bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3078: The Stimulator Of Interferon Genes (STING) Modulates Adverse Cardiac Remodeling Induced By Cardiac Pressure Overload: New Role Of Endothelial Cell Sting In Systolic Dysfunction
Authors: Sanders Erin , Wagner Noah , Smolgovsky Sasha , Bayer Abraham , Theall Brandon , Aronovitz Mark , Kaur Kuljeet , Alcaide Pilar
Published: October 2023

Introduction:
Cardiac inflammation is a hallmark of heart failure (HF) defined by T cell infiltration and increased expression of pro-inflammatory cytokines. Type I Interferon (IFN-I), a cytokine induced upon activation of STING, is increased in the heart after myocardial infarction and transverse aortic constriction (TAC). We reported that absence of endothelial cell (EC) STING results in an IFN-I dependent decline in T cell extravasation to inflammatory sites. We hypothesized that STING contributes to adverse cardiac remodeling induced by TAC and EC STING regulates systolic dysfunction through modulating cardiac inflammation.

Methods:
Male wildtype (WT), STING
-/-
and inducible EC specific STING
-/-
mice (Cad5
ERTCre2+/-
STING
fl/fl
treated i.p. with 85mg/kg Tamoxifen), or oil treated and Cre
-
littermate WT controls were subjected to TAC or Sham surgery. Systolic function by echocardiography and left ventricular (LV) hypertrophy, fibrosis, and capillary density by histology were analyzed four weeks post-surgery and T cell activation was characterized in mediastinal draining lymph nodes (MdLN) by flow cytometry.

Results:
In contrast to WT, STING
-/-
mice did not show a decline in ejection fraction (EF). LV hypertrophy was decreased in TAC STING
-/-
mice compared to TAC WT mice. Isolectin staining showed that capillary density was higher in STING
-/-
mice in response to TAC as compared to the expected capillary loss observed in WT TAC mice. The frequency of CD4+CD62
lo
CD44
hi
cells in the MdLN, indicative of effector T cell activation, was decreased in STING
-/-
mice compared to WT mice in response to TAC. Similar analyses of Cad5
ERTCre2+/-
STING
fl/fl
mice showed that EC STING
-/-
mice did not show a decline in EF in response to TAC, in contrast to their littermate WT controls. However, no changes were observed in LV hypertrophy, fibrosis or capillary density between groups and activation of MdLN effector T cells was also comparable in both groups.

Conclusion:
STING contributes to adverse cardiac remodeling induced by TAC. While EC STING contributes to systolic dysfunction through a yet unknown mechanism, STING in other cell compartments contributes to cardiac hypertrophy, capillary density and T cell activation in response to TAC.
Abstract P3088: Hexokinase 3 Regulates Macrophage Polarization And Cardiac Remodeling Likely Through Serine Metabolism
Authors: Mahmoodzadeh Amir , Keykhaei Mohammad , Akrami Ashley , Spencer Duncan Spencer , Ardehali Hossein
Published: October 2023

Myocardial infarction (MI) is associated with activation of macrophages in the myocardium. Hexokinase 3 (HK3) is the mainly expressed hexokinase in macrophage. