bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract We056: Hydroxychloroquine cures autoimmune myocarditis by inhibiting innate immune via the CXCL16-CXCR6 axis between macrophages and T cells
Authors: Xuan Yunling , Chen Chen , Wang Dao Wen
Published: October 2024

Background:
Myocarditis is a live-threatening inflammatory disease of the heart and is lack of effective treatment measures. Hydroxychloroquine (HCQ), a classic antimalarial drug, has been widely used in the treatment of rheumatic diseases. This study investigated whether HCQ can treat chronic autoimmune myocarditis.

Methods and Results:
Murine autoimmune myocarditis was induced, and therapeutic effects of oral HCQ on acute and chronic myocarditis were evaluated using echocardiography, cardiac catheterization, and analysis of inflammatory scores in cardiac tissue. Results showed that HCQ treatment significantly improved cardiac function, inhibited ventricle enlargement and reduced cardiac inflammation. Single-cell transcriptomic analysis in the heart showed that HCQ treatment significantly reduced infiltration of macrophage, neutrophiles and T-cells, inhibited inflammatory pathway, especially the secretion of CXCL16 from macrophages, thereby reducing the chemotaxis of T cells, especially NKT cells and Th17 cells. Finally, both in vitro and in vivo experiments confirm the inhibitory effect of HCQ on CXCL16-CXCR6 axis between macrophages and T cells. Additionally, treatment with anti-CXCL16 antibody can also improve cardiac function and attenuate cardiac fibrosis in myocarditis.

Conclusions:
HCQ treatments clinically cure mice with autoimmune myocarditis through inhibiting innate immune and infiltration of macrophages majorly via CXCL16-CXCR6 axis.
Abstract We063: CD73 EXPRESSION ON NEUTROPHILS PROTECTS AGAINST CARDIOMYOCYTE DAMAGE DURING INVASIVE
STREPTOCOCCUS PNEUMONIAE
INFECTION
Authors: Bhalla Manmeet
Published: October 2024

In recent years,
Streptococcus pneumoniae
(pneumococcus)-induced cardiac events have emerged as one of the most serious and life-threatening infection outcomes of invasive pneumococcal disease (IPD), leading to major adverse cardiac events (MACE) with high mortality rates.
S. pneumoniae
has the ability to invade myocardium and damage cardiomyocytes through the release of bacterial factors and formation of bacterial-filled cardiac microlesions. We previously found that polymorphonuclear cells (PMNs) or neutrophils are crucial for host defense against
S. pneumoniae
lung infection and that extracellular adenosine (EAD) production, by the exonucleosidase CD73, controlled the anti-bacterial functions of these cells. The objective of this study was to explore the role of PMNs and CD73 in cardiac damage during invasive pneumococcal infection. Upon intra-peritoneal (
i.p
.) injection with TIGR4, an invasive pneumococcal strain, C57BL/6 mice showed an increased influx of PMNs into the cardiac tissue as determined by flow cytometry. However, the increased PMN numbers failed to contain bacterial burden in the heart and showed a positive correlation with serum levels of the cardiac damage marker Troponin-1 as determined through ELISA. Depletion of PMNs prior infection reduced pneumococcal burden in the heart and lowered the Troponin-1 levels suggesting that PMNs induce cardiac damage during infection. While exploring the mechanisms underlying the detrimental PMN response, we found that the late stage of IPD was associated with reduced CD73 expression on PMNs. The role of CD73 in regulating cardiac damage was tested
in vivo
using CD73
-/-
mice which had significantly higher bacterial burden and cardiac damage compared to wild-type mice despite similar PMN numbers. The role of CD73 expression on PMNs was also tested
ex vivo
using the HL-1 cardiomyocyte cell line which showed increased LDH release upon
S. pneumoniae
infection in presence of CD73
-/-
PMNs, a phenotype reversed by supplementation of exogenous adenosine. Similar iincreased damage in presence of CD73
-/-
PMNs was seen with C57BL/6 Mouse Primary Cardiac Microvascular Endothelial Cells which lead to increased migration of beads across endothelium. Our findings suggest that dysregulation of CD73 on PMNs during invasive pneumococcal infection leads to an inability to control bacterial numbers and increased cardiac damage, and that the EAD-pathway can be a potential pharmacological target to limit disease severity.
Abstract We065: Sex Differences in Autoimmune Signatures After Myocardial Infarction in Mice
Authors: Mouton Alan , Taylor Erin , Mendoza Luciano , Aitken Nikaela , Sandifer Jayla , Raman Indu , Zhu Chengsong , Morato Jemylle , da Silva Alexandre , Docarmo Jussara , Omoto Ana Carolina , Wang Zhen , Li Xuan , Spitz Robert , Hall John
Published: October 2024

Introduction:
Myocardial infarction (MI) is caused by ischemic injury to the left ventricle (LV), leading to an inflammatory response, scar formation, and loss of LV function. The role of the autoimmune (AI) response during chronic MI, such as B cell activation and autoantibody production, is not well understood. As AI disease is much more prevalent in females, we hypothesized that female mice are more susceptible to MI-induced development of AI signatures.

Methods:
Permanent MI was induced by left coronary artery ligation in adult male and female C57BL/6J mice for 7, 28, or 56 days. LV function was tracked using echocardiography (VEVO 3100). Cytokines were measured by qPCR. LV IgG and IgM antibody deposition was measured by immunofluorescence. Plasma IgG and IgM were measured by ELISA, and plasma autoantibodies (AABs) (IgG and IgM subclasses) were measured with an autoantigen (AAN) array.

Results:
In both sexes, MI led to progressive LV hypertrophy, dilation, and wall thinning. MI increased spleen mass in D7, 28, and 56 females, but decreased spleen mass in D28 and 56 males. LV infarct cytokines (IL-1β, IL-6, IL-18, IFN-γ, TNF, CCL2, IL-10) were increased at D7, while IL-6 and IL-18 remained elevated at D28 and 56 to a higher degree in females. Several cytokines in the remote LV were increased in females relative to males, including TNF, IL-6, CCL2, and IL-18. Splenic cytokines were also differentially expressed between sexes, with IL-1β, IL-12a, IL-10, IFN-γ, IL-18, CCL2, and IL-4 increased in females at several time points. Infarct IgG and IgM deposition occurred in both sexes, but to a higher degree in females. Remote IgG and IgM deposition occurred at D56 in both sexes. For plasma AABs, we detected 34 AANs against which IgG AABs were significantly elevated (p<0.05) at D56 in females vs 9 in males, and 30 IgM AABs vs 1 in males. Of note, anti-IL-6 IgM AABs were elevated in D56 females (9.8±0.8-fold), likely due to persistently elevated IL-6 in the female infarct and remote LV vs males. Total levels of plasma IgM were increased only in females at D56 (1.79±0.15-fold).

Conclusions:
MI promotes development of AI signatures which is exacerbated in females, including increased spleen mass, LV and splenic cytokines, LV infarct and remote IgG and IgM antibody deposition, increased plasma IgG and IgM AABs, and increased total levels of plasma IgM. Targeting the AI response may be a viable option for treating chronic MI outcomes, particularly in females.
Abstract We066: Macropinocytosis is essential for macrophage activation in acute ischemia.
Authors: Wen Jinhua , Mangione mariasanta , Luo Xiang , Cao Dian
Published: October 2024

Macrophages (Mφ) play central roles in inducing the robust inflammatory response to acute ischemia and promoting anti-inflammatory actions necessary for myocardial repair. The broad range of functional repertoire stems from Mφ plasticity dictated by their interaction with the dynamic ischemic milieu. The limited understanding of such interactions contributes to the lack of means to mitigate inflammatory injury in MI patients. We hypothesize that the dynamic Mφ plasma membrane-mediated process macropinocytosis (MP) is essential for Mφ’s activation and function evolvement in ischemia. By MP, Mφs engulf large amounts of soluble contents from the ischemic environment, and the macropinosomes formed from MP become signaling hubs of DAMP (Damage-Associated Molecular Pattern)-PRR (Pattern Recognition Receptor). We aim to determine if ischemia activates MP in Mφs, the signaling pathways that MP promotes, and the role of this process in post-ischemia repair. The rationale is based on our data demonstrating
only soluble DAMP (sDAMP), not tissue debris, activates Mφs
. We tested our hypothesis using the murine MI model and bone marrow-derived Mφs (BMDM) and used unpaired t-tests for statistical analyses. We uncover that ischemia markedly increases MP in Mφs; blocking MP by the inhibitors or siRNA-mediated knock-down of the Na+H+ exchanger
abolished
the production of potent cytokines such as IL1β from Mφs
in vitro
and
in vivo
. Furthermore, MP activates NF-κB and MAP kinase triggered by sDAMP and ischemia, critical pathways to produce inflammatory cytokine and chemokine. Intriguingly, mTORC1 activation in BMDM and Mφs in the ischemic myocardium depends on MP. These data suggest MP organizes the activation signal and the pathway required for metabolic rewiring in inflammatory Mφs. Suppressing MP at the earlier stage of ischemia (72h) when sDAMP is abundant in the ischemic tissue significantly reduces mortality and improves cardiac function. In conclusion, we identify MP functionalizes the danger signals in bulk and integrates activation signals with metabolic rewiring to promote and sustain the inflammatory function. Targeting MP is better equipped to reduce inflammatory injury as it modifies the dynamic interaction of Mφs with the ischemic environment.
Abstract We074: Macrophages Endocytose the Extracellular Matrix Component Hyaluronan
Authors: Howard Caitlin , Pendergraft Emma , Belcher Sarah , Jones Steven
Published: October 2024

Background:
Following myocardial infarction, macrophages clear cellular debris during remodeling of the extracellular matrix (ECM). The presence of macrophages coincides with a significant increase in the ECM component hyaluronan (HA). HA is produced in copious amounts following injury, such as myocardial infarction, and HA may interfere with myocardial wound healing. Because macrophages express hyaluronan receptors and are known to facilitate ECM remodeling, it is possible that macrophages remove HA early after MI. The extent to which macrophages clear HA is not known.

Goal:
To determine the extent to which macrophages endocytose hyaluronan (HA).

Methods and Results:
Bone marrow derived macrophages (BMDMs) were polarized into a pro-inflammatory (M1) phenotype using LPS and INF-g, or pro-reparative phenotype (M2) using IL-4 and IL-13. In addition to the polarization factors, cells were treated with 5,000 ng/ml of a fluorescein-labeled HA or a vehicle for 24 hours. At the time of collection, cells were stained with trypan blue to quench surface fluorescence and flow cytometry was used to measure the uptake of labeled HA. An increase in cellular fluorescence intensity after HA treatment indicates the extent of endocytosis of HA. Mean fluorescence intensity (MFI) values were recorded and used to calculate the change in fluorescence between the treated and control groups (DMFI). A significant increase in DMFI was observed when both M1 and M2 macrophages were compared to naïve (M0) controls.

Conclusion:
Macrophages endocytose HA, and polarized macrophages endocytose HA at a greater level than naïve macrophages. Hence, polarized macrophages may play a role in clearing HA in the heart following injury. We speculate that enhancement of macrophage-mediated clearance of HA may improve acute scar formation following myocardial infarction.
Abstract We076: Humoral profile of individuals with Chagas Cardiomyopathy
Authors: Venturinida Silva Gabriela , Kula Tomasz , Bes Taniela , Shrock Ellen , Elledge Stephen , Gorham Joshua , Sabino Ester Cerdeira , Pereira Alexandre , Seidman Jonathan , Seidman Christine
Published: October 2024

Chronic Chagas cardiomyopathy (CCC) is the most severe manifestation of Chagas disease and one of the main causes of cardiomyopathy in Latin America. The number of individuals infected with Trypanosoma cruzi (T. cruzi), the causative parasite of Chagas disease, exceeds 6 million, with an increasing presence in non-endemic regions such as Europe, Japan, Canada, and the USA. While approximately 70% of infected individuals remain asymptomatic and are categorized as indeterminate (IND), the remaining 30% develop cardiac manifestations several decades after the initial infection. Despite extensive efforts to elucidate the triggers and predictors underlying the progression from IND to CCC, the exact causative factors remain elusive. Hypotheses have been proposed that focus on the specific humoral response, including the production of antibodies that effectively neutralize the parasite or demonstrate cross-reactivity between parasite and cardiac proteins. Here, using PhIP-seq technology we comprehensively characterized the IgG profile of 897 T. cruzi seropositive individuals. We identified 628 epitopes with increased IgG reactivity in chronic T. cruzi seropositive individuals. Mucin TcMUCII, trans-sialidase, and MASP emerged as the primary classes of proteins showing elevated IgG response. Remarkably, the serum of CCC individuals showed robustly elevated reactivity against 84 epitopes compared to IND individuals, while IgG response against 23 epitopes was increased in the serum of IND individuals. Trans-sialidase and mucin-like glycoproteins were the main classes of proteins distinguishing these two groups. Among all epitopes with increased reactivity in the serum of CCC individuals compared to IND, 36 epitopes showed linear sequence similarity to 210 human proteins highly expressed in human heart. These included sarcomere structural proteins, suggesting potential cross-reactions between the antibodies targeting T. cruzi epitopes and proteins with key roles in cardiac function. Overall, our study describes the humoral landscape of Chagas disease, expanding the pool of potential candidates for future vaccine development or as targets for infection/trials efficiency monitoring. Additionally, we identified potential sources of antibody cross-reaction that may contribute to CCC progression, including epitopes sharing sequence similarities with key proteins involved in cardiac structure.
Abstract We077: Mechanisms of T-lymphocyte Dysfunction in Dilated Cardiomyopathy Patients
Authors: Angelotti Austin , Bansal Shyam
Published: October 2024

Preclinical studies have shown that T-cells infiltrate the heart during heart failure (HF), promoting remodeling. However, it remains unclear whether activated T-cells infiltrate the human failing hearts also. Importantly, cellular mechanisms that are involved in T-cell activation during human HF are unknown. To address this question, we analyzed published single-cell RNA sequencing data from patients with dilated cardiomyopathy (DCM) and compared it with control hearts. Our analysis focused on characterizing the gene expression of cardiac T-cells to uncover potential mechanisms underlying their role in human HF. We found that patients with DCM had significantly more cardiac CD3+ T-cells (%CD45+ cells; 35.5±5.9 vs 22.0±0.8 p < 0.01), with gene signatures indicative of higher TCR dependent antigenic activation, proliferation, and exhaustion. Although there were no differences in the proportion of CD4+ helper and CD8+ cytotoxic T-cells between groups, patients with DCM had a higher proportion of activated (%CD45+ cells, 6.6±0.8 vs 3.1±0.3 p = 0.03) and proliferative CD4+ T-cells (%CD45+ cells, 8.0±0.8 vs 3.1±0.3 p < 0.01). Additionally, genes involved in TNF and TGFβ signaling were significantly enriched in cardiac T-cells derived from patients with DCM (normalized enrichment scores = 2.35, p
adj
< 0.01, and 1.72, p
adj
< 0.01, respectively),consistent with preclinical studies published by us and others. Recently, we also showed that dysfunctional T-cells exhibit increased estrogenic signaling with activation of Estrogen Receptor (ER)α in mice. Thus, we also looked at estrogen signaling in T-cells infiltrated into the DCM hearts and found that, indeed, cardiac T-cells in DCM patients exhibit significant upregulation of genes involved in estrogen signaling (normalized enrichment score = 1.69, p
adj
< 0.01). This comprehensive analysis, for the first time, shows that T-cells are antigenically activated, actively infiltrate the failing human hearts and exhibit increased estrogenic signaling during DCM, providing novel insights and potential avenues for targeted immunotherapies.
Abstract We085: Origin and fate of CX3CR1
+
cells in the heart: their unique contribution to cardiovascular cells
Authors: Cho Kyuwon , Andrade Mark , Bae Seongho , Lee Christine , Kim Sangsung , Kim Jin Eyun , Yoon Young-sup
Published: October 2024

Introduction:
During development, various cells intricately collaborate to form embryonic and fetal hearts. We can harness this naturally occurring generative mechanism to advance regenerative medicine for cardiac repair. Studies reported that C-X3-C Motif Chemokine Receptor 1 (CX3CR1)
+
cells play vital roles in cardiac development and disease. However, the origin and the fate of CX3CR1
+
cells during cardiogenesis remain unclear. Specifically, the existence of CX3CR1
+
cells before E8.5 and their commitment other than to macrophages have not been investigated.

Methods:
We investigated the origin and fate of CX3CR1
+
cells using various knock-in reporter mice with CX3CR1 drivers for spatiotemporal genetic lineage tracing of CX3CR1
+
cells. In addition, we developed a method to generate CX3CR1
+
cells from differentiating mouse embryonic stem cells (mESCs). mESC-derived CX3CR1
+
cells were characterized
in vitro,
in the fetal mouse heart
ex vivo
, and in the adult heart
in vivo
. Their heterogeneity and trajectory were analyzed via single cell RNA sequencing (scRNA-seq).

Results:
Genetic fate mapping of CX3CR1
+
cells identified epiblasts at E6.5, the parietal endoderm at E7.0, yolk sac cells at E8.0, and cardiomyocytes at E9.5. Temporal genetic labeling (Cx3cr1-CreERT2;R26-tdTomato) showed that CX3CR1
+
cells at E6.5 can contribute to cardiomyocytes (CMs) and endothelial cells (ECs) during prenatal development via both
de novo
differentiation and fusion with pre-existing CMs or ECs, respectively. In the adult heart, cells that have ever expressed Cx3cr1 comprised 13.5 ± 0.2% of CMs and 30.9 ± 1.3% of ECs. In addition, CX3CR1
+
cells, which were differentiated from mESCs generated CMs, ECs and macrophages
in vitro,
in the fetal mouse heart
ex vivo
, and in the adult heart
in vivo
. Single cell RNA sequencing showed that Cx3cr1
+
cells represent an intermediate cell population differentiating into the mesoderm from pluripotent stem cells.

Conclusions:
Our data demonstrate that CX3CR1 serves as a marker for a unique subset of progenitors that contribute to the formation of CMs and ECs as well as macrophages, providing new insights into the versatile role of CX3CR1
+
cells in cardiogenesis.
Abstract We090: Antagonistic and Cooperative Dynamics of
Irx3
and
Irx4
in Ventricular Compaction
Authors: Abid Rimshah , Oh Yena , Liu Wei Fan , Bakr Marwan , yin wen-chi , ZHOU YU-QING , Zhang Xiaoyun , Kim Riyoun , Mo Rong , Puviindran Vijitha , van Eede Matthijs , Henkelman Mark , Miquerol Lucile , Bruneau Benoit , Hui Chi-Chung , Kim Kyoung-Han
Published: October 2024

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart condition characterized by excessive trabeculae and intertrabecular recesses in the left ventricle of the heart. It is believed to be caused by abnormal trabecular compaction process, which involves trabecular remodelling to form a thick myocardial wall. Embryonic trabeculae consist of bipotent progenitors that differentiate into the ventricular conduction system (VCS) and contractile cardiomyocytes, yet it remains to be further understood how the pathways involved in trabecular cell specifications play a role in the compaction process. Here we present two transcription factors,
Iroquois
homeobox 3 (
Irx3
) and
Irx4
, playing antagonistic and cooperative roles during trabecular specification and maturation, thereby facilitating proper ventricular compaction. Our single-cell RNA sequencing analysis revealed that the differentiation of trabecular cells into VCS cells was accompanied by increased
Irx3
but decreased
Irx4
expression. In addition, we found that the loss of
Irx4
led to both an increased expression level of
Irx3
and an expansion in the number of
Irx3
-expressing cells. Conversely, the loss of
Irx3
resulted in an upregulation of
Irx4
expression within VCS cells. Next, to investigate the functional importance of the antagonistic relationship between
Irx3
and
Irx4
in trabecular differentiation, we generated
Irx3
and
Irx4
compound knockout (KO) mice and examined the structure and function of their hearts. Notably, unlike
Irx3
KO hearts showing normal ventricular myocardium,
Irx4
KO hearts exhibited mild LVNC, and
Irx3
and
Irx4
double knockouts (
Irx3
/4dKO) hearts displayed severe LVNC. Multiome sequencing of E14.5 ventricles revealed that
Irx3
/4dKO ventricles contained an increased number of trabecular cells, expressing higher levels of cell proliferation markers and altered trabecular identity genes, compared to littermate controls. Collectively, our results demonstrate that balanced antagonism between
Irx3
and
Irx4
is crucial for establishing trabecular identity. This highlights the importance of proper differentiation of trabeculae into the VCS and non-VCS contractile cells during ventricular compaction, thereby preventing noncompaction cardiomyopathy.
Abstract We091: Maternal hyperglycemia disrupts human cardiac cell lineage differentiation
Authors: Contreras Javier , Wang Cankun , Ye Shiqiao , Yu Yang , Alonzo Matthew , Ma Qin , Zhao Mingtao
Published: October 2024

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.
Abstract We097: Dual Targeting CXCR4 and CXCR7 Prevents Doxorubicin-Induced Cardiotoxicity by Eliminating Cardiac Inflammation and Preserving Cardiomyocyte Function
Authors: Hui Xiaodan , Gu Chunjie , Abdelbaset-Ismail Ahmed , Xu Jianxiang , Tse William , Raj Ashok , Deng Zhongbin , Zhu Wuqiang , Cai Lu , Tan Yi
Published: October 2024

Background:
Doxorubicin (DOX)-induced cardiotoxicity (DICT) has become a serious health burden for cancer survivors. The detrimental effects of CXCR4-mediated excessive inflammation in myocardium and the protective effects of CXCR7 in cardiomyocytes have been demonstrated.

Aims:
We tested the protective effects against DICT of a dual-targeting compound TC14012, which has specific antagonistic activity against CXCR4 and potent agonistic activity on CXCR7.

Methods:
Eight-week-old male C57BL/6J mice were injected with DOX for consecutive 5 weeks, during which mice were treated with TC14012 twice a week. One week after the last dose injection, mice were euthanized after heart function examination. Markers of cardiac inflammation and fibrosis were evaluated. Cardiac RNA-seq was performed to explore the underlying molecular pathways.

Key findings:
TC14012 supplement significantly prevented DOX-induced cardiac systolic dysfunction, reflected by improved ejection fraction and fraction shortening, along with remarkable attenuation of DOX-induced cardiac perivascular and interstitial collagen deposition and neutrophils and macrophage infiltration. RNA-seq revealed that TC14012 prevention of DICT was accompanied with a remarkable stimulation of signals involving in cardiac contraction and energy reserve metabolic process, particularly glycogen and glucan metabolism that are crucial for meeting the cardiac energy demand in response to DOX induction. These signals could be further enriched into AMPK signaling pathway and validated by upregulation of AMPK phosphorylation. Besides, TC14012 prevention of DICT was accompanied with a significant reduction in cardiac mast cell activation and degranulation. These signals could be further enriched into Rap1 signaling pathway, and TC14012 was validated to inhibit Rap1 activity and mast cell activation. Most importantly, TC14012 treatment did not affect the suppressive effects of DOX on tumor growth in a syngeneic EL4 lymphoma-bearing, immunocompetent mouse model.

Conclusions:
We preliminary conclude that TC14012 prevents DICT by activating cardiac CXCR7 to reprogram cardiomyocyte metabolism and prevent cardiac damage and dysfunction via activating AMPK, and by antagonizing CXCR4 to inhibit cardiac mast cell infiltration and activation via blunting Rap1 activity but does not affect the anti-tumor activity of DOX.
Abstract We098: Target-Specific Novel Therapeutic Approach for Prevention of Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia
Authors: Bavishi Shreya , Meadows Stryder
Published: October 2024

Background:
Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disease with no cure. HHT is characterized by abnormalities in blood vessels that occur in mucocutaneous tissues (telangiectasias) and deep tissues (arteriovenous malformations, AVMs). The tangled, enlarged, fragile, and aberrant vasculature in HHT patients is prone to chronic bleeding or hemorrhage and may cause serious complications, especially in the brain.

Problem Statement:
While diagnosed AVMs can be surgically treated, there are no FDA-approved drugs to prevent or manage them. Developing targeted therapeutics to manage AVMs has been incredibly challenging as the mechanism of AVM formation is not clearly understood.

Rationale:
Dysregulation of pro-angiogenic signaling pathways is considered to play a major role in AVM development. For instance, VEGFA-VEGFR2, ANG2–TIE2 and MAP Kinase 1/2 signaling have been implicated individually in HHT pathogenesis. While MAPK 1/2 signaling is difficult to target because of its universal presence, its upstream activators VEGFA and ANG2 are lucrative targets for their relative specificity primarily affecting vasculature (Figure 1).

Innovation:
We use a novel approach to target VEGFA and ANG2 simultaneously using IgG CrossMab bispecific inhibitor, allowing superior efficacy by inhibiting dual specific targets to prevent genetic vasculature abnormalities.

Approach:
We use vascular latex casting and molecular quantification techniques to assess the benefits of inhibiting abnormal pro-angiogenic signaling in HHT mice models. Our preliminary data shows improved vasculature development with small doses of MAPK 1/2 inhibitor, but early mortality due to its damaging side effects. Preliminarily, the combined effect of the bispecific inhibitor in smaller doses improves the vasculature defects in the mutant brain more robustly than the individual inhibitors alone, while not negatively affecting the wild-type model (Figure 2). We further expect this dual inhibition to normalize the transcriptomic signature of mutants to mimic the wild-type setting.

Societal Impact:
The expected beneficial effects of the bispecific inhibitor will provide a pre-clinical assessment of the cerebrovascular AVM management strategies for 1.4 million HHT patients. It will further the mechanistic understanding of AVM development and prevention in HHT, the 2
nd
most common bleeding disorder.
Abstract We101: Targeted gene silencing with small interfering RNA-loaded lipid nanoparticles to treat cardiac fibrosis
Authors: Liu Mengrui
Published: October 2024

Bromodomain-containing protein 4 (BRD4) is an effector of transforming growth factor-β (TGF-β) signaling. Upon injury, BRD4 stimulates the differentiation of quiescent cardiac fibroblasts into activated fibroblasts. These activated fibroblasts unfavorably remodel the myocardium by depositing excess extracellular matrix. Here, we reported lipid nanoparticles (LNPs) loaded with small interference RNA against BRD4 (siBRD4) to deactivate fibroblasts after cardiac injury. Because fibroblast activation protein (FAP) is a biomarker of activated fibroblasts, we conjugated anti-FAP antibodies on the LNPs for targeted delivery to activated fibroblasts. Systemic administration of these targeted siBRD4-loaded LNPs inhibited fibroblast activation, reduced fibrosis, and improved cardiac functions in rodent models of myocardial infarction and cardiac fibrosis. Single-nucleus RNA sequencing and cytokine analysis revealed that this therapy further converted the pro-fibrotic and pro-inflammatory microenvironment to a pro-resolution and pro-regeneration one. The safety and toxicity of this nanomedicine were tested in nonhuman primates.
Abstract We109: AAV9-Mediated Overexpression of Mixed Lineage Kinase 3 After Transaortic Constriction Improves Left Ventricular Function
Authors: Pande Suchita , Martin Gregory , Blanton Robert
Published: October 2024

Introduction:
Mixed-lineage kinase 3 (MLK3) is a cGMP-dependent protein kinase 1α (PKG1α) substrate, preserving left ventricular (LV) function without affecting blood pressure (BP). MLK3 kinase-dependent effects maintain LV function, while kinase-independent signalling regulates BP. Thus, enhancing MLK3 kinase activity may improve LV function in heart failure (HF) while circumventing PKG1-induced hypotension. No MLK3-activation strategies exist currently, thus hindering progress in MLK3 research and potential therapeutics.

Hypothesis:
Augmenting MLK3 after pressure overload by transaortic constriction (TAC) would prevent LV hypertrophy and dysfunction in mice.

Goal:
To test the effect of adeno-associated virus 9 (AAV9)-mediated MLK3 overexpression on LV function, when administered in the setting of pre-existing LV hypertrophy and dysfunction.

Methods:
Male mice 12 weeks of age underwent 26G TAC or sham surgery. At day 5 post-TAC we administered by retro-orbital injection AAV9 encoding MLK3 or TdTomato negative control, each under control of the CM-specific cTNT promoter (AAV-MLK3: n=12 TAC, 7 sham; AAV-TdTomato: n=10 Tac, 8 sham. At day 28 post-AAV9 injection, we measured cardiac structure and function by echocardiography, invasive LV hemodynamics, and organ weights.

Results:
Echocardiography 5 days post-TAC confirmed TAC-induced LV hypertrophy and reduced systolic function prior to AAV9 treatment. At day 28, AAV-MLK3-treated TAC mice demonstrated improved LV ejection fraction and fractional shortening compared with TAC-AAV-Td controls, indicating improved systolic function. MLK3-treated TAC mice maintained higher LV systolic pressure post-TAC vs. Td controls, further indicating improved cardiac performance. Diastolic indices, including ratio of mitral E/A inflow velocities, tau, and LV relaxation rate were also improved in AAV-MLK3 TAC versus AAV-Td controls. AAV9-MLK3 administration had no effect on LV wall thickness or on LV or whole heart mass normalized to tibia length. MLK3 expression was increased 3-fold in AAV9-MLK3 TAC LVs compared with AAV9-Td controls.

Conclusion:
AAV9-mediated MLK3 overexpression after the onset of LV hypertrophy and dysfunction improves LV function in mice.
Abstract We115: Modified mRNA Therapeutics Inhibits Cardiomyocyte Apoptosis and Induces Cardiac Protection in Ischemic Heart Diseases
Authors: Magadum Ajit , Mallaredy Vandana , Roy Rajika , Gurrala Charan Thej , Joladarashi Darukeshwara , Cimini Maria , Gonzalez Carolina , Truongcao May , Benedict Cindy , Kishore Raj
Published: October 2024

Introduction:
Cardiovascular diseases are the leading cause of mortality worldwide. After myocardial infarction (MI), there is a permanent loss of cardiomyocytes (CMs), and as the mammalian heart has limited regenerative capacity, it leads to Heart Failure. Extracellular vesicles (EVs) derived from induced pluripotent stem cells (iPSCs) have emerged as potential therapeutic agents, yet their precise mechanism of CM survival and cardiac repair remains elusive.

Hypothesis:
The myocardial delivery of hiPSC-EV-specific protein improves cardiac function and mice survival post-MI by inhibiting CM apoptosis and oxidative stress molecular pathways.

Methods and Results:
Our preliminary studies confirmed that hiPSC-EVs promote CM survival post-MI in mice, and through proteomic analysis, we identified a novel protein uniquely expressed in hiPSC-EVs. We showed that the myocardial delivery of hiPSC-EVs specific protein in the form of modRNA inhibited CM apoptosis and induced CM survival post-MI. This increase in the CM's survival by hiPSC-EVs specific protein expression was associated with reduced scar size, improved cardiac function, and mice survival 28 days post-MI. Furthermore, using the siRNA and small molecule inhibition of hiPSC-EVs specific protein, we found enhanced MI-induced CM apoptosis post-MI. Mechanistic insights reveal the hiPSC-EVs specific protein induced STAT3 pathway, while STAT3 pathway inhibition by small molecule repressed hiPSC-EVs specific protein induced CM survival
in vitro
. Additionally, our preliminary study shows this protein translocates to the nucleus post-MI or ischemic injury.

Conclusion:
Taken together, the myocardial injection of hiPSC-EVs specific protein through a highly therapeutic modRNA tool improves cardiac function by inhibiting CMs apoptosis, and reactivating cardiac repair post-injury. Moreover, our studies demonstrate that the modRNA (protein) approach in mouse models is potentially adaptable for mRNA therapeutics in a clinical setup.
Abstract We123: Cardiomyocyte Specific Sustained Atg7 Overexpression Activates Mitochondrial Autophagy in the Heart
Authors: Abdullah Chowdhury S. , Aishwarya Richa , Remex Naznin Sultana , Orr Wayne , Sadoshima Junichi , Bhuiyan Md Shenuarin
Published: October 2024

Introduction:
Accumulating evidence indicates that autophagy-related protein 7 (Atg7) is essential in cellular autophagy. Our earlier studies showed that Atg7 is required to promote macro-autophagy in the heart. However, it is unknown if the activation of Atg7 has a role in the heart to regulate selective mitochondrial autophagy (mitophagy).

