tbuckley/cabot-search huggingface dataset:
Abstract Mo075: Cigarette smoking causes somatic mutations in cardiac endothelial cells
Authors: Zinan Zhou, August Yue Huang, Shulin Mao, Nazia Hilal, Brian Chhouk, Christopher Walsh, Eunjung Alice Lee, Ming-Hui Chen, Sangita Choudhury
Journal: Circulation Research (2024)

Cigarette smoking (CS) is a major contributor to cardiovascular diseases (CVDs), responsible for one in every four CVD-related deaths. Despite this, the precise mechanisms through which smoking induces CVDs remain elusive. The chemicals in cigarette smoke, particularly oxidants, are known to instigate oxidative stress and activate the vessel wall. Alongside the dysregulation of cellular functions, oxidative stress can lead to DNA damage. Accumulation of DNA damage and somatic mutations is a recognized hallmark of aging, a significant risk factor for various age-related diseases, including CVDs, neurodegenerative diseases, and cancer. Recent studies have indicated a substantial increase in somatic mutations, with mutational signatures reflective of oxidative stress, in bronchial epithelial cells from smokers. Given their proximity in the blood circulation, these pathophysiological findings could naturally extend to the coronary system. Therefore, we hypothesized that CS induces somatic mutations and injuries in cardiac endothelial cells (ECs), laying the mechanisms for cigarette smoking-induced CVDs. To test this hypothesis, we conducted single-cell whole-genome sequencing (scWGS) on 105 cardiac endothelial cells isolated from postmortem hearts of 6 normal individuals, 3 smokers, and 4 smokers with atherosclerotic cardiovascular disease (ASCVD). Our results revealed a significant increase in somatic single nucleotide variants (SNVs) and insertions and deletions (indels) in cardiac ECs from smokers and those with ASCVD. These excess somatic mutations exhibited Catalogue Of Somatic Mutations In Cancer (COSMIC) mutational signatures, including SBS4, SBS29, SBS40, SBS92, and ID3, indicative of DNA damage and mutagenesis associated with cigarette smoking, oxidative stress, and formaldehyde exposure. Further analysis of single-nucleus RNA sequencing (snRNA-seq) data from postmortem hearts of 3 normal individuals, 4 smokers, and 1 smoker with ASCVD demonstrated upregulated pathways involved in apoptosis, EC migration, and vascular development. In conclusion, our study demonstrates that chemicals in cigarette smoke exert direct genotoxic effects on cardiac ECs, mirroring their impact on bronchial epithelial cells. Cigarette smoking significantly accelerates the accumulation of somatic mutations in cardiac ECs, resulting in apoptosis and vascular damage. Consequently, this damage leads to a secondary activation of angiogenesis for vascular repair.
Abstract Mo040: Enhanced neovascularisation does not account for preservation of cardiac function by 11βHSD1 inhibition in a pig model of myocardial infarction
Authors: Sara Al Disi, Raimondo Ascione, Tom Johnson, Eva Sammut, Vito Domenico Bruno, Shazia Khan, Trisha Singh, Daniel Baz-Lopez, Carol-Ann James, Brian Walker, Nicholas Mills, Scott Webster, Adrian Freeman, Andrew Whittaker, Ruth Andrew, Gillian Gray
Journal: Circulation Research (2024)

While acute post-myocardial infarction (MI) survival has improved thanks to early intervention, the myocardium still sustains damage that increases the risk of heart failure. Adrenal glucocorticoids (GCs) protect cardiomyocytes immediately after MI, but inhibition of GC regeneration within the heart by 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) during infarct repair prevents subsequent deterioration of ventricular structure and function. In the 11βHSD1-knockout mouse this outcome is associated with enhancement of peri-infarct neovascularisation and prevention of infarct expansion. Here, we tested the hypothesis that pharmacological 11βHSD1 inhibition after MI can preserve function in a translational pig model and investigated the role of neovascularisation. MI was induced in female minipigs via temporary coronary occlusion. A standard clinical therapy (SCT, n =9) group received statin, anti-platelet, βadrenoreceptor antagonist and ACE inhibitor immediatley after MI, and a 11βHSD1i group (n=11) received SCT +11βHSD1 inhibitor (50mg/kg) from 48h after MI until the end of the study. Prior to 11βHSD1i treatment, short-axis cine and late gadolinium-enhanced MRI showed no difference in either infarct mass (4.2±0.4g SCT vs 4.0±0.6g 11βHSD1i) or ejection fraction (EF; 60.6±2.9% SCT vs 56.6±1.8% 11βHSD1i). However, by 28 days after MI, while EF was reduced in SCT pigs to 48±4%, it was maintained in pigs given additional 11βHSD1i treatment (57.9±1.8%, p &lt;0.05 vs SCT). Infarct mass did not differ between the two treatments, suggesting that inhibition of infarct expansion did not account for the improvement in function, this was confirmed by a similar area of infarct collagen in histological analysis. Angiogenesis, represented by an increase in CD31 +ve capillaries, was not enhanced in the infarct or the border zone (BZ) after 11βHSD1i treatment, relative to SCT. While vessel maturation (αSMA +ve vessels) was improved in the infarct compared to the remote myocardium, there was no difference between the two groups. Proteomic pathway analysis of the BZ indicated extracellular matrix organisation as the main pathway regulated by 11βHSD1i compared to SCT. In conclusion, 11βHSD1i after MI successfully preserves cardiac function and structure but the mechanism is independent of scar size reduction or enhanced neovascularisation. Instead, the beneficial effects of 11βHSD1i are likely to result from favourable regulation of collagen processing during scar formation.
Abstract Mo041: Bile Acid Dysregulation in Response to Right Heart Failure and Hepatic Congestion
Authors: Arick Park, Bin Yang, Adeesh Parvathaneni, Christina Fu, Kathleen Byrnes, Rebecca Haeusler, Joel Schilling
Journal: Circulation Research (2024)

Introduction: Right heart failure (RHF), the result of impaired RV contractility and/or severe right-sided valvulopathies, is a clinical syndrome that commonly results in chronically elevated central venous pressures and hepatic congestion. Hepatic dysfunction in patients with heart failure portends a poor prognosis. In part, this may be due to the liver’s pivotal role in bile acid synthesis and processing. In addition to the classic role of a digestive surfactant, bile acids serve as important and dynamic signaling molecule. The present study characterizes a unique bile acid profile found in patients with RHF. Research Question: We hypothesize that RHF and hepatic congestion results in a dysregulation of bile acids. These untoward changes in bile acids concentrations may provide both important prognostic value and novel mechanistic pathways in congestive liver disease. Aims: 1) To characterize systemic and hepatic vein bile acid concentrations in patients with right heart failure. 2) To phenotype a mouse model of hepatic congestion and understand the mechanistic effects of specific bile acids on liver biology Methods: We enrolled a total of 60 patients who were referred for right heart catheterization (RHC). Patient demographics, echocardiograms, and routine laboratory tests were captured at enrollment. Blood was sampled from the IVC and hepatic vein during RHC. Bile acids were quantified from serum using liquid chromatography-tandem mass spectrometry. To model hepatic congestion, mice underwent ligation of the IVC. Blood and tissue samples were harvested and underwent mass spectrometry evaluation for the bile acid composition and immunohistochemistry was performed to characterize the immune landscape, respectively. Results: We captured a total of 12 control samples from patients with normal filling pressures, 25 patients with left heart failure (LHF), and 23 patients with RF. Patients with RHF had poorer survival outcomes compared to control or LHF. Patients with RHF had significant increases in specific conjugated bile acids when compared to control or LHF (Figure 1). Conclusions: In this study, we identified several conjugated bile acids that are uniquely elevated in patients with RHF compared to LHF and control. Additionally, patients with RHF had poorer survival. Collectively, these data suggest bile acids may serve as important biomarkers and signaling molecules in the pathogenesis of cardiogenic liver disease.
Abstract We062: Characterization of Transcriptional Responses to Streptococcus pneumoniae Infection in the Heart versus Sterile Cardiac Injury
Authors: Daniel Minassian, Garrett Wilson, Casey Weaver, Carlos Orihuela
Journal: Circulation Research (2024)

Background: Streptococcus pneumoniae ( Spn ) remains the leading cause of community-acquired pneumonia (CAP). As many as 30% of patients hospitalized for CAP experience a major adverse cardiac event (MACE). Importantly, the elevated risk for MACE is not only limited to the time of acute infection but also extends several years post-hospitalization. Research Question: Is the host response to Spn- mediated injury in the heart different from the response to sterile injury? Goals: To identify defining features of the host response to Spn in the heart via differences in gene expression. Methods: We performed a bioinformatic analysis of bulk RNAseq data collected from hearts of mice acutely infected with Spn and compared this to publicly available data sets of mice that received experimental myocardial infarction, i.e. ischemia reperfusion and permanent ligation. Using DeSeq2, we identified 2,971 genes with differential expression between the infected and infarcted conditions. To identify gene expression differences that might be relevant to human patients, we subsequently used the Reactome GSA software to map our differential expression results onto predicted human pathways. Results: Categorization of the gene roles identified those in the classical antibody-mediated complement activation as the dominant pathway with differential expression. Reactome analysis of the top 5 differentially expressed genes (p &lt; 0.05) at the individual level identified BAIAP2L1 , CXCL9 , IRF7 , IL18BP , RPL12 , and PSMB10 as significantly upregulated in Spn infected hearts versus infarcted hearts. As one feature of Spn mediated damage is the recruitment of immune cells to the affected sites and cardiac remodeling, we validated upregulation of CXCL9 as well as CXCL10, potent recruiters of T-cells, as enhanced during Spn infection. Conclusions: Our analyses provide information on how the host response to disseminated pneumococcal infection in the heart is distinct from sterile injury and identified mediators that may be contributing to adverse cardiac remodeling.
Abstract We055: Role of Classical Dendritic Cells in the Sterile Inflammatory Response Following Myocardial Infarction
Authors: Michael Katsnelson, Ameen Ismahil, Sumanth Prabhu
Journal: Circulation Research (2024)

Myocardial infarction and the subsequent development of chronic ischemic cardiomyopathy is the most common cause of congestive heart failure. Infarction is associated with an intense sterile inflammatory response which can lead to adverse remodeling over the long term. Dendritic cells (DCs) are antigen presenting cells that function as a bridge between innate and adaptive immunity. Precursor DCs give rise to several distinct classical DC (cDC) subsets that mediate a variety of functions. The cDC1 subset is essential for cross-presentation of antigens to CD8 + T cells, while the cDC2 lineage is a potent inducer of CD4 + T cell proliferation. The aim of the study was to characterize the profile of the cDC populations present during the various stages of the development of chronic ischemic cardiomyopathy. We hypothesized that cDCs are essential mediators of chronic LV remodeling in the period following ischemic myocardial injury. We sought to determine which specific cDC subtype (cDC1 vs cDC2) forms the predominant population in the heart and secondary lymphoid tissues following ischemic injury. Adult male C57BL/6 (WT) mice underwent non-perfused myocardial infarction by ligating the left anterior descending artery (LAD) to induce sterile injury, with sham operated mice used as controls. Left ventricular (LV) remodeling was assessed by echocardiography at 1 w and 8 w following surgery. Alterations in cDC1 (CD45+CD11c+CD103-XCR1-) and cDC2 (CD45+CD11c+CD103+XCR1+) profile were examined by flow cytometry. When compared with the sham operated mice, mice with ischemic cardiomyopathy following LAD ligation exhibited the following features. There is an initial influx of cDC2 into the myocardium 1 w after MI (mean of 3.0% vs 0.26% of CD45 + leukocytes, p=0.02). This is followed by a subsequent influx of cDC1 into the myocardium 4 weeks after MI (mean of 1.98% vs 0.16% of CD45 + leukocytes, p=0.001). This influx of cDC into the heart is accompanied by an egress of cDC from the spleen (mean of 0.37% vs 0.76% of CD45 + leukocytes, P=0.001) at 4 weeks following MI. In conclusion, the findings of our study suggest that there is an initial influx of cDC2 followed by a more sustained influx of cDC1 into the myocardium in the weeks following an infarct. Antigen presentation by cDC likely plays a critical role in driving the inflammatory response and inducing chronic LV remodeling in the period following MI.
Abstract Mo054: Long Noncoding RNA LINC00667 Downregulation Leads to SCN5A Alternative Splicing and Arrhythmogenesis
Authors: Gyeoung-Jin Kang, Eun Ji Kim, An Xie, Samuel Dudley
Journal: Circulation Research (2024)

Background: The voltage-gated sodium channel SCN5A, which encodes the Nav1.5 protein, is integral to the propagation of electrical signals in cardiac tissue. SCN5A expression is diminished in cardiomyopathy, significantly increasing the susceptibility to arrhythmias. This reduction in channel activity can be attributed to decreased mRNA stability coupled with aberrant mRNA splicing. The underlying mechanisms driving the dysregulation of mRNA splicing under pathophysiological conditions remain poorly understood. Objective: This study explores the role of long noncoding RNA LINC00667 in the abnormal alternative splicing of SCN5A mRNA. Methods: LINC00667 expression was analyzed using public databases, human heart tissue, iPSC-derived cardiomyocytes (iPSCMs), and cell lines. The impact of SCN5A on action potentials and sodium currents was studied in iPSCMs. LINC00667 and SCN5A expressions were measured using qRT-PCR, while Nav1.5 protein levels were assessed through Western blot. Alternative splicing of SCN5A mRNA was examined using PCR with exon-specific primers. Gene ontology and in silico analysis identified RNA binding proteins (RBPs) potentially interacting with LINC00667. RNA immunoprecipitation (RIP) assays confirmed direct binding with key splice factors. Group differences were assessed using 2-tailed Student’s t-tests, with P &lt; 0.05 indicating significance. Results: LINC00667, found at chromosome 18p11.31, is expressed in the human heart and associated cells, including cardiomyocytes. Its levels are inversely related to arrhythmic risk, decreasing in heart failure and ischemic cardiomyopathy as well as under hypoxic conditions. Gene ontology linked RBPs to SCN5A mRNA splicing, with RBM25, RBM5, and TIA1 identified as LINC00667 interactors. LINC00667 reduction affected regions of SCN5A mRNA encompassing Exons 6, 24, and 28, which could potentially be linked to decreased function/expression or increased unfolded protein response (UPR). Abnormal RNA splicing decreased SCN5A-encoded sodium current, reduced action potential upstroke velocity, and shortened AP duration without altering resting membrane potential. Conclusion: Decreased LINC00667 releases splicing factors that result in abnormal SCN5A mRNA splicing and reduced sodium current. Since LINC00667 is reduced in cardiomyopathy, overexpression may be antiarrhythmic by preventing sodium channel downregulation.
Nonmodifiable Risk Factors Predict Outcomes in Brugada Syndrome
Authors: Deni Kukavica, Alessandro Trancuccio, Andrea Mazzanti, Carlo Napolitano, Massimo Morini, Gianluca Pili, Mirella Memmi, Patrick Gambelli, Raffaella Bloise, Jannì Nastoli, Barbara Colombi, Alessio Guarracino, Maira Marino, Carlo Ceriotti, Paola Galimberti, Luca Ottaviano, Massimo Mantica, Silvia G Priori
Journal: Journal of the American College of Cardiology (2024)
Abstract Mo063: Hacking the ubiquitin code to distinctively modulate ion channel functional expression
Authors: Sri Karthika Shanmugam, Scott Kanner, xinle zou, Papiya Choudhury, Rajesh Soni, Robert Kass, Henry Colecraft
Journal: Circulation Research (2024)

Ubiquitin is a powerful modulator of ion channel fate, yet the mechanistic basis and scope of its regulation has remained elusive due to the complexity of the ubiquitin code and an inability to achieve substrate-specific control of the ubiquitome. To circumvent this and to specifically interrogate the role of distinct ubiquitin linkages on the cardiac repolarizing I Ks channel KCNQ1 (Q1), we developed a toolkit of engineered deubiquitinases (enDUBs) featuring a GFP/YFP-targeted nanobody fused to catalytic domains of DUBs with preferences for hydrolyzing distinctive polyubiquitin linkage types: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K27/K33; and USP21 - non-specific. Co-expressing each of the enDUBs with YFP-tagged Q1 channels in HEK293 cells led to decreased channel ubiquitination but produced quantitative and qualitative differences in Q1 expression. NanoCezanne and nanoTRABID significantly increased Q1 surface density and ionic currents, whereas nanoOTUD1 and nanoUSP21 yielded more moderate effects. In sharp contrast, nanoOTUD4 downregulated Q1 surface density and currents. The impact on steady-state Q1 surface density was achieved by divergent trafficking mechanisms – nanoTRABID and nanoCezanne increased channel forward trafficking to the surface, while nanoOTUD4 reduced channel forward trafficking. The E3 ligases NEDD4L and ITCH both eliminate Q1 functional expression but with different polyubiquitin signatures as assessed by mass spectrometry. The enDUBs nanoOTUD1, nanoCezanne and nanoUSP21 protected Q1 from NEDD4L; whereas nanoTRABID, nanoOTUD1, nanoCezanne and nanoUSP21 shielded Q1 from ITCH. In adult guinea ventricular cardiomyocytes, the enDUBs displayed context-specific regulatory effects, where nanoOTUD1, nanoCezanne and nanoUSP21 significantly enhanced Q1 surface expression and stability, with nanoTRABID having more moderate effects, while nanoOTUD4 sharply decreased Q1 trafficking to the sarcolemma. Finally, linkage-specific enDUBs demonstrated varying capabilities to rescue distinct trafficking-deficient mutant Q1 channels that cause long QT syndrome and predispose to sudden cardiac death. The results reveal a rich multi-faceted role for linkage-specific ubiquitination in regulating ion channel trafficking with implications to develop targeted therapies for trafficking-deficit ion channelopathies.
Abstract Mo088: <i>RBFOX2</i> haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism
Authors: Mengmeng Huang, Xinlei Gao, Michael Trembley, Maksymilian Prondzynski, Yashasvi Tharani, Farina Nawar, Rongbin Zheng, Stefan Aigner, Gene Yeo, William Pu, Kaifu Chen, C. Geoffrey Burns, Caroline BURNS
Journal: Circulation Research (2024)

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.
Abstract We142: Identification of the Molecular Determinants for the Circadian Regulation of the Human KCNH2 promoter
Authors: Ezekiel Rozmus, Alexander Alimov, Isabel Stumpf, John McCarthy, Brian Delisle, Elizabeth Schroder
Journal: Circulation Research (2024)

Background: KCNH2 encodes a voltage-gated potassium channel (Kv11.1), which conducts the rapid delayed rectifier potassium current (IKr) critical for ventricular repolarization in the heart. Circadian clock factors regulate the expression of the KCNH2 mRNA transcript in the heart. However, the molecular mechanisms for the circadian expression of KCNH2 transcripts have yet to be defined. Hypothesis: A conserved cis-regulatory element in the core KCNH2 promoter drives circadian regulation of human KCNH2 (hKCNH2) promoter activity. Objective: Identify the molecular determinants of the circadian regulation of hKCNH2 promoter activity and determine the impact of single nucleotide polymorphisms (SNPs) in this region. Methods: Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays were employed to identify critical cis elements that regulate the circadian expression of the human KCNH2 promoter using promoter-luciferase reporter constructs. We studied several deletion and mutant hKCNH2 promoter reporter vectors transiently transfected in a myogenic cell line (C2C12 cells). Bioluminescence was measured at 10-minute intervals for 7-10 days. Data were analyzed for circadian characteristics (period, phase and amplitude). Mutant constructs included both engineered and constructs containing naturally occurring rare and common SNPs. Results: Lumicycle data demonstrated that the hKCNH2 promoter reporter showed robust circadian and overall activity in C2C12 cells. Deletion and mutational analysis of the hKCNH2 promoter-reporter construct identified a highly conserved tandem E-box element within 1 Kb of the exon 1 start site that was critical for circadian and overall regulation of hKCNH2 promoter activity. Cells expressing hKCNH2 promoter-reporter constructs with different SNPs in this tandem E-box element showed SNP-specific effects. Surprisingly, a relatively common SNP decreased hKCNH2 circadian promoter activity by 66% (n = 6, p=0.0049). Conclusion: We identified a conserved tandem E-box in the proximal promoter of hKCNH2 Exon1 necessary for circadian and overall hKCNH2 promoter activity. Several SNPs in this region suggested a functional impact that might lead to decreased levels of IKr.