tbuckley/cabot-search huggingface dataset:
Abstract Mo066: A Bifunctional Actuator Reverses Na <sub>V</sub> 1.5 Dysfunction Linked To Cardiac Arrhythmias
Authors: Lucile Fossier, Marc Yehya, Ryan Mahling, Sandra Gabelli, Henry Colecraft, Manu Ben Johny
Journal: Circulation Research (2024)

Cardiac voltage-gated sodium channels (Na V 1.5) are critical for the initiation of action potentials within the heart. Alterations in Na V 1.5 function significantly contribute to diverse inherited and acquired arrhythmias, such as Long QT syndrome (LQT), Brugada syndrome (BrS), mixed-syndrome, and heart failure. These pathologies are closely associated with two distinct modifications in Na V 1.5 function: (1) a gain-of-function phenotype, characterized by incomplete inactivation of Na V 1.5 leading to a sustained Na + current ( I NaL ), and (2) a loss-of-function phenotype resulting in diminished peak Na + current ( I NaP ). Despite the broad understanding of Na V 1.5 implication in cardiac pathophysiology, the development of effective therapeutic strategies to reverse these dysfunctions remains challenging. Here, we introduce a novel bifunctional actuator that inhibits I NaL and upregulates I NaP , as a common molecular strategy to reverse pathophysiological changes in Nav-linked cardiac arrhythmias. To do so, we use a genetically encoded approach by engineering a selective nanobody (nb82) targeting Na V 1.5, linked to (1) a peptide inhibitor targeting I NaL (FixR), and (2) the catalytic domain of a deubiquitinase (DUB) to enhance Na V 1.5 membrane expression. Changes in I NaP and I NaL were assessed using whole-cell and multichannel electrophysiology techniques in both wild-type and channelopathic mutant channels. Our results demonstrate that NbFixR-DUB effectively reduces I NaL while concurrently increasing I NaP for multiple channelopathic mutations. Overall, this approach holds considerable promise for rectifying both loss-of-function and gain-of-function phenotypes in Nav channelopathies.
Abstract We073: CLEC4E Signaling in Myocardial Ischemia-Reperfusion Injury
Authors: Michiel Algoet, Matic Pusovnik, Ellen Caluwe, Hilde Gillijns, Willy Gsell, Uwe Himmelreich, Wouter Oosterlinck, Stefan Janssens
Journal: Circulation Research (2024)

Background: The innate immune receptor CLEC4E (C-type lectin domain family 4 member E) in cardiac resident cells and circulating leukocytes contributes to ischemic myocardial damage and adverse LV remodeling following ischemia reperfusion injury (I/R). Mice with loss of CLEC4E gene function show reduced cardiac injury and better LV remodeling but the mechanisms of this cardioprotection remain unknown. Aims: We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury. Methods: We generated chimeric mice by IV infusion of 5 x 10 6 bone marrow cells originating from C57Bl6/J CLEC4E -/- and C57Bl6/J wild-type (WT) donor mice in 8w-old lethally irradiated (9.5 Gy) WT (n=36) and CLEC4E -/- (n=50) receptor mice, respectively. After 4 w, mice were subjected to ischemia reperfusion injury (I/R) by 60 min LAD occlusion. Troponin levels (TnI) were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 h and 28 d post I/R. Mice were euthanized at 28 days for immunohistochemistry and analysis of BM chimerism. Results: Immunohistochemistry for CLEC4E expression on bone marrow smears confirmed successful BM chimerism. At 24h post I/R, TnI levels and LGE-assessed infarct size on MRI were not significantly different between groups (42.1 vs 48.4 ng/ml, and 25.2% vs 31.5% of LVmass in CLEC4E -/- &amp; WT BM versus in WT &amp; CLEC4E -/- BM, respectively) suggesting similar myocardial damage . After 28 d, LV end-diastolic volumes (43µL vs 55µL), and end-systolic volumes (13µL vs 25µL) were significantly smaller in CLEC4E -/- &amp; WT BM than in WT &amp; CLEC4E -/- BM mice (P&lt;0.05 for both), resulting in better preserved global LV function (LVEF 62% vs 47%, P&lt;0.05) . Infarct size, semiquantitatively assessed using planimetry on Sirius red stains, was smaller in CLEC4E -/- &amp; WT BM than in WT &amp; CLEC4E -/- BM (6.9% vs 19.8%, P&lt;0.01). Conclusion: CLEC4E gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodelling after myocardial I/R. Inhibition of CLEC4E signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.
Abstract We081: β1 integrins regulate cellular behavior and cardiomyocyte organization during ventricular wall formation
Authors: Lianjie Miao, Yangyang Lu, Anika Nusrat, Luqi Zhao, Micah Castillo, Yongqi Xiao, Hongyan guo, Yu Liu, Preethi Gunaratne, Robert Schwartz, Alan R Burns, Ashok Kumar, C. Michael DiPersio, Mingfu Wu
Journal: Circulation Research (2024)