Objectives:
We employed
in vitro
and
in vivo
approaches to delineate the molecular function and mechanisms of Atg7-mediated activation of mitophagy in the heart.

Methods and Results:
Subcellular fractionation and quantum dot-labeled electron microscopy revealed Atg7’s localization on the mitochondrial fraction in the heart. Atg7 co-localization to Mitotracker-labeled mitochondria was confirmed using isolated heart mitochondria. To quantitate the mitophagy, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye to enumerate mitophagy dots by microscopy. We found significantly increased mitophagy dots in Atg7 overexpressed cardiomyocytes than in β-gal expressing control. To assess the activation of mitophagy
in vivo
, we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria from Atg7 Tg mice hearts post-CCCP treatment. To assess the extent of mitochondrial lysosomal delivery in Atg7 overexpressing hearts, we crossed αMHC-Atg7 Tg mice with cardiac-specific Mito-Keima transgenic (Mt-Keima Tg) mice. Mt-Keima Tg mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the measurement of mitophagy activation
in vivo
. We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to Mt-Keima mice.

Conclusions:
Our biochemical and genetic approaches revealed that Atg7 localizes to the mitochondria, leading to the recruitment and initiation of mitophagy machinery. Our data further confirm that genetic activation of Atg7 leads to increased mitophagy activity in the heart.
Abstract We125: HIV-1 Nef protein induces aging and cardiac dysfunction through autophagy dysregulation
Authors: Kondrachuk Olena , Gupta Manish
Published: October 2024

Background:
Despite highly active antiretroviral therapy (ART), HIV-associated cardiovascular disease and accelerated aging cause increased mortality in people living with HIV/AIDS (PLWHA). Nef protein has been detected in HIV patients even with ART therapy. We hypothesized that the Nef protein may compromise heart function and accelerate aging by dysregulation of autophagy.

Methods:
We generated a Nef transgenic (Nef-TG) mouse model by cloning the HIV-Nef gene in the CAG-Lox-CAT vector. We used Western blotting to determine protein expression in cardiac tissue. Heart function was evaluated by echocardiography. Nef protein function was also determined by plasmid DNA transfection in the HEK293 cells.

Results:
Nef-TG mouse shows cardiac dysfunction, increased cardiac fibrosis, and early mortality. Expression of autophagy marker LC3II was significantly decreased in the heart of Nef-TG mice. The data suggest Nef inhibits autophagy in the cell. Mechanistically, we found that senescence-associated markers p21, Bcl2, and Beclin1 levels significantly increased in Nef-TG mice's hearts, which may negatively regulate heart function and promote aging.

Conclusion:
Our new Nef-TG mouse model revealed the cause of HIV-associated aging in PLWHA. Our mouse model can also be used to elucidate the role of Nef protein on other organ systems and test novel therapeutics for HIV.
Abstract We127: Infective Endocarditis in COVID-19 - An Unusual Suspect
Authors: Masi Sean , Venugopal Darshine , Delvecchio Stefanie
Published: October 2024

Introduction:
The COVID-19 pandemic globally overwhelmed healthcare systems and the complications associated with this illness have contributed to poor outcomes. The hypercoagulable state associated with COVID-19 is well recognized in the literature. Thromboembolic complications such as left ventricular thrombus (LVT), deep venous thrombosis (DVT) and pulmonary embolism (PE) have been increasingly reported. Dhakal et al. published an association between COVID-19 and cardiovascular disease, including possible complication by infective endocarditis (IE) which was confirmed by Amir et al. reporting the first case. Since then, there have been few reported cases. We present a case of COVID-19 in a patient who developed LVT, complicated by non-valvular IE by an underreported organism, Granulicatella adiacens.

Case Presentation:
A 55-year-old male with history of dental caries, provoked DVT/PE, NSTEMI x2 and STEMI in-stent restenosis requiring thrombectomy presented to the hospital with a 2-day history of shortness of breath, fatigue, cough, change in mentation and right-sided weakness. He tested positive for COVID-19 by NAAT. CXR revealed cardiomegaly and multiple bilateral infiltrates. CT head demonstrated acute ischemic stroke of the left occipital, parietal and posterior temporal lobes, also confirmed on MRI. CTA neck showed 30% stenosis of left ICA. Blood cultures x2 resulted positive for G. adiacens. Echocardiogram was obtained, demonstrating reduced EF of 35% and the presence of a large, 2.7 x 2.2cm mobile thrombus in the apical-septal region of the left ventricle, not visualized on previous echocardiogram 6 months earlier.

Methods:
Data was collected from direct patient care and electronic medical records.

Discussion:
G. adiacens is a fastidious gram-positive coccus, part of the nutritionally variant streptococci (NVS) species, and part of the normal oral and gastrointestinal flora. It is also found in dental plaques and endodontic abscesses. NVS account for less than 5% of cases of bacteremia and IE. G. adiacens account for even fewer cases. Two separate meta-analyses conducted by Quintero-Martinez et al. and George et al. failed to reveal any cases of IE attributable to G. adiacens in the setting of COVID-19 infection.

In conclusion, we believe that the hypercoagulable state of COVID-19 infection led to endocardial damage and development of LVT, thereby creating a nidus for microorganism adherence from underlying dental carries resulting in thromboembolic stroke.
Abstract We128: Effect of Coronavirus Disease 2019 on the Incidence of Stress-induced Cardiomyopathy; A Single Center Experience
Authors: Mohammed Fawaz , Ali Khan Zaina , Mohammed Muna , Gubitosa James , H. Cox Anna , D. Kamaluldeen Ahmed , A. Nour Aisha , Long Paige , M. Osborn Amanda , Howard Kathryn , Sedhai Yub Raj , Saleem Sameer , AMIN AKHTAR , Dawson Dowe Jacqueline , Abdul Waheed Mohammad , Akbar Muhammad , Kazimuddin Mohammed
Published: October 2024

Introduction:
Takotsubo Syndrome, or stress-induced cardiomyopathy (SIC) is a transient dysfunction of the left ventricle. Emerging data has shown a rise in the number of cases in recent times. In January of 2020 the Centers for Disease Control and Prevention (CDC) declared Coronavirus disease 2019 (COVID-19) a public healthcare emergency. Given the impact of the COVID-19 on economic and psychosocial factors in individuals across the globe, we sought to study the effect of COVID-19 on the incidence of SIC at our institution.

Research Question:
Has economic and psychosocial stress associated with COVID-19 led to an increase in the incidence of stress-induced cardiomyopathy?

Aim:
To study the impact of COVID-19 on the incidence of stress-induced cardiomyopathy.

Methods:
A retrospective review of cardiac catheterization laboratory data was performed from January 2011 to August 2022. We identified a total of (n=121) cases of stress induced cardiomyopathy. Diagnosis was adjudicated based on the findings on cardiac catheterization, left ventriculography, and echocardiography. Clinical and demographic data of interest was obtained from individual patient electronic medical records into a secure database management tool. Additional data of interest was obtained by performing a post-discharge questionnaire via a telephone survey.

Results:
The mean age of the population was 69 years (SD± 11.5). 93% (112) of the study population were females. A preceding physical stressor as a triggering factor was present in 59% (72) of individuals, 25% (31) individuals reported a preceding emotional stressor. An increase in the number of cases of SIC was noticed from January of 2020. Only 11% (8) of the patients who were admitted from Janurary 2020 had COVID-19 [Figure 1]. 36% (44) patients participated in the survery. Patients graded their disability from SIC as the following: Mild in 11% (4) , moderate in 7% (3) and severe in 2% (1) of the individuals [Figure 2]. All the patients were referred to Cardiac Rehabilitation at the Medical Center; a American Association of Cardiovascular and Pulmonary Rehabilitation (AACVPR) certified program that provides patients with educational, preventional and rehabilitative resources.

Conclusion:
A rise in the incidence of SIC was noted in our single center retrospective study when the CDC declared COVID-19 a healthcare emergency. This suggests an indirect economic and psychosocial stress related process from COVID-19; the "Long COVID" that is not talked about.
Abstract We129: Endothelium-Targeted MiR-122 Inhibition Improves Vascular Endothelial Function In a High-Fat Diet Fed mice
Authors: Gaddam Ravinder , Dhuri Karishma , Pathuri Mounika , Amalkar Veda , KIM YOUNG-RAE , Jacobs Julia , Sali Veeresh Kumar , Nakuluri Krishna , Irani Kaikobad , Bahal Raman , Vikram Ajit
Published: October 2024

Background:
MicroRNA-122-5p (miR-122), a liver-specific miR, levels are increased in the blood during obesity and diabetes and is taken-up by the extra-hepatic tissues including vascular endothelium. Despite miR-122’s role in maintaining hepatic homeostasis, its uptake (from circulation) by other tissues disrupts their function and increases the risk of cardiovascular disorders. Hence, inhibiting extra-hepatic miR-122 is a viable approach to mitigate vascular disorders associated with diabetes.

Hypothesis:
Endothelium-targeted miR-122 inhibitor selectively targets miR-122 in endothelial cells and improves vascular endothelial function in high-fat-diet (HFD) fed pre-diabetic mice.

Methods:
To selectively inhibit miR-122 in the endothelial cells, we synthesized gamma-peptide nucleic acid miR-122 inhibitor (γP-122 I) tagged with vascular cell adhesion molecule 1 (VCAM1) targeted peptide (e-γP-122 I). The purity was assessed using HPLC. Pre-diabetic condition was induced by feeding mice with a HFD (for 8 weeks) and e-γP-122-I was injected to the HFD-fed mice (2 weeks after dietary intervention; 5 mg/kg/day, i.p., 6 weeks). MiR-122 expression was measured by real-time PCR. The vascular endothelial function was assessed by determining endothelium-dependent and independent relaxation of phenylephrine-induced precontracted aortic rings. Results were expressed as mean ± s.e.m.

Results:
The e-γP-122-I was >95% pure. The HFD-fed mice show higher levels of serum and aortic miR-122 expression and impaired vascular endothelial function. e-γP-122-I treatment selectively inhibited aortic miR-122 expression while it has no effect on other organs (heart, kidney etc.) and improved vascular endothelial function and inflammation.

Conclusions:
e-γP-122-I selectively inhibits miR-122 in the endothelium and improves endothelial function in pre-diabetic mice.