bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3018: SCARNA20 Promotes M2 Macrophage Polarization Under Endotoxemia And Reduces Inflammation
Authors: Perales Selene , Sigamani Vinoth , Rajasingh Sheeja , Vembuli Hemanathan , Pushparaj Rahul , Rajasingh Johnson
Published: October 2023

Background:
Macrophage polarization plays a key role in the inflammatory process. M1 pro-inflammatory and M2 anti-inflammatory phenotypes can down-regulate each other and may contribute to the pathogenesis of various diseases. Small Cajal Body Associated RNAs (SCARNAs) play a key role in alternative splicing and maturation of mRNAs. Our recent studies found that a combination of Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli.

Hypothesis:
We hypothesize that the P+T treatment increases the expression of SCARNA20 hence reducing the proinflammatory response and promoting M2 macrophage polarization after LPS challenge.

Methods and Results:
To elucidate SCARNAs of interest during the inflammatory response, we treated macrophages with LPS (1μg/mL) and P (400nM/mL) + T (2.5μM/mL) for 1hr and performed RNA sequencing. Based on the sequencing results and qRT-PCR validation, SCARNA20 was the most significantly differentially expressed SCARNA between conditions
(Fig. A)
. The expression of TNFα was significantly reduced with the P+T treatment
(Fig. B)
. SCARNA20 overexpression (OE) showed an increase in the expression of the M2 marker CD163 and a reduction in the expression of the M1 marker CD80
(Fig. C)
. Additionally, TNFα expression was significantly reduced in the SCARNA20 OE samples after the LPS challenge without treatment with P+T
(Fig. D)
. Finally, SCARNA20 knockdown macrophages
(Fig. E)
treated with P+T did not suppress TNFα expression
(Fig. F)
.

Conclusions:
We report for the first time that SCARNA20 is an important regulator of M2 macrophage polarization and pro-inflammatory response.
Abstract P3023: Endothelial Pparδ Deletion Exacerbates Endotoxemia-Induced Vascular Injury And Inflammation
Authors: Hong Huiling , Wu Yalan , Tian Xiao-Yu
Published: October 2023

Rationale and Background:
Impairment of vascular endothelial integrity is one of the hallmarks of acute lung injury, which underlies leukocyte infiltration and edema formation and contributes to organ dysfunction. The peroxisome proliferator-activated receptor delta (PPARδ) is a transcription factor regulating vascular endothelial cell (EC) function. Our previous study revealed that selective deletion of PPARδ in the endothelium worsens ischemic vascular injury, disrupts endothelial integrity, and exacerbates vascular inflammation in a mouse model of hindlimb ischemia.

Methods and Results:
In this study, we investigated whether PPARδ is protective for EC function in acute vascular injury and inflammation in the mouse lung. We performed lipopolysaccharide (LPS)-induced endotoxemia in the EC-selective PPARδ knockout mice (Ppard
EC-KO
) and their wild-type littermates (Ppard
EC-WT
). The mortality of Ppard
EC-KO
mice was higher after LPS injection. The survived Ppard
EC-KO
mice showed more severe lung vascular inflammation suggested by sustained leukocyte infiltration in the lung tissues and in the bronchoalveolar fluid, more pro-inflammatory cytokine production, and higher expression of EC activation markers CCL2, ICAM1, VCAM1, and E-selectin. In addition, the lung endothelium of LPS-treated Ppard
EC-KO
mice has degraded endothelial glycocalyx and discontinuous EC junctions, resulting in increased vascular leakage.
In vitro
experiments using human lung microvascular endothelial cells (HLMVECs) revealed that silencing PPARδ downregulated the expressions of ZO-1, claudin-5, and VE-cadherin, and altered their membrane distribution, leading to hyperpermeability of EC monolayer, at basal level and also in the presence of LPS or TNFα. Silencing of PPARδ also induces more activation of pro-inflammatory transcription factors including STAT1, IRF3, and IRF7.

Conclusion:
In summary, our study showed the protective role of endothelial PPARδ against endotoxemia-induced disruption of vascular barrier function and vascular inflammation in the lung endothelium. (Supported by Hong Kong Research Grant Council GRF 14107822 and 14105321)
Abstract P3035: Incidence, Complications And Outcomes Of Cerebral Venous Sinus Thrombosis In Patients With Acute Ischemic Stroke
Authors: Parajuli Tilachan , Niroula urza , Mir Tanveer , Hassan Shahzad , Qureshi Waqas , Dutta Abhishek , Mehta Shivani
Published: October 2023

Introduction:
Cerebral venous sinus thrombosis (CVST) is known to occur in patients with acute ischemic stroke. The incidence, complications and outcomes of CVST in acute ischemic stroke is unknown. We aimed to analyze a national database to evaluate the epidemiological characteristics of CVST in acute ischemic stroke patients.

Method:
We included weighted 624,046 adult patients (age ≥18 years) with acute ischemic stroke hospitalized in 2019 from the Nationwide Readmission Database (NRD). Baseline demographics, comorbidities, and outcomes were identified using International Classification of Diseases (ICD) codes.

Results:
Of the total, 1,156 (0.19%) stroke patients (mean age 50 ± 25) years and 55.9% females) were admitted with CVST. Of these, 144(12.4%) patients died in the CVST group and 41,372(6.64%) in the non CVST group (p <0.001). Complications were noted to be higher in CVST group vs. non CVST; common complications that were significant included but not limited to; intracranial hemorrhage OR 6.11, CI (4.86-7.67), p <0.001, resp failure OR 1.89, CI (1.47-2.44), p<0.001, seizure OR 4.34, CI (2.74-6.87), cranial nerve palsy OR 8.15, CI (4.14-16.10), p<0.001. The cost of index hospitalization and length of stay were higher for patients with CVST as well. Increased mortality was associated with diagnosis of CVST on presentation.

Conclusion:
In hospitalized acute ischemic stroke patients, cerebral venous sinus thrombosis was associated with increased risk of morbidity, mortality, increased length of stay and higher cost of hospitalization.
Abstract P3038: Novel MicroRNAs Targeting Myosin Binding Protein-C3 Gene In Hypertrophic Cardiomyopathy
Authors: Ananthamohan Kalyani , Baby Akhil , Jegga Anil , Sadayappan Sakthivel
Published: October 2023

Background:
Mutations in cardiac myosin binding protein-C3 (
MYBPC3
) constitute a significant cause of hypertrophic cardiomyopathy (HCM) with incomplete penetrance, and later age of onset.
MYBPC3
variant with 25 base pair deletion in intron 32 (

MYBPC3
Δ25bp

) leads to exon 33 skipping and 3’-untranslated region truncation. Independent transcriptomics studies in HCM individuals carrying c.927-2A>G, c.2373insG, c.3624delC and c.3369_3370insC
MYBPC3
variants reported altered levels of cardiac and circulating microRNAs (miRNAs). However, the miRNA-mediated post-transcriptional regulation of the
MYBPC3
and

MYBPC3
Δ25bp

in the HCM phenotype remains largely unknown.

Methods:
Using a computational consensus approach, top-scoring cardiac-specific miRNAs predicted to target wild-type
MYBPC3
(WT) and

MYBPC3
Δ25bp

were identified. To further characterize the potential regulators, the miRNAs with earlier reports on cardiac and skeletal diseases were selected and their targets were intersected with a compiled set of HCM and heart failure (HF) candidate genes. The overlapping genes were subjected to enrichment analysis for biological processes.

Results:
The computational analyses predicted 7 high scoring miRNAs for
MYBPC3,
among which miR-146b-3p, miR-2355-5p, miR-4508, miR-4800-5p and miR-214-5p were predicted to regulate both the WT and

MYBPC3
Δ25bp

variant, whereas the binding sites for miR-5588-5p, and miR-6500-3p were lost in
MYBPC3

Δ25bp

variant. In specific, miR-146-3p, miR-214-5p, and miR-2355-5p have been reported to be associated with skeletal muscle dysfunction, HCM and myocardial ischemia-reperfusion injury, respectively. The putative targets of these 3 miRNAs intersected with HCM and HF candidate genes, in addition the intersected targets displayed enrichment for cardiac disease-associated biological processes.

Conclusions:
Computational prediction suggests that predicted
MYBPC3
miRNAs affect biological processes and pathways associated with HCM and HF. Importantly,

MYBPC3
Δ25bp

show a loss of miRNA regulation compared to the WT. Further studies and experimental validations are required to determine the influence of loss of miRNA regulation in the

MYBPC3
Δ25bp

and their impact on HCM interventions.
Abstract P3041: Stage-specific Alternative Rna Splicing Attunes Cardiomyocyte Proliferation And Contraction
Authors: Gan Peiheng , Wang Zhaoning , Bassel-Duby Rhonda S , Liu Ning , Olson Eric N
Published: October 2023

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.
Abstract P3043: Mir92b-3p: A Key Regulator In Vascular Homeostasis And Remodelling
Authors: Köster Jan Michael , Kalies Katrin , Boettcher Solveig , Daniel Fiene , Hehl Laura , Guerlach Simon , Knoepp Kai , Dutzmann Jochen , SEDDING Daniel
Published: October 2023

In vascular disease, cells undergo important alterations in cellular function, influencing vascular homeostasis. Apoptosis is one of these functions, affecting vascular tone and plaque stability. Influencing this process for example, by targeting microRNAs is key for therapeutic strategies tackling vascular diseases in a cell-specific manner. We identified the critical role of miR92b-3p on the functional properties of vascular cells. Studying human primary coronary artery smooth muscle cells (SMCs) and human umbilical vein endothelial cells (ECs), methods included expression analysis in mice tissue after restenosis by wire-induced injury and in atherosclerosis. The role of miR-92b on cellular function and target regulation was determined by pre- and antagomir transfection. Initial analysis in vivo showed miR92b-3p to be significantly upregulated in atherosclerosis (p<0,01) and regulated in restenosis (p<0,05). Under growth conditions in both ECs and SMCs, miR-92b-3p was significantly higher expressed (p<0,05). In SMCs, upregulation of miR92b-3p resulted in increased proliferation (p<0,01) and significantly enhanced cell migration in SMCs (p<0,001) but hampered migration in ECs (p<0,01). Downregulation of miR-92b-3p in SMC resulted in decreased migrational (p<0.001) and proliferative capacity (p<0.001), accompanied by a reduction in metabolic activity (p<0.05) and, exclusively in SMCs, increased apoptosis (p<0.05). In ECs, proliferation was enhanced following downregulation (p<0,01). Multiple targets were identified, using Western blot and qPCR including KLF4 (p<0,01), DKK3 (p<0,05), Notch1 (p<0,01), PTEN (p<0,05), and especially in SMC, Bim (p<0,001). Regulation of Bim by miR92b-3p and subsequent apoptosis was then evaluated by siRNA-knockdown experiments, confirming their strong interaction. We further verified the crucial role of miR-92b-3p by reversing cell death in apoptotic SMCs through miR-92b-3p overexpression experiments (p<0.05). This study reports miR 92b-3p to be a crucial regulator of vascular function. Therapeutic regulation might present a promising strategy for stabilizing critical plaques, restoring the function of vascular cells, and thereby preventing vascular remodeling.
Abstract P3056: Cardiac Fibroblast GSK-3α Contributes To Chronic Inflammation In The Ischemic Heart
Authors: Umbarkar Prachi , Ejantkar Suma , Ruiz Ramirez Sulivette , Tousif Sultan , Toro Cora Angelica , Zhang Qinkun , Lal Hind
Published: October 2023

Background:
Myocardial Infarction (MI) is the leading cause of death worldwide. Glycogen Synthase Kinase-3 (GSK-3) has been identified as a promising therapeutic target to treat cardiovascular diseases. The GSK-3 family has 2 isoforms, α and β. Previously, our lab has identified cardiac fibroblast (FB) GSK-3β acts as a negative regulator of fibrosis in the ischemic heart. However, the role of FB-GSK-3α in MI-induced adverse cardiac remodeling is not defined.

Methods and Results:
To determine the role of FB-GSK-3α in MI-induced adverse cardiac remodeling, GSK-3α was deleted specifically in the activated fibroblast/myofibroblast using tamoxifen (TAM)-inducible Periostin promoter-driven Cre recombinase system. At 12 weeks of age, mice were fed with the TAM diet. After 1 week of TAM treatment, control (GSK-3α
fl/fl
Cre
-/-
) and KO (GSK-3α
fl/fl
Cre
+/-
) mice were subjected to permanent LAD ligation (MI) surgery. Serial echocardiographic analysis revealed that FB-GSK-3α KO mice were protected from MI-induced systolic dysfunction and dilative remodeling. To investigate the role of FB-GSK-3α in MI-induced chronic inflammation, hearts were harvested at 4 weeks post-MI, and expression of signature inflammatory genes (TNFα, IL-6, IL-1β) was analyzed by qPCR. Surprisingly, the KO heart displayed remarkably low levels of inflammatory gene expression. To delineate underlying mechanisms, the effect of FB-GSK-3α deletion on the pro-inflammatory Nuclear factor kappa B (NF-κB) pathway was examined
in vitro
. Western blot analysis confirmed a significant reduction in NF-κB activation in GSK-3α-KO FBs. Importantly, the co-culture experiment demonstrated that the expression of inflammatory genes was downregulated in immune cells when cultured with GSK-3α KO FBs. These results indicate that GSK-3α can modulate immune cell phenotype through FB-immune cell crosstalk.

Conclusion:
Our findings suggest a critical role of FB-GSK-3α in pathological cardiac remodeling and heart failure that could be therapeutically targeted for future clinical applications.
Abstract P3058: Myocardial Infarction Impairs T Lymphopoiesis In Mice
Authors: Ma Yonggang , Shane Danielle X , Konovalova Daria , Yuan Sarah Y
Published: October 2023

T lymphocytes, especially CD4
+
T cells, play beneficial roles in regulating cardiac repair after acute myocardial infarction (MI). Some patients with MI show lymphopenia, a reduction in blood lymphocyte count. More importantly, lymphopenia negatively correlates with patient outcome. We have previously shown that circulating lymphocyte trafficking to the bone marrow contributes to lymphopenia post-MI. However, whether MI affects T cell production remains uninvestigated. The aim of this study was to investigate whether MI impairs T lymphopoiesis, the process of T cell development. Three to six months old male C57BL/6J mice underwent MI surgery by permanent ligation of the left anterior descending coronary artery. Sham controls were subject to the same procedure with the exception of artery ligation. At 7 days post-MI, we quantified thymic mass, cellularity, developmental thymocytes, and hematopoietic stem/progenitor cells in the bone marrow and thymus using multiparameter flow cytometry. Thymocyte apoptosis was evaluated using an Annexin V assay kit. Compared to sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, and less thymocytes at different developmental stages, including double negative CD4
-
CD8
-
, double positive CD4
+
CD8
+
, and single positive CD4
+
thymocytes. Early thymic progenitors (ETP) that generate thymocytes in the thymus were also lower in MI mice than sham counterparts. Interestingly, common lymphoid progenitors (CLP) in the bone marrow, the ancestors of ETP, were not affected by MI. To determine the reason that MI mice show less thymocytes, we assessed thymocyte apoptosis. The data showed that thymocytes in MI mice exhibited higher apoptotic rate than sham controls. In conclusion, our study, for the first time, demonstrates that MI impairs T lymphopoiesis, and this impairment mainly occurs in the thymus, but not in the bone marrow. The underlying mechanisms are related to increased thymocyte apoptosis.
Abstract P3060: Modeling Immune Checkpoint Inhibitor Associated Myocarditis
In Vitro
Authors: Jensen Garrett , Wang Xinjie , Zhao Wei , chen Zhishi , Yu Wei , Palaskas Nicolas L , Stephan Clifford , Powell Reid , Wu Joseph C , Luo Weijia , Chang Jiang
Published: October 2023

Introduction:
Immune checkpoint inhibitor-associated myocarditis (ICI myocarditis) is a severe side effect of treating malignancies with immune checkpoint inhibitors. Patients suffering from ICI myocarditis often display an extraordinarily high mortality rate. Establishing a clinically relevant model to address the lack of identifiable risk factors, screening tests, or effective treatments is critically urgent.

Hypothesis:
Human ICI myocarditis can be accurately modeled in vitro.

Methods and Results:
We present the first cell culture-based model of ICI myocarditis. We use a novel method to co-culture human iPSC-derived cardiomyocytes (hiPSC-CM), human peripheral blood mononuclear cells, and immune checkpoint inhibitors to recapitulate critical aspects of clinical ICI myocarditis. Using this platform, we show rapid, significant cardiomyocyte necrosis after model initiation. We observe substantial disruption to hiPSC-CM cell morphology and sarcomere structure, and we use powerful multi-electrode array technology to define contractile functions and electrophysiological changes, demonstrating the critical similarities between this cell-based ICI myocarditis model and patient myocarditis. Our model also reveals an increased risk of arrhythmia, recapitulating,
in vitro
, the primary cause of death in patients suffering from ICI myocarditis. Finally, we demonstrate that this hiPSC-CM-based “model in a dish” can be rescued pharmacologically and identify specific leukocyte populations required for myocarditis development. In summary, we present the first
in vitro
model of ICI myocarditis and demonstrate its scalability, validity, patient relevance, and reproducibility. Utilizing the model can aid in accomplishing precision medicine objectives and holds significant potential for preventing and treating ICI myocarditis.
Abstract P3061: Development Of Molecular Targeted Therapy Against Right Ventricular Failure: Involvement In Complement C3- Factor D- C3a Pathway
Authors: Ito Shogo , Yuasa Shinsuke , YAMAKAWA Hiroyuki , Komuro Jin , Hashimoto Hisayuki , Kimura Mai , Nakao Eichi , Fukuda Keiichi , Fukumoto Yoshihiro
Published: October 2023

Backgrounds:
Right ventricular (RV) failure plays a critical role in right heart failure and left heart failure. However, there is no specific therapy developed for RV failure. To elucidate a novel therapeutic target against RV failure, we focus on RV specific pathway.

Methods:
Microarray analysis using several parts of adult murine heart was conducted and differentially expressed genes (DEGs) were applied to pathway analysis. Molecular mechanism was examined by using neonatal rat ventricular cardiomyocyte (NRVM) in vitro. To understand the function of target molecule in vivo, we induced RV failure by pulmonary artery constriction (PAC) in mice and inhibition experiments were performed using RV failure model.

Results:
In microarray analysis elucidated 995 genes as DEGs in RV. Pathway analysis showed that alternative complement pathway-related genes were significantly up-regulated in RV. Moreover, complement factor D (Cfd) and C3a was a potential upstream factor attributable to unique feature of RV. Administration of C3a recombinant protein to primary cultured ventricular myocyte indicated that phosphorylation of several MAP kinases were enhanced. In vivo, in C3KO PAC mice, RV dysfunction was significantly suppressed, and histological study showed that RV fibrosis was significantly suppressed. In wild type mice with PAC, administration of C3a receptor antagonist dramatically improved RV dysfunction and reduced RV fibrosis. Additionally, in vivo electrophysiological study revealed that the inducibility of ventricular arrhythmia was increased in wild type PAC mice, but ventricular arrhythmia was significantly attenuated in C3KO PAC mice. Furthermore, electrophysiological analysis by isolated cardiomyocyte showed that calcium overload was also attenuated in RV cardiomyocyte of C3KO PAC mice.

Conclusion:
We revealed that complement C3a was highly produced in RV and chemical or genetical blockade of C3a ameliorates RV dysfunction and RV fibrosis in PAC mice. C3a was to be a potent bioactive protein for immunocompetent cells that played an important role in modulating cardiac function. Accordingly, we demonstrated that the blockade of C3a had a potential role for novel therapeutic target to RV failure.
Abstract P3063: Selective Release Of Mitochondrial Dna After Brief Myocardial Ischemia
Authors: Cucinotta Daniel M , Rolland Tyler , Zahra Sumbule , Weil Brian R
Published: October 2023

Objective:
Mitochondrial DNA (mtDNA) is one of several pro-inflammatory damage-associated molecular patterns released from necrotic myocytes during myocardial infarction (MI). Based on our recent observation of a ~50-fold rise in circulating mtDNA after brief whole-body ischemia in a porcine model of cardiac arrest without ongoing MI, we hypothesized that ischemia-induced mtDNA release can occur in the absence of necrosis. Accordingly, we measured circulating mtDNA levels before and after localized myocardial ischemia of insufficient duration to produce myocyte necrosis in swine.

Methods:
Swine (n=6) were subjected to a 10-minute occlusion of the left anterior descending (LAD) coronary artery based on prior evidence that this duration of ischemia elicits myocardial injury without necrosis. Peripheral venous and coronary sinus (CS) blood samples collected at baseline (BL) as well as 1-hour and 24-hours after reperfusion were analyzed via qPCR to quantify mtDNA and nuclear DNA (nucDNA) release. Results were compared to a separate group of swine (n=6) subjected to a 75-minute LAD occlusion to produce MI.

Results:
Prolonged ischemia led to a significant rise in CS levels of mtDNA (6.2 ± 3.6-fold vs. BL; p<0.05) and nucDNA (5.4 ± 2.0-fold vs. BL; p<0.05) 1 hour after reperfusion, consistent with rapid non-selective DNA release after myocyte necrosis. In contrast, brief ischemia was associated with detectable elevations in CS mtDNA (4.6 ± 1.4-fold vs. BL; p<0.05) but not nucDNA (2.1 ± 0.5-fold vs. BL; p=0.35) at this timepoint. However, the magnitude of DNA release after brief or prolonged ischemia was not sufficient to increase circulating mtDNA or nucDNA, as peripheral venous levels of mtDNA and nucDNA did not differ from BL at 1-hour or 24-hours post-reperfusion in either group.

Conclusion:
Brief myocardial ischemia is associated with selective release of mtDNA into circulation, providing a potential mechanism by which immune activation may occur after myocardial injury even when myocyte necrosis is absent. The magnitude of mtDNA release after brief myocardial ischemia is ~10-fold lower than that detected in peripheral blood after resuscitation from cardiac arrest, suggesting that mtDNA is released from other organs after brief whole-body ischemia.
Abstract P3067: Emerging Immune Checkpoint Therapies Reshape The Cardiac Immune Landscape And Promote Myocardial Vulnerability
Authors: Ma Pan , Villanueva Ana , Liu Jing , Egawa Takeshi , Lin Chieh-Yu , Moslehi Javid , Lavine Kory J
Published: October 2023

Immune checkpoint therapies have shown remarkable efficacy across multiple tumor types by amplifying cancer-immunity and boosting T-cell immune response against tumors. As an increasing number of immune checkpoint molecules have been identified, numerous combination therapeutic strategies are under clinical investigation. For instance, the combination of an OX40 (TNFRSF4) agonist and PD-1 (Programmed cell death protein 1) inhibitor has been shown to have promising anti-tumor effects in pre-clinical studies and early clinical trials. Despite remarkable therapeutic benefit, it has become evident with increasing clinical usage that these agents elevate the risk of immune-related adverse events (irAEs), especially cardiovascular sequelae. Mitigating irAEs is critical for the establishment of efficacious and safe interventions. Currently, little is understood regarding the cardiac effects of combined OX40 activation and PD-1 inhibition. In this study, we observed that PD-1 deletion upregulated OX40 expression on cardiac T-cells. Combined PD1 inhibition (Pdcd1
-/-
or PD-1 blocking antibody) and OX40 activation (OX40 agonistic antibody) increased the frequency of splenic and cardiac effector memory CD4
+
and CD8
+
T-cells, inflammatory monocyte and macrophage infiltration into the heart, and enhanced myocardial expression of numerous chemokines and cytokines in both naïve and tumor bearing mice. Intriguingly, persistent increases in cardiac effector memory CD4
+
and CD8
+
T-cells and inflammatory macrophages was observed at chronic time points suggesting long term effects on the cardiac immune landscape. Furthermore, OX40 activation aggravates angiotensin II and phenylephrine (AngII/PE) infusion induced cardiac hypertrophy, fibrosis and inflammation in Pdcd1
-/-
mice suggesting that the reshaped T-cell and macrophage landscape imparts myocardial vulnerability. This study will also uncover mechanism-based therapeutic targets to prevent or treat cardiac adverse events elicited by combined OX40 activation and PD-1 inhibition.
Abstract P3070: Aortic Valve Calcification Is Accelerated By DNMT3A And TET2 Gene Driver Mutations Associated With Clonal Hematopoiesis
Authors: Abplanalp Wesley T , Schuhmacher Bianca , Mas-Peiro Silvia , Zuriaga Maria A , Matesanz Nuria , Fuster Jose J , John David , Muhly-Reinholz Marion , Vasa-Nicotera Mariuca , Merten Maximilian , Dimmeler Stefanie , Zeiher Andreas M
Published: October 2023

Background:
Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations associates with heightened inflammation and a worse prognosis among patients with severe calcific aortic valve stenosis (AS). However, it is unknown what role CH plays in the pathogenesis of AS. AS is the most common age-related heart valve disease, with no medical therapy to halt progression.

Methods and Results:
Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), having a mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, which were associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) along with paracrine pro-calcific factors (Oncostatin M (OSM), S100A9), signifying that CH might be causally involved in AS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced supernatants induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic transformation could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving Tet2-/- bone marrow transplants demonstrated increased total calcified area (p<0.05, 1.49-fold) along with increased numbers of calcification deposits (p<0.05, 2.33-fold) as shown by von Kossa staining, along with enhanced OSM and S100A9 levels.

Conclusion:
This is the first study to show that somatic CH-driver mutations in monocytes may accelerate AS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the development of AS in patients harboring CH mutations.
Abstract P3079: Integrating Existing Cardiac Single-cell RNA Sequencing Datasets To Uncover The Role Of B-cells In Heart Disease
Authors: Bermea Kevin , Bhalodia Aashik , Cohen Charles D , lovell jana , Rousseau Sylvie , Zita Marcelle D , Adamo Luigi
Published: October 2023

Introduction:
The role of B-cells in human heart failure remains unclear. To address this knowledge gap, we analyzed publicly available datasets of scRNAseq and snRNAseq from patients with various forms of cardiomyopathy.

Methods and Results:
We used two public datasets including single cells or single nuclei RNAseq data collected from the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), dilated cardiomyopathy (DCM), idiopathic cardiac diseases (ICD), or healthy controls. The first dataset comprised 30 healthy controls and 22 non-ischemic DCM; the second contained 18 controls and 61 with DCM, ARVC, or ICD. Data were normalized and scaled, a UMAP plot was generated, and cells were classified using ScType. B-cells were merged into a single matrix, and the final dataset contained 1100 cells from 32 healthy controls, 59 DCM, and 8 ARVC patients. Four different B-cell clusters were identified. The top 100 biomarkers from each cluster were used to identify B-cell subtypes. Cluster 1 was identified as transitional splenic B-cells; Cluster 2 as plasma cells; Cluster 3 as follicular splenic B-cells; Cluster 4, present only in DCM, as immature B cells. Ingenuity Pathway Analysis (IPA) of the genes enriched in each cluster highlighted that different B cell clusters displayed distinct biological activity, ranging from phagosome formation to B cell receptor signaling and cytokine production. Differentially expressed gene analysis comparing healthy controls to DCM and ARVC showed both similar and disease-specific gene expression patterns in the various B cell clusters. For instance, cluster 2 showed upregulation of oxidative phosphorylation in both DCM and ARVC. On the other hand, DCM cluster 2 showed downregulation of CD40 signaling, while in ARVC the same cluster showed downregulation of IL-6 signaling (Z score >2, p<0.05).

Discussion:
Our findings suggest that human heart failure is associated with perturbations in B cell gene expression, with both shared and disease-specific features. This data supports the notion that B cells likely play a role in human cardiomyopathy. Further, this data suggests that B-cells play distinct roles in disparate forms of cardiac disease.
Abstract P3083: Trim29 Deficiency Ameliorates Viral Myocarditis By Reducing Perk Mediated Er Stress And Ros Responses
Authors: Wang Junying , Lu Wenting , Du Yong , Zhang Jerry , Minze Laurie J , Xing Junji
Published: October 2023

Infections by viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could cause viral myocarditis. Endoplasmic reticulum (ER) stress is associated with several metabolic diseases such as cardiovascular disease, diabetes and cancer. However, the immune mechanisms of ER stress regulation in controlling the pathogenesis of viral myocarditis are unclear. Here, we found that knockdown or knockout of TRIM29 significantly inhibited PERK (Protein kinase RNA-like endoplasmic reticulum kinase) mediated ER stress and apoptosis induced by cardiotropic viruses CVB3 (coxsackievirus B3) or EMCV (encephalomyocarditis virus) in human and mouse cardiomyocytes. Additionally, TRIM29 deficiency relieved ROS (Reactive oxygen species) mediated STING (Stimulator of interferon genes) inhibition to produce more type I interferon for restricting cardiotropic viruses in cardiomyocytes. Importantly, cardiomyocytes specific TRIM29 deficiency exhibited dramatic protective effects on cardiac damage, cardiac dysfunction, viral replication and promoted survival in CVB3-induced viral myocarditis model
in vivo
. Mechanistically, TRIM29 was shown to interact with PERK and catalyze SUMOylation for maintaining its stability, thereby promoting PERK mediated ER stress and ROS responses to induce pathogenesis of viral myocarditis. Our results indicate that TRIM29 loss-of-function in cardiomyocytes ameliorates viral myocarditis by reducing PERK mediated ER stress response and unleashing ROS-suppressed interferon response. Our data suggest the TRIM29-PERK axis is a potential therapeutic target for viral myocarditis and other ER stress-associated cardiovascular disease.
Abstract P3084: Neutrophil Expansion And Netosis Are Essential For Adverse Left Ventricular Remodeling And Dysfunction In Chronic Ischemic Heart Failure
Authors: Rokosh Gregg , Antipenko Sergey , Mayfield Nicolas , Jinno Miki , Gunzer Matthias , Ismahil Ameen , Hamid Tariq , Prabhu Sumanth D
Published: October 2023

Chronic inflammation contributes significantly to disease progression in heart failure (HF) with the expansion and adverse activation of macrophages, dendritic cells, and T cells in the failing heart. Neutrophils play a well-known role in the initial acute immune response to myocardial infarction (MI), however, whether and how they contribute to inflammation and adverse LV function and remodeling in chronic ischemic HF is unknown. We tested the
hypothesis
that activated neutrophils are required for tissue remodeling and disease progression in HF in mice studied 4 and 8 weeks after MI during established ischemic HF (vs sham). CD11b
+
Ly6G
+
neutrophils were expanded in peripheral blood (PB), heart, spleen and bone marrow 8 w post-MI. Neutrophils in the failing heart were localized to the MI border zone and augmented in remote zone myocardium by immunofluorescent staining. CXCL1 and CXCL5 expression was increased in the failing heart, consistent with chemotactic signals for myocardial neutrophil recruitment. Plasma from HF mice elicited greater chemotaxis in naïve neutrophils (vs naïve plasma) by time-lapse video microscopy. Further, PB neutrophils from HF mice exhibited significantly increased neutrophil extracellular trap (NET) production, with concomitant increase in plasma NETs (histone-DNA complexes) in HF mice. The direct pathological impact of NETs was demonstrated by intramyocardial injection of isolated NETs into naïve hearts mimicking HF with decreased EF, LV dilation, and fibrosis. Finally, the in vivo role of neutrophils in chronic HF (4 and 8 w post-MI) was determined via depletion studies using genetic (Ly6G-diptheria toxin receptor mice) and antibody (1A8) based approaches. Neutrophil depletion significantly improved LV EF and reduced end systolic and diastolic volumes. 1A8 treated HF mice also exhibited reduced remote zone fibrosis. The necessity of NETosis in HF development was confirmed using neutrophil and myeloid – specific PAD4 deletion where the decline in LV EF and increase in volumes in HF were attenuated.
In conclusion
, neutrophil expansion and NETosis are important contributors to chronic inflammation in HF and play an obligatory role in the development of LV dysfunction and remodeling in ischemic cardiomyopathy.
Abstract P3087: Macrophage Fatty Acid Metabolism Promotes Splenic Hematopoiesis Through Mitochondrial Signaling During Heart Failure With Preserved Ejection Fraction
Authors: Filipp Mallory , Ge Zhi-Dong , Lantz Connor , Shah Sanjiv J , Thorp Edward B
Published: October 2023

Rationale:
Diastolic dysfunction, including during heart failure with preserved ejection fraction (HFpEF), is a widespread morbidity with limited therapeutic options. Though heterogeneous in etiology, major risk factors include hyperlipidemia, hypertension, and inflammation. Recent studies have also shown significant changes in systemic metabolite availability during HFpEF, further suggesting that metabolic changes may be an important indicator of HFpEF pathophysiology. Macrophages, which have previously been implicated in the promotion of diastolic dysfunction, utilize specific metabolic pathways to drive inflammation or tissue repair. However, little is understood about myeloid metabolism during HFpEF.

Objective:
We set out to define the functional and metabolic heterogeneity of the cardiac immune response during diastolic dysfunction.

Methods/Results:
Diastolic dysfunction was induced by HFD and eNOS inhibition in mice. scRNAseq showed a specific induction of pro-fibrotic and lipid handling pathways in cardiac macrophages, including the fatty acid receptor Cd36. In live animals, myeloid deficiency of Cd36 prevented diastolic dysfunction and was associated with dampened cardiac inflammation. Investigating immune cell sourcing during HFpEF revealed that a splenic hematopoietic niche formed in wild-type mice. Further, mitochondrial metabolism in splenic macrophages was associated with increased expression of VCAM-1, the latter of which has been shown to play a role in retention of HSCs in the spleen. Indeed, blocking VCAM-1 or genetic deletion of VCAM-1 in myeloid cells was sufficient to disrupt retention of HSCs within the spleen during HFpEF. Culturing splenic macrophages with saturated fatty acids (SFAs) in vitro was sufficient to induce expression of
Vcam1.
Increased cellular citrate levels were correlated with increased histone acetylation at the Vcam1 promoter in splenic macrophages cultured with SFAs. Inhibition of mitochondrial uptake of fatty acids through CPT1a siRNA or inhibition of ACLY were sufficient to prevent this upregulation of
Vcam1
expression.

Conclusions:
Our data suggest a critical role for myeloid metabolism in the sourcing of detrimental-monocyte-derived macrophages from the spleen to the heart.
Abstract P3102: Sex Differences In Outcomes Among Heart Failure Hospitalizations
Authors: Appunni Sandeep , Rubens Muni , Saxena Anshul , Ramamoorthy Venkataraghavan , Roy Mukesh , Ahmed Md Ashfaq , Zhang Zhenwei , Zhang Yanjia
Published: October 2023

Background:
Both men and women have a comparable overall lifetime chance of developing heart failure, but there are clear sex differences in the nature of this condition that are both significant and yet underexplored. In this study, we investigate gender differences in clinical characteristics and in-hospital outcomes of heart failure hospitalizations using a large national database.

Methods:
This study is a retrospective analysis of National Inpatient Sample data, 2016-2019. Hospitalizations ≥41 years of age, admitted for heart failure were included in the study and stratified by sex. The outcomes of the study were in-hospital mortality rate, prolonged hospital length of stay, mechanical ventilation, mechanical circulatory support, vasopressors use, and adverse disposition. Propensity score matching with conditional logistic regression analysis was done to compare outcomes between males and females.

Results:
A total of 4,704,684 primary heart failure hospitalizations were included for the analysis. Of these 2,447,784 (52.0%) were males and 2,256,899 (48.0%) were females. In-hospital mortality (
P
<0.001), mechanical ventilation (
P
<0.001), mechanical circulatory support (
P
<0.001), and vasopressors use (
P
<0.001) were significantly lower among females, while prolonged length of stay (
P
<0.001) and disposition other than home (
P
<0.001) were higher among females. Conditional logistics regression analysis showed that females had significantly lower odds for mortality (aOR, 0.92, 95% CI: 0.89-0.94), mechanical ventilation (aOR, 0.89, 95% CI: 0.86-0.92), mechanical circulatory support (aOR, 0.54, 95% CI: 0.50-0.58), vasopressor use (aOR, 0.70, 95% CI: 0.66-0.74), and significantly higher odds for prolonged length of stay (aOR, 1.05, 95% CI: 1.04-1.06) and disposition other than home (aOR, 1.32, 95% CI: 1.31-1.34)

Conclusion:
Results of our study show that there are significant differences in outcomes between males and females hospitalized for heart failure. Further research is required to delineate how sex affects several aspects of heart failure, including epidemiology, risk factors, pathogenesis, response to medication, and eventually outcomes.
Abstract P3108: Mouse
In Utero
Plus Neonatal Sertraline Exposure May Result In A Sex-dependent Risk Of Myocardial Infarction In Adulthood
Authors: Lu Yongjun , Kenkel Elizabeth , Zimmerman Kathy , Weiss Robert M , Roghair Robert , Haskell Sarah E
Published: October 2023

Background:
We previously reported that sertraline reduced left ventricular size, acutely slowed down mouse pup growth, and significantly reduced calcium oscillations in cardiomyocytes (p<0.05), and that the sertraline-induced up-regulations of certain miRNAs might epigenetically modulate cardiac functional gene expression of serotonin signaling pathway with down-regulation of
Htr2a, Htr2b
and
Slc6a4
mRNAs and corresponding proteins (p<0.05). Here we hypothesize that sertraline effect is gender related when subjected to a stress at adult.

Method:
C57BL/6 adult female mice received sertraline (5 mg/kg/d IP) or saline 5 days before mating, throughout pregnancy until time of delivery, and the pups were given 1/3 adult dose before weaning. At age of 10 weeks, offspring mice were subjected to echocardiograms before myocardial infarction (MI) induced by left anterior descending coronary artery ligation. Mice were divided into groups according to sex, and treatments (saline vs sertraline, & MI vs sham). Ten weeks post-MI, echocardiograms were performed again for the survived mice.

Results:
Female mice before MI had significantly reduced heart rates (649±18 vs 691±7 bmp), increased end systolic volumes (4.5±0.6 vs 3.2±0.1 μL), and decreased ejection fractions (EF) (80.6±0.7% vs 83.9±0.6%) (n=34, p<0.05). Male mice did not show the significant effects on baseline. Two weeks post-MI, Females had survival rates reduced by sertraline (74.3% vs saline 100%, p=0.0833). Males exhibited MI intolerance with lower survival rates (66.8% vs saline 67.2%), and ODD ratio was deteriorated (24.35 vs female 1.00, p=0.0317). At the end point, the survived mice showed variable MI-induced ischemia zones (47.5±13.2%) corresponding to variable EF (30.0±14.9%). The cardiac functions became not significant different in both male and female survived mice.

Concluding Remarks:
The cardiac potential risk was observed in the baseline and in the earlier MI phase of female mice with
in utero
plus neonatal sertraline exposure, while male mice showed different pattern of sertraline effect. Such sex-dependent cardiac effects of maternal SSRI exposure in adult offspring deserve to be detailly studied by using mouse models for heavy use of SSRIs in pregnant women.
Abstract P3111: Sex Differences In The Proteomic Response To Chronic Myocardial Ischemia In Swine
Authors: Broadwin Mark , Harris Dwight , Sabe Sharif A , Xu Cynthia , Sabra Mohamed , Abid Ruhul , Sellke Frank W
Published: October 2023

Intro:
There are known sex differences in response to ischemic insult yet the mechanisms behind these differences have not been fully elucidated. Previous work in our lab has demonstrated female sex confers a relative protective effect on CO in the setting of chronic ischemia. The aim of this study was to use total proteomic analysis in order to uncover the mechanisms behind sex specific responses to myocardial ischemia in a porcine model.

Methods:
Eight 11-week old pigs (3 female, 5 male) underwent surgical placement of an ameroid constrictor to the left circumflex artery via left thoracotomy in order to induce chronic myocardial ischemia. After seven weeks, pigs were euthanized and myocardial tissue was harvested for total proteomic analysis.

Results:
When compared with non-ischemic male tissue non-ischemic female tissue demonstrated upregulation of two variants of LMOD2 and down regulation of five variants of ACADS (Figure 1A). However, when comparing ischemic female tissue with ischemic male tissue there were 626 upregulated proteins and 194 down regulated proteins (Figure 1B). Enrichment analysis revealed upregulation of certain metabolic pathways including the citric acid cycle and glycolysis/gluconeogenesis (Figure 1C) while there was down regulation in pathways involved in cardiac muscle contraction and dilated cardiomyopathy (Figure 1D). There was no difference in serum glucose, HDL, LDL, or total cholesterol (P > 0.05 for all) (Figure 1E).

Conclusion:
There were clear sex differences in response to chronic cardiac ischemia. Proteins involved in metabolic pathways and myocyte function appear to be particularly affected.
Abstract P3123: Sex-related Differences In Cardiac Biomarkers And Ventricular Fibrosis In A Minipig Model Of Hypertrophic Cardiomyopathy
Authors: Castro Coronado Aileen , Geist Gail E , Smit Trisha , Rogers Christopher , Rohret Frank , Del Rio Carlos L , Garcia Ricardo , Ryba David M
Published: October 2023

Hypertrophic Cardiomyopathy (HCM) is characterized by ventricular hypertrophy and stiffness resulting in part from interstitial fibrosis. While associated with autosomal dominant mutations in sarcomere proteins, it has been reported that males and females may have different clinical presentations and disease courses. We have previously shown that minipigs carrying the R403Q mutation in MYH7 demonstrate a phenotype that recapitulates human non-obstructive HCM with increased hypertrophy, markers of cardiac stress, and diastolic dysfunction. Under the hypothesis that sex-dependent differences in cardiac physiology can independently modulate disease phenotype, we evaluated ventricular fibrosis and cardiac biomarkers in both male and female mutant pigs. Purpose-bred Yucatan R403Q MYH7 heterozygous mutant minipigs of either sex (M: n=8, F: n=6) and wild-type littermates (M: n=4, F: n=14) were used; for the purposes of these comparisons, cloned male pigs were also included (R403Q: 8). Males were castrated, and all pigs received soybean-based feed. Circulating levels of both cardiac troponin T (cTnT) and brain natriuretic peptide (BNP), markers of cardiac injury and stress, were assessed. Interstitial fibrosis was assessed in left ventricular free wall (LV-FW) and apical tissue samples utilizing picrosirius red staining and semi-automated quantification (HALO). Male HCM pigs died earlier (<9M of age, P<0.05) and had higher serum biomarkers than females (cTnT levels (M: 143±17 vs F: 44±7 ng/L, P<0.05; BNP 1536±335 vs 4881±414 ng/L, P<0.05) Consistent with the higher cTnT levels, male HCM pigs had greater fibrosis areas than females in both LV-FW (11±3% vs 7±2%, P<0.05) and apical regions (13±1% vs. 8±2%, respectively, P<0.05). All HCM pigs had significantly increased fibrotic area vs WT controls (R403Q: 10±3% vs WT: 3±2%, P<0.05). Our findings demonstrate that despite a common genotype, female and male carriers of the HCM pathogenic R403Q mutation present different disease phenotypes, with castrated males showing greater fibrotic burden. Future studies to understand the mechanistic drivers of these differences should not only help elucidate the role of sex in HCM disease progression, but also the development of sex-informed therapies.