Introduction: Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. However, the mechanisms through which these cells organize themselves to establish the tissue architecture of trabeculae and the ventricular wall remain inadequately elucidated. Integrins are the primary cell surface receptors for adhesion to the ECM and mediate both inside-out and outside-in signal transduction pathways that control various cell functions, including proliferation, survival, migration, and gene expression. We found that the β1 integrin subunit (β1), encoded by Itgb1 , is highly expressed in all cardiac cell types. Hypothesis: β1 integrins play a crucial role in regulating cardiomyocyte behavior and organization during ventricular wall morphogenesis in mice. Methods and Results: We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. β1 and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1 , which encodes the β1, was deleted via Nkx2.5 Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts lack a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of β1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. Conclusions: β1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of β1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.
Abstract Mo077: Cardiac Troponin I3 Kinase in Viral Myocarditis
Authors: Kelsey Tjen, Nick Farahani, Lauren Parrow, Jake Ricci, Katelyn Bruno, Henry Sucov
Journal: Circulation Research (2024)

Myocarditis is a leading cause of heart failure and sudden cardiac death. Often caused by viral infection, myocarditis is defined histopathologically as mononuclear immune infiltration in the heart muscle. While most patients recover with no long-term effects, some deteriorate rapidly. It is unknown what controls this variable susceptibility to severe disease. Without this knowledge, the ability to develop targeted therapies for acute myocarditis is limited. Previous studies utilizing mouse genetics identified a locus that controls susceptibility to viral myocarditis. Cardiomyocyte-specific kinase TNNI3K is one candidate gene within this locus, but its role in myocarditis has not been established. Importantly, millions of people throughout the world carry natural variants of TNNI3K. My overall hypothesis is that TNNI3K confers resistance to viral myocarditis in a kinase-dependent manner. In an initial study, we infected strain background-matched wild-type, Tnni3k knockout, and Tnni3k kinase-dead mice with heart-passaged coxsackievirus B3. After 10 days, during the peak of acute myocarditis, mice missing functional Tnni3k protein were found to have higher viral genomes and greater presence of immune cell markers (CD45+), clearly demonstrating the role of Tnni3k in the response to viral myocarditis. H&amp;E-stained heart sections show large clusters of hematoxylin-stained nuclei, indicating infiltration of ventricular tissue by immune cells, with more of these clusters in Tnni3k knockout hearts. Because kinase-dead mutants are similarly compromised as fully null mice, this indicates that the kinase activity of TNNI3K is critical in the antiviral response. These results are the first demonstration of the in vivo activity of a specific gene that modifies viral myocarditis sensitivity. Future work will expand on this study by examining the effects of coxsackievirus B3 infection of Tnni3k mutant mice at early and late time points. Additionally, efforts are currently underway to identify the specific mechanisms by which TNNI3K regulates the immune response to viral infection.
Abstract Mo060: SPEG mediates increased atrial fibrillation incidence in chronic kidney disease
Authors: Jose Alberto Navarro Garcia, Satadru Lahiri, Yuriana Aguilar, Joshua Keefe, Jia Song, Na Li, Xander Wehrens
Journal: Circulation Research (2024)

Introduction: Chronic kidney disease (CKD) is a major public health problem. CKD is a risk factor of cardiovascular morbidity and mortality due to the increased incidence of atrial fibrillation (AF). AF onset is known to be the consequence of cardiomyocyte intracellular calcium (Ca 2+ ) mishandling where increased ryanodine receptor type 2 (RyR2) activity causes triggered activity. RyR2 activity is regulated by phosphorylation of serine 2367 (S2367) by the ‘striated muscle preferentially expressed protein’ (SPEG). Reduced RyR2 phosphorylation at S2367 increases the risk of AF. We hypothesized that changes in atrial SPEG underly the increased risk of AF in CKD patients. Methods: The experimental 5/6 nephrectomy model was used to mimic CKD disease in mice. AF inducibility was determined using intracardiac programmed electric stimulation. Echocardiography was used to analyze cardiac structure. RYR2 phosphorylation and SPEG levels were measured by western blotting. Intracellular Ca2+ dynamics were assessed by confocal microscopy. Results: CKD mice showed increased serum phosphate (14.1±1.4 vs 9.3±0.4mg/dL; p&lt;0.01), creatinine (0.4±0.1 vs 0.8±0.3 mg/dL; p&lt;0.001) and urea (30.5±1.1 vs 20.9±0.4 mg/dL; p&lt;0.001). AF incidence was 4-fold higher in CKD respect sham mice (60 vs 14.2%; p&lt;0.05). CKD mice showed longer P waves (14.3±0.4 vs 12.5±0.3; p&lt;0.01) and greater increase of left atrial size than Sham mice (35.0±7.5 vs 13.9±4.6; p&lt;0.05). Ejection fraction was reduced in CKD mice after 6 weeks of CKD progression (57.9±0.6 vs 61.7±0.8%; p&lt;0.001) while no changes were observed in Sham mice. SPEG levels were decreased in atria from CKD mice compared to sham (0.64±0.11 vs 1.19±0.13; p&lt;0.05). RyR2 phosphorylation at S2367 was reduced in CKD mice atria (0.66±0.16 vs 1.25±0.26 in sham; p=0.098). Atrial cardiomyocytes from CKD mice exhibited altered Ca 2+ handling, with decreased SERCA activity (p&lt;0.05), increased spark-mediated diastolic Ca 2+ leak (p&lt;0.01) and increased NCX activity (p&lt;0.05). Finally, CKD-induced AF onset was prevented in a mice with a mutation that mimics RYR2 phosphorylation at SPEG site (S2367D mice) Conclusion: This study shows for the first time that SPEG levels are decreased in atria from CKD mice what induces increased RyR2 activity leading to increased AF incidence. By conserving RYR2 phosphorylation at Ser2367, CKD-induced AF was prevented. Thus, we propose SPEG as a possible therapeutic target to reduce the increased incidence of AF in CKD patients.
Abstract Mo048: Cereblon as a potential biomarker for early detection of diabetic cardiomyopathy
Authors: Sung Woo Cho, Hyoung Kyu Kim, Byung Gyu Kim, Jin Han
Journal: Circulation Research (2024)

Type 2 diabetes mellitus (T2DM) patients have a higher risk of heart failure and cause cardiomyopathy without other cardiac disease. There is no specific biomarker for discriminating patients with diabetic cardiomyopathy (DCM). In this study, we investigated the cereblon (CRBN) level in mice and human samples to determine its potential as a biomarker for early detection of diabetic cardiomyopathy (DCM). C57BL/db/db male mice at 6 and 8 weeks of age, as the early DCM model with obesity and T2DM were investigated. We confirmed that mitochondrial function was decreased in the 8 weeks old db/db mice without alterations of cardiac function and structures. Compared to the control mice, CRBN protein level was significantly increased and phosphorylation of AMPK, a negative regulator by CRBN, was significantly decreased in the heart tissue of 8 weeks old db/db mice. Blood CRBN level was also significantly increased in both 6 and 8 weeks old db/db mice compared with control mice. In blood of diabetic patients, CRBN level was significantly correlated with IL-6, IL-1b, TNF-a, IGFBP-7, and adiponectin, which are previously reported biomarkers of DCM. However, CRBN level was not correlated with cardiac troponin I, NT-proBNP, and ST2, which are biomarkers of myocardial injury and heart failure. Furthermore, the CRBN level showed a higher tendency in patients with diastolic dysfunction without systolic dysfunction compared to patients with normal function and systolic dysfunction, although the difference was not statistically significant. In conclusion, cardiac mitochondrial dysfunction occured and CRBN levels of heart and blood increased prior to cardiac functional and structural changes in the early DCM mouse model, and CRBN might serve as a potential biomarker for the early detection of DCM in T2DM patients.
Abstract We070: The role of neutrophil YAP in cardiac inflammation and injury
Authors: Jamie Francisco, Satvik Mareedu, Jin Guan, Eun-Ah Sung, Peiyong Zhai, Che-Ming Hu, Dominic Del Re
Journal: Circulation Research (2024)

