mature cardiomyocyte (CM) phenotype is necessary to create a physiologically relevant environment and accurately model and study cardiac diseases. Increased success in maturation of human induced pluripotent stem cell derived CMs (hiPS-CMs) has been achieved in 3D
in vitro
models in the form of engineered heart tissues (EHTs). Digital light processing (DLP) offers a high throughput and efficient method to fabricate hydrogels that recapitulate properties of native tissues. In this study, we use DLP-printed EHTs to seed and mature differentiated hiPS-CMs and test their potential for modeling pathological cardiac hypertrophy. To further encourage maturity of CMs, we utilized a low glucose maturation medium supplemented with fatty acids. DLP-printed EHTs displayed high reproducibility and increased CM maturity. To determine degree of maturity of our system, we performed RT-qPCR, western blot analysis, and immunofluorescence (IF) microscopy. We found increased expression of fatty-acid transport and beta oxidation genes and improved sarcomere alignment compared to 2D hiPS-CMs. To model pathological cardiac hypertrophy, we used two different approaches: 1. acute adrenergic agonism with isoproterenol and phenylephrine; 2. chronic culturing of EHTs in pathologically stiff conditions. To assess pathology, we conducted RT-qPCR, western blot, ELISA, IF microscopy, and functional analyses. We found that agonist-treated EHTs displayed increased pathology-associated gene expression compared to standard 2D hiPS-CMs. Both agonist-treated and stiff EHTs had increased secreted B-Type Natriuretic Peptide (BNP) and phosphorylation of proteins involved in pathological remodeling, including ERK and P38. Additionally, we observed increased nuclear area, increased calcium handling, and increased contractile force in the treatment groups. In conclusion, we have established a high throughput, reproducible, mature EHT platform that can be used to model pathological cardiac hypertrophy. This platform can be easily adopted by other laboratories to study a wide array of cardiac diseases.
Abstract We014: Creating Cell-specific Computational Models of Stem Cell-derived Cardiomyocytes Using Optical Experiments
Authors: Yang Janice , Daily Neil , Pullinger Taylor , Wakatsuki Tetsuro , Sobie Eric
Published: October 2024

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction as a powerful model in cardiac disease and therapeutics research, since iPSCs are self-renewing and can be derived from healthy and diseased patients without invasive surgery. However, current iPSC-CM differentiation methods produce cardiomyocytes with immature, fetal-like electrophysiological phenotypes, and the variety of maturation protocols in the literature results in phenotypic differences between labs. Heterogeneity of iPSC donor genetic backgrounds contributes to additional phenotypic variability. Several mathematical models of iPSC-CM electrophysiology have been developed to help understand the ionic underpinnings of, and to simulate, various cell responses, but these models individually do not capture the phenotypic variability observed in iPSC-CMs. Here, we tackle these limitations by developing a computational pipeline to calibrate cell preparation-specific iPSC-CM electrophysiological parameters.

We used the genetic algorithm (GA), a heuristic parameter calibration method, to tune ion channel parameters in a mathematical model of iPSC-CM physiology. To systematically optimize an experimental protocol that generates sufficient data for parameter calibration, we created simulated datasets by applying various protocols to a population of
in silico
cells with known conductance variations, and we fitted to those datasets. We found that calibrating models to voltage and calcium transient data under 3 varied experimental conditions, including electrical pacing combined with ion channel blockade and changing buffer ion concentrations, improved model parameter estimates and model predictions of unseen channel block responses. This observation held regardless of whether the fitted data were normalized, suggesting that normalized fluorescence recordings, which are more accessible and higher throughput than patch clamp recordings, could sufficiently inform conductance parameters. Therefore, this computational pipeline can be applied to different iPSC-CM preparations to determine cell line-specific ion channel properties and understand the mechanisms behind variability in perturbation responses.
Abstract We029: Common and Divergent Cellular Etiologies Underlying Hypoplastic Left Heart Syndrome and Hypoplastic Right Heart Syndrome
Authors: Yu Yang , Wang Cankun , Ye Shiqiao , Qin Hannah , McNutt Megan , Alonzo Matthew , Texter Karen , Garg Vidu , Zhao Mingtao
Published: October 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 We033: Heart-Specific Histone Methyltransferase SMYD1 Promotes Cardiac Maturation in the Direct Conversion of Human Fibroblasts into Cardiomyocytes.
Authors: Woldemariam Anteneh Getachew , Li Hui , Wang Meimei , Peng William , Bridges Justin , Lu Rui , Zhou Yang
Published: October 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 We043: MYCN is a regulator of cardiomyocyte proliferation and regeneration in the mammalian heart
Authors: Das Satyabrata , Larson Thijs , Sierra-Pagan Javier , Alexy Tamas , li qinglu , Ma Xiao , Gong Wuming , Sadek Hesham , Garry Mary , Zhang Jianyi , Garry Daniel
Published: October 2024

The identification of new factors and mechanisms that can promote the repair and regeneration of the adult injured mammalian heart are necessary as the adult heart has limited ability to repair itself. We identified MYCN as a downstream effector of the sonic hedgehog (SHH) signaling cascade during cardiomyocyte proliferation in the neonatal heart. Although the protooncogene, MYCN, has been shown to be an essential factor during embryonic heart development, its role in regenerating the adult heart has not been established. Here, we define MYCN as a critical regulator of cardiomyocyte (CM) proliferation and regeneration using genetic mouse models and in vivo modified RNA (modRNA) delivery. In these studies, we conditionally deleted
Mycn
in the neonatal heart (P0). Immunohistochemical (IHC) analysis of P3 neonatal hearts demonstrated significantly (p < 0.05) decreased expression of Ki67 and pH3 in the absence of
Mycn
compared to control (no 4-OHT) hearts. This observation was further validated in isolated CMs and neonatal P7 hearts pulsed with EdU which demonstrated a significant decrease in EdU+ CMs in the absence of
Mycn
compared to control. Single-nucleus RNA-sequencing results from control and
Mycn
CKO P4 ventricles revealed a severe depletion of the proliferative and regenerative cardiomyocyte population in the
Mycn
CKO hearts. We then ligated the left anterior descending coronary artery (LAD) to produce an ischemic cardiac injury in control and
Mycn
CKO P2 neonatal mice. Echocardiography one-month following the LAD surgeries demonstrated that the
Mycn
CKO failed to recover baseline cardiac function compared to their control counterparts who achieved complete recovery. RNAseq analysis of control and
Mycn
CKO hearts 1 month following LAD ligations demonstrated that muscle developmental pathways were significantly downregulated in the
Mycn
CKO group compared to the control. We then delivered either GFP (control) or
Mycn
modRNA intramuscularly into the border zone following LAD coronary artery ligation surgeries in adult (2-6 month old) mice. We observed that the
Mycn
modRNA treated mice had significantly improved cardiac function at one month and two months following injury/delivery compared to controls. These results identify MYCN as an important regulator of CM proliferation and heart regeneration.
Abstract We050: Elevated Cell-Free RNA in Maternal Circulation during Single Ventricle Heart Defect Pregnancies Dysregulate Human Cardiomyocyte Proliferation
Authors: Alonzo Matthew , xu zhaohui , Ye Shiqiao , Wang Cankun , McNutt Megan , Yu Yang , Ma Qin , Texter Karen , Garg Vidu , Zhao Mingtao
Published: October 2024

Congenital single-ventricle heart defects (SVHDs) are life-threatening conditions in newborns, where one of the heart's ventricular chambers is severely underdeveloped. While medical advancements have improved survival rates, many SVHD patients still suffer lifelong cardiac complications and comorbidities. Clinical diagnosis of fetal SVHD occurs at around 20 weeks of gestation through echocardiogram. In this study, we aimed to identify novel maternal blood biomarkers that could signal SVHD development in fetuses. The study's premise is based on the limited regenerative capacity of adult human cardiomyocytes, like those in pregnant mothers, compared to the extensive proliferation seen in fetal cardiomyocytes that form the functional heart. We hypothesized small signaling molecules secreted by proliferating fetal cardiomyocytes enter the maternal circulatory system and could indicate abnormal ventricular growth in SVHD-affected fetuses. Both clinical and
in vitro
approaches were employed to identify potential cell-free miRNAs released by the developing fetal heart into maternal circulation. Blood plasma samples from pregnant women carrying healthy and SVHD-affected fetuses were screened for differentially expressed cell-free miRNAs and were compared with those from the supernatants of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) grown from healthy and SVHD donors. This comparative approach led to the identification of an miRNA panel (miR-487b, miR-433, mi-134, and miR-889) that were elevated in pregnant women carrying SVHD-affected fetuses and in proliferating iPSC-CMs from SVHD patients compared to healthy controls (p.adj<0.05). From this panel, forced-expression of miR-487b and mir-134 modulated the proliferation profiles of human cardiomyocytes
in vitro
. Results from this study establish these circulating cell-free miRNAs in maternal blood as potential non-invasive biomarkers for predicting abnormal fetal cardiac development leading to SVHDs. These findings could potentially revolutionize SVHD screening as an alternative diagnostic tool to echocardiogram.
Abstract We053: Altered inflammatory state and mitochondrial function identified by transcriptomics in paediatric congenital heart patients prior to surgical repair
Authors: Bartoli-Leonard Francesca , Harris Amy , Caputo Massimo
Published: October 2024

Background:
Congenital heart disease (CHD) remains the most common birth defect, with surgical intervention required in complex cases. In contrast to adult heart disease which disproportionately affects the left ventricle, complex CHD can be characterized by increased right ventricular (RV) pressure, leading to RV hypertrophy and eventually failure, with RV function known to be a major predictor in sustained cardiac health in these patients.

Methods:
RV samples were collected from discarded myocardial tissue from paediatric patients (0-16 years) undergoing their first cardiac surgery at Bristol Royal Hospital for Children or obtained from autopsy subjects. RNA sequencing and subsequent cellular deconvolution was conducted in CHD samples and integrated with publicly available control dataset. Cellular deconvolution, pathway network analysis and gene ontology were conducted and changes in expression of markers was confirmed via histological analysis.

Results:
Presence of CHD drove divergence of transcriptome with 511 differentially expressed genes (log fold change≥±2, padj≤0.05), with 82% shared homology. Known CHD genes;
TBX5
, JAG1,
NOTCH2
and
NOTCH1
were reduced in CHD compared to controls (padj=2.24
E-07
, 1.38
E-27
, 7.76
E-31
, 9.11
E-7
)
.
Conversely, mitochondrial dysfunction genes
RPPH1
and
RMPR
(padj=4.67
E-132
, 2.23
E-107
) were increased. Gene set enrichment identified mitochondrial dysfunctional pathways.Negative regulation of mitochondrial functions and metabolism was identified in functional network analysis. T helper cell markers
CD4
and
SELL
were significantly downregulated (p<0.0001, p=0.003). Cytotoxic T cell markers
CD8a, LAGE3
and
CD49a
were significantly increased in CHD compared to control (p=0.0006, p<0.0001, p=0.0118) as was proinflammatory marker
Caspase1
(p=0.0055). Histological analysis confirmed an increase in CD45 and CD8 positive cellular bodies with the CHD tissue, which was absent in control. Deconvolution of bulk RNAseq data suggests a reduction in CD4+ T cells (p=0.0067) and an increase in CD8+ T cells (p=0.0223). Network analysis identified positive regulation of the immune system and cytokine signalling clusters within the inflammation functional network as were lymphocyte activation and leukocyte differentiation.

Conclusions:
Utilizing RV tissue from paediatric patients undergoing CHD cardiac surgery this study identifies dysfunctional mitochondrial pathways and an increase in inflammatory T cell presence prior to reparative surgery.
Abstract We054: Mitochondria-containing Extracellular Vesicles Mediate Heart Failure Sterile Inflammation
Authors: Wang Dennis , Huynh Kim Anh , O'Brien Kevin , Tian Rong
Published: October 2024

Introduction:
Sterile inflammation has long been postulated to exacerbate heart failure (HF); however, a lack of viable biological targets and thorough understanding of the underlying mechanism posed formidable challenges in the development of novel therapeutic interventions.

Hypothesis:
Given proinflammatory mitochondrial DNA (mtDNA) are known to be elevated in HF plasma; and that cells release mitochondrial components in extracellular vesicles, we hypothesize that proinflammatory circulating Mitochondria-containing Extracellular Vesicle (MitoEV) is a mediator of HF sterile inflammation.

Methods/Results:
Using plasma samples of 12 Stage-D HFrEF and 12 healthy participants, plasma microparticles (MP) are co-stained with mitochondrial- and cytoplasmic-specific markers then quantified by volume-metric flow analysis. We found that there are significantly more mitochondrial-cytoplasmic double-positive MP (designated MitoEV, accounts for >70% of total MP) in HF relative to control. Next, using size-exclusion chromatography, we found MitoEV fractions are significantly more potent than soluble protein fractions to induce cytokine production in macrophage, implicating MitoEV as the source of HF plasma immunogenicity. Surface marker analysis by flow suggests that
CD14+ monocytes
is a major source of HF plasma MitoEV. Using THP-1 monocytes, we found that TLR9 activation by mtDNA results in injured mitochondria to be released as MitoEV (rather than removed by mitophagy), a pathway negatively regulated by beclin-1. Finally, boosting NAD by nicotinamide riboside preserves mitochondrial dysfunction and significantly reduces MitoEV release in primary monocytes.

Conclusion:
Our findings implicate circulating MitoEV released by monocytes upon mtDNA activation as a pivotal mediator of sterile inflammation in HF, demonstrating a promising avenue for potential therapeutic targeting. The study also provides a potential cellular mechanism to the anti-inflammatory effect of boosting NAD.

Model:
In HF circulation, mtDNA activates TLR9 of monocytes, injuring mitochondria, which are released as MitoEV (containing mtDNA) to amplify and propagate inflammation in an autocrine manner. Becn1, a key mediator of mitophagy, negatively regulates MitoEV release. Activated monocytes also release cytokines to further injure the heart, resulting in a vicious cycle. Boosting NAD protects against mtDNA-induced mitochondrial dysfunction, attenuates the MitoEV release, and ameliorates inflammation.
Abstract We060: Hypoxia Inducible Factor-2α Enhances Neutrophil Survival and Promotes Cardiac Injury Following Myocardial Infarction via cIAP-1 Signaling
Authors: Piccolo Enzo , Ge Zhi-Dong , Thorp Edward , Sumagin Ronen
Published: October 2024

Rationale:
Heart failure is a major cause of mortality following myocardial infarction (MI). Neutrophils (PMNs) are among the first immune cells to accumulate in the infarcted region. While some long-term beneficial functions of PMN in heart injury are now appreciated,
en mase
PMN accumulation in injured hearts is still associated with worsened resolution of inflammation and disease outcomes. MI causes extensive hypoxic injury to heart tissue, where hypoxia-inducible factors (HIFs) play critical roles in cellular functions. While HIF1α regulation of cell survival, migration and metabolism have been well-studied, the role of HIF-2α in these processes is not well-understood. Even less is known of HIF-2α contributions to PMN effector functions, especially in the context of MI.

Objectives:
The current study aimed to 1. Mechanistically explore how HIF-2α regulates survival of tissue infiltrating PMNs post-MI and 2. Determine how HIF-2α activity in PMNs impacts heart injury resolution post-MI, and whether specific HIF-2α deletion in PMNs can alleviate MI-induced tissue injury.

Methods & Results:
Infarct size, cardiac functions monitored by echocardiography, and scar tissue formation after left anterior descending coronary ligation injury model were compared between littermate control and specific PMN HIF-2α depletion (Ly6G-cre
+/-
HIF-2α
fl/fl
) mice. Myocardial infarction size, cardiac dysfunction, scare tissue deposit and survival of tissue infiltrating PMNs post-MI were substantially attenuated in mice with PMN HIF-2α deletion. A significantly reduced lifespan of HIF-2α knockout PMNs was further confirmed by fate-mapping EdU pulse chase experiments. Mechanistically, molecular studies revealed that HIF-2α transcriptionally regulates expression of the pro-survival factor cIAP1 in PMNs binding to an HRE located in the promoter region of
BIRC2
, in a hypoxia-inducible manner.
Ex-vivo
pharmacological inhibition of cIAP1 expression in PMNs using Birinapant resulted in dramatic cell death, establishing the critical role of cIAP1 in PMN survival. Finally, our data demonstrate that the protective effect of HIF-2α deletion in PMNs on MI outcomes was due to elevated recruitment of reparative macrophages into infarcted hearts.

Conclusion:
HIF-2α, through cIAP1 regulation, promotes PMN survival and tissue retention post-MI, leading to worsen cardiac functions and scar formation.
Abstract We067: A meta-analysis of RNA sequencing data to characterize immune cell activation among patients with cardiac sarcoidosis
Authors: Banga Natasha , Huang Yuhsin , Chou Harrison , Vitale Maria Rosaria , Baker Matthew , Witteles Ronald , Zhu Han
Published: October 2024

Introduction:
Cardiac sarcoidosis (CS) is a life-threatening auto-immune disorder that causes the formation of non-caseating granulomas in cardiac tissue. Th17.1 cell have been found to be upregulated in patients with pulmonary sarcoidosis (PS) although data is lacking among CS patients. This meta-analysis integrates all publicly available granuloma single-cell and single-nuclei RNA sequencing data to identify patterns in immune cell activation across different granulomatous diseases while focusing on CD4+T cell activation among CS patients.

Methods:
Five studies were included, spanning cardiac tissue from healthy controls and DCM (Koenig 2022, PMID:35959412), cardiac tissue from ICM and cardiac sarcoidosis (Liu 2022, PMID:36111531), BAL from beryllium sensitized, chronic berylliosis, and PS (Liao 2021, PMID: 33602861), peripheral blood samples from sarcoidosis (Garman 2020, PMID: 33363531), and lesional and nonlesional skin tissue from sarcoidosis (Krausgruber 2023, PMID: 36750099) for a total of 122 patients. All integrative analysis was performed using the R package Seuratv5 on Stanford’s computing cluster, Sherlock. After quality control filtering, a total of 127,335 cells were integrated using Seurat’s reference-based integration. After labeling the UMAP with general immune cells (figure 1A), we subsetted T cells into CD4+ and CD8+ and examined the landscape of CD4+ T cells (figure 1B).

Results:
We found significantly more Tregs in CS than PS (P=0.013, figure 1C). Additionally, patients with CS had significantly more Th17.1 cells in CS in comparison to PS (P=0.0041). However, significantly less Th2 cells were found in CS than PS patients (P=0.0152). These differences remained statistically significant across various analyses comparing Tregs and Th17.1 cell as a proportion of CD4+, CD3+, and CD45+ cells.

Conclusions:
In comparison to patients with PS, pathways which differentiate naive CD4+ cells into TH17.1 and Tregs are upregulated among CS patients while the TH2 differentiation pathway is downregulated. Our findings indicate distinct CD4+ T cell patterns among CS and PS patients. Our ongoing work aims to further evaluate T cell proliferation and expression to elucidate mechanistic pathways as potential drug targets for CS.
Abstract We072: Effects of electronic cigarettes on mouse cardiac lymphatic vessels after TAC surgery
Authors: Tao Siqi , Qian Jiang , Shi Wei , Xu Jian , Lien Ching-Ling
Published: October 2024

Introduction:
Heart failure is a public health concern worldwide. It is known that tobacco smoking is a risk factor of heart failure. Tobacco smoking also causes inflammation reaction and lymphatic dysfunction in the lung. However, the effects of electronic cigarettes (ECIG), as a new method of smoking, on the heart and cardiac lymphatic vessels (lymphatics) are not clear yet. The lymphatic vessels play an important role in maintaining cardiac function by modulating fluid homeostasis and transporting macromolecules. It has been reported that cardiac lymphatic vessel formation limits pressure overload-induced heart failure in C57Bl/6 but not in Balb/c mice. How cardiac lymphatics might contribute to the pathogenesis induced by ECIG is not known.

Research Questions:
The roles of cardiac lymphatic vessels in responses to ECIG exposure are not investigated. I hypothesize that the toxic effects of ECIG on the heart are partially caused by damaging the cardiac lymphatic vessels.

Aim:
To determine the effects of ECIG exposure on cardiac lymphatics after Transverse aortic constriction (TAC).

Methods:
I perform TAC surgery on mice as a heart hypertrophy and chronic heart failure model. Mice after TAC or Sham surgery are treated with different vaping components from ECIG: ECIG, propylene glycol and glycerin (PGVG) as vehicle, and air. After 4 weeks of treatment, hearts were harvested for histological analyses. The lymphatic vessel coverage percentage and morphology were examined by immunostaining.

Results:
Prior research suggested that genetic background in mice might affect the responses of cardiac lymphatic vessels to TAC-induced heart failure. I have established the TAC model using CD-1 mice. My results showed that CD-1 mice treated with PGVG after TAC do not show a significant difference in cardiac lymphatic vessel density compared to Sham group (p value=0.5951). ECIG treatment also did not affect the lymphatic density significantly compared to PGVG treated mice after sham operation (p value=0.8937). ECIG treated mice after TAC surgery showed a trend of increased lymphatic coverage and more mice are still being processed and will be presented at the conference.

Conclusion:
PGVG treated CD-1 mice do not show changes in lymphatic vessel density 4 weeks post TAC. Longer time points and more mouse numbers are needed to compare the effects of PGVG and ECIG after TAC induced hypertrophy and heart failure.

This research is supported by California TRDRP grant.
Abstract We079: Program Macrophage Phenotypes and Cardiac Remodeling After Myocardial Infarction with Interleukin-10
Authors: Chen William CW , Zhang Jing , Li Xiaoping , Kim Kang , Huard Johnny , Rizk Rodrigue , Wang Yadong
Published: October 2024

Introduction:
Macrophages (Mφ) and their phenotypic polarization play essential roles in modulating cardiac inflammation and recovery after myocardial infarction (MI). Interleukin-10 (IL-10) is a potent immunomodulatory cytokine that directs Mφ polarization to beneficial reparative phenotypes and is widely investigated in diseases involving extensive inflammation. However, high-dose IL-10 treatment may not lead to the expected therapeutic outcome, raising the question of whether IL-10 has dose-dependent effects on Mφ polarization and cardiac remodeling post-MI.

Hypothesis:
IL-10 can program Mφ phenotypes under inflammation and has functional impact in cardiac remodeling post-MI in dose-dependent manner.

Goals:
We use RAW264.7 Mφ and mouse permanent MI models to test our hypothesis.

Approach:
We applied 25-1000 ng/mL IL-10
in vitro
before or after inducing M1 (with interferon-g) and M2 (with interleukin-4) polarization for pro-inflammatory and anti-inflammatory phenotypes, respectively. We intramyocardially administered 25-2000 ng IL-10
in vivo
immediately after inducing MI and examined cardiac function, inflammatory responses, fibrosis and revascularization for 6 weeks (n=5/group).

Results:
IL-10 at 100-250 ng/mL directed Mφ phenotype shift from M1 to M2 most effectively after inducing polarization
in vitro
(both
p
<0.01); groups with ≥500 ng/mL IL-10 exhibited high M1/low M2 phenotypes, similar to no IL-10 controls. Cells treated with ≥100 ng/mL IL-10 before polarization exhibited a notable reduction in M1 phenotype after induction (all
p
<0.05). Echocardiography showed that with single IL-10 administration, only 250 ng IL-10 notaly improved left ventricular (LV) function and reduced LV chamber size compared with the saline control (
p
<0.05) whilst ≥1000 ng caused a transient negative impact in LV function at 5 days post-MI (
p
<0.05), Phagocyte infiltration at MI was notably diminished in ≥100 ng groups at 6 weeks post-MI (all
p
<0.05). Only 250 ng IL-10 led to a notable 53% reduction in LV fibrosis (
p
=0.05) and nearly 200% increase in CD31+ cell counts at MI (
p
=0.013).

Conclusion:
Our results suggest an optimal range of IL-10 doses for macrophage polarization and cardiac benefits post-MI, with risks of side effects from IL-10 overdose.
Abstract We086: Identifying Gene Regulatory Subnetwork Drivers of Cardiomyocyte Chamber Specification
Authors: Clark Alexander , Saucerman Jeffrey
Published: October 2024

Introduction:
Fetal heart development is a highly coordinated process directed by complex gene regulatory networks (GRN). Imbalances in such networks can lead to congenital heart defects, a leading cause of morbidity and mortality in children. Many of these defects are related to the improper specification of cardiac progenitor cells to chamber-specific (i.e., atrial, or ventricular) cardiomyocytes, but the GRNs involved in this process are not well characterized.

Hypothesis:
We hypothesize that cardiomyocyte chamber specification depends on discordant atrial and ventricular regulatory subnetworks within heart field cardiac progenitor cells.

Approach:
We used CellOracle, a GRN inference software, and publicly available fetal mouse heart single cell RNA and ATAC sequencing data to infer cell type specific GRNs during cardiomyocyte differentiation. Each of the first heart field and two second heart field networks included 4,000 directed interactions and more than 600 genes. We performed a systematic
in silico
knockdown study with these networks to identify TFs that differentially modulate atrial and ventricular gene expression. Correlations in the downstream responses to TF perturbations were used to extract chamber specification subnetworks.

Results:
The most central genes within the heart field networks included several previously identified cardiac developmental TFs (Nkx2-5, Mef2c, Tbx5). Perturbations of these heart field GRNs identified 12 TFs that increased ventricular gene expression while also decreasing atrial gene expression (ventricular program), as well as 20 genes with the opposite effect (atrial program). Previous
in vitro
knockdown studies during iPSC-CM differentiation validated one of the ventricular (Hey2) and two of the atrial programming TF (Nr2f2, Tead2) predictions. Discordant atrial and ventricular regulatory subnetworks were identified, including one network with opposing effects on the expression of Wnt2 by ventricular (repressing) and atrial (activating) TFs.

Conclusions:
We identified a set of TFs organized within regulatory subnetworks that drive discord specification of ventricular vs. atrial cardiomyocyte gene expression within cardiac progenitor cells in mouse development. These predictions will be validated through
in vitro
and
in vivo
studies.
Abstract We089: Numb Family Proteins in Epicardium Regulate Epicardial Cells Differentiation and Ventricular Patterning During Development
Authors: Nusrat Anika , Zhao Luqi , Miao Lianjie , Wu Mingfu
Published: October 2024

Background:
Numb Family Proteins (NFPs), specifically two mammalian homologs Numb (denoted by
Nb
) and Numblike (denoted by
Nl
), have been evident to regulate essential cardiac functions during development. Previous studies reported NFPs as integral for epicardial cells (EpiCs) epithelial mesenchymal-transition (EMT). However, whether NFPs in epicardium regulate EpiCs differentiation to affect cardiac morphogenesis have not been explored. In this study, we investigated how NFPs in epicardium regulate EpiCs EMT and differentiation to modulate cardiac development and subsequently affect cardiac maintenance in adult stages.

Method:
Using mCherry:Numb knockin mouse model we observed enrichment of Numb in epicardium. We deleted
Nb
and
Nl
in epicardium via

Tbx18
Cre/+

lineage and refer to this as EDKO. We harvested hearts at embryonic day 15.5/18.5 and assessed morphological and mechanistic differences between control and EDKOs. Conventional and Speckle tracking Echocardiography were used to examine adult heart structure and functions.

Results:
Our findings revealed that EDKO embryonic hearts display specific morphology with extended left ventricles. We found a surprising difference between female and male EDKOs in terms of survival to adulthood. Reduced EpiCs EMT and Cardiac Fibroblasts (CFs) migration distances, changes in CFs distribution have been observed in EDKO developing hearts. Reduced expression of smooth muscle cells (SMCs) and pericytes also was noticed in EDKOs. Strikingly, EDKO embryonic hearts show defects in ventricular compaction, ventricular wall patterning and in cell proliferation. Furthermore, when survived to adult stage, EDKO hearts revealed significant structural and functional changes over their aging. While digging into regulatory pathways, we found changes in Fibroblast growth factor receptors (Fgfrs)/p-Erk pathway and in activation of Notch1 in EDKO hearts.

Conclusion:
NFPs in epicardium regulate EpiCs differentiation into CFs and SMCs, and regulate ventricular wall morphogenesis during development. NFPs are also required to maintain cardiac structure and function at adult stages. Fgfrs/p-Erk pathway and Notch1 could be the possible mechanism behind cardiac morphogenetic regulation by NFPs in epicardium.
Future aims:
Further studies will identify downstream targets of NFPs’ regulation of EpiCs differentiation and ventricular wall patterning in embryonic hearts, and mechanistic pathways of epicardial NFPs’ role in adult hearts.
Abstract We099: Antisense Oligonucleotide Treatment of Calmodulinopathy
Authors: Bortolin Raul , NAWAR FARINA , Trembley Michael , Prondzynski Maksymilian , Sweat Mason , Wang Peizhe , chaehyoung park , Lu Fujian , Keating Erin , Yoshinaga Daisuke , Pavlaki Nikoleta , Cavazzoni Cecilia B. , T. Sage Peter , D. Whitehill Robert , Abrams Dominic , Carreon Chrystalle , Putra Juan , Alexandrescu Sanda , Guo Shuai , Tsai Owen , Rubart Michael , Kubli Dieter , Mullick Adam , Bezzerides Vassilios , Pu William
Published: October 2024

Introduction:
Antisense Oligonucleotides (ASOs) are an emerging class of drugs that target RNAs to impede their production, stability, and translation. Calmodulinopathies are severe cardiac arrhythmias caused by dominant mutations in
CALM1
,
CALM2
, or
CALM3
. Each of these genes encodes the identical calmodulin (CaM) protein. Leveraging this genetic redundancy, we hypothesized that ASO-mediated knockdown of the affected
CALM
gene would treat arrhythmia while functional redundancy of the other
CALM
genes would maintain CaM protein level and prevent toxicity.

Methods:
Cells from a patient expressing the heterozygous
CALM1
- F142L mutation were reprogrammed into induced pluripotent stem cells (iPSCs;

P-CALM1
F142L/+

). Additionally, the variant or a nonsense codon were introduced into the wild-type reference line, WTC11, to yield isogenic disease

(E-CALM1
F142L/+
)

and
CALM1
null (

CALM1
Δ/Δ

) lines respectively. iPSCs were differentiated into cardiomyocytes (iPSC-CMs) to assess their action potentials and Ca
2+
transients using multielectrode arrays and fluorescent voltage- and calcium-sensitive dyes.

Calm1
N98S/+

mice, previously shown to develop catecholamine-induced ventricular tachycardia, were used for
in vivo
studies. Using these model systems, we tested ASOs that selectively depleted human or murine
CALM1
.

Results:
Both

E-CALM1
F142L/+

and

P-CALM1
F142L/+

iPSC-CMs exhibited prolonged action potentials, consistent with QT prolongation observed in the proband.

CALM1
Δ/Δ

iPSC-CMs expressed normal levels of CaM protein and exhibited no change in action potential duration. ASO that selectively depleted
CALM1
transcripts by ~50% did not alter CaM protein level but normalized action potential and Ca
2+
transient duration in

CALM1
F142L/+

iPSC-CMs. In

Calm1
N98S/+

mice, ASO selectively depleted
Calm1
transcripts by ~90% and prevented catecholamine-induced ventricular tachycardia.
Calm1
ASO did not alter CaM protein level, cardiac function, or treadmill endurance, or cause significant histopathological changes.

Conclusion:
ASO-based therapy safely and effectively treated arrhythmias caused by dominant
CALM1
variants, suggesting that ASOs are a promising potential therapy for calmodulinopathy.
Abstract We103: Integration of deep learning assisted high-content screening and deep tissue-phenotyping to identify cardioprotective compounds in dilated cardiomyopathy
Authors: Wiest-Daessle Nicolas , Sharkova Krasimira , Weiss Amelie , Sommer Peter , Zimmermann Wolfram , Gkatzis Konstantinos
Published: October 2024

With a prevalence of 4:10,000, dilated cardiomyopathy (DCM) is the most common hereditary heart disease, characterized by dilatation of one or both ventricles and impaired systolic and diastolic function. Numerous mutations have been identified to cause DCM. TITIN-truncating variants (TTNtv) are commonly observed in 20% of the genetic DCM cases. Progress in drug discovery for DCM has been hampered by the lack of robust high-throughput/high-content analysis pipelines of disease-specific cell and tissue platforms for deep structural and functional phenotyping.

To address this challenge, we have developed a two-tiered human induced pluripotent stem cell (iPSC)-based screening approach, comprised of high-content cardiomyocyte imaging with artificial intelligence (AI)-driven image and data analyses as well as deep tissue phenotyping in engineered heart muscle (EHM) models. As a DCM use case, we have developed several isogenic human iPSC-lines with and without a previously reported and pathologically relevant A-band-TTNtv mutation (c.70692_70693insAT; p.T23565SfsX5; Gerull et al. 11788824, Gramlich et al. 25759365, Fomin et al. 34731013)

Using fixed weights from a convolutional deep neural network trained on ImageNet, we generated unbiased deep embeddings from each 2D cell model and applied these to train machine learning (ML) models to detect morphological disease phenotypes. Our ML model is able to confidently separate healthy controls from
TTNtv
cells with high accuracy (>93%), specificity (WT >79%) across different batches/plate layouts and generate concentration-response curves, demonstrating platform robustness and sensitivity. Building upon this work, we will present our latest efforts applying the platform technology for a chemogenomic screening campaign to identify novel cardioprotective drugs using a library of 7,100 bioactive compounds and validation assays, including measuring functional changes in EHMs.

The phenotypic profiling platform, as presented here, can be readily adapted to other disease-relevant cell types and pathologies. Our results demonstrate the power of combining iPSC-CMs with cytological profiling and deep learning as well as tissue-level phenotyping to accelerate drug discovery for patients with DCM.