Background: There is a growing appreciation that neutrophils demonstrate plasticity, not unlike the macrophage, and can modulate both inflammation and repair following organ injury. Neutrophils are rapidly recruited to the heart after acute myocardial infarction (MI) and participate in cardiac inflammation and remodeling. Nevertheless, mechanisms regulating neutrophil function during heart injury remain largely unknown. Research Question/Aim: Previous work demonstrated that Yes-Associated Protein (YAP) regulates macrophage function during heart injury. However, whether YAP regulates neutrophil function in any context, including reperfused MI (I/R), remains unexplored. Approach: We generated neutrophil-specific YAP KO mice (MRP8 Cre ) and subjected them to I/R to elicit inflammation and injury. Cell-based experiments were performed using primary isolated mouse neutrophils. We utilized flow cytometry, single-cell RNAseq, and histological and biochemical assays to investigate underlying mechanisms. Results: Neutrophils isolated from the blood and heart of WT mice subjected to I/R revealed increased YAP expression. Compared to control mice, neutrophil YAP KO hearts had decreased infarct size after 24 hours reperfusion, and better cardiac function after 2 weeks. Single-cell RNAseq of heart leukocytes indicated suppression of inflammatory, migratory, and superoxide production pathways in YAP KO neutrophils. Characterization of neutrophils by flow cytometry indicated a shift toward reparative polarization in YAP KO mice during early reperfusion. Bone marrow-derived neutrophils (BMDNs) from KO mice had attenuated inflammatory gene expression and ROS production compared to control cells. Pharmacological inhibition of YAP using verteporfin produced comparable results. Ongoing experiments are investigating how YAP modulates neutrophil inflammatory function, and will determine the therapeutic potential of targeted YAP inhibition for I/R injury. Conclusion: These results suggest that YAP regulates neutrophil function during I/R injury in the heart and that suppression of neutrophil YAP affords cardioprotection.
Abstract We060: Hypoxia Inducible Factor-2α Enhances Neutrophil Survival and Promotes Cardiac Injury Following Myocardial Infarction via cIAP-1 Signaling
Authors: Enzo Piccolo, Zhi-Dong Ge, Edward Thorp, Ronen Sumagin
Journal: Circulation Research (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 &amp; 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 Mo062: Cpne5 is Necessary for Normal Sinoatrial Node Function
Authors: Katherine Dang, Lu Ren, Felix Atzert, Phung Thai, Sruthi Mantri, Lauren Duan, Benjamin Beyersdorf, Nipavan Chiamvimonvat, Sean Wu, William Goodyer
Journal: Circulation Research (2024)

Background: Cpne5 (Copine 5) is a Ca2+-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing (scRNA-seq) of the developing mouse heart, we found Cpne5 gene expression to be highly enriched throughout the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesize that Cpne5 is necessary for normal CCS function. Objective: Determine whether Cpne5 is necessary for the normal development and/or function of the CCS. Methods: To assess the necessity of Cpne5, homozygous systemic knockout (KO) mice were generated using CRISPR/Cas9. Cpne5KO and littermate wild-type (WT) control mice were characterized using surface electrocardiogram (ECG), continuous telemetry and echocardiogram. Protein expression of Cpne5 and CCS-related ion channels were assessed by immunofluorescence of tissue sections, isolated adult murine SAN cells and whole mount immunostaining with 3D volumetric imaging (iDISCO+). Further, CRISPR-mediated deletion of CPNE5 was performed in human induced pluripotent stem cells (hiPSC). Differentiated SAN-like cells were phenotyped via calcium transient (CaT) dynamics and microelectrode array. Results: Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, sinus pauses, and increased heart rate variability compared to WT controls on long term telemetry. Additionally, CPNE5KO hiPSC-derived SAN-like cells exhibited irregular CaT as well as significant beat period variability compared to isogenic WT controls. Conclusion: Systemic loss-of-function of Cpne5 in mice resulted in sinus node dysfunction, consistent with an hiPSC-derived SAN model and human GWAS data, implicating a novel, cell-intrinsic role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies are ongoing to further elucidate the underlying mechanism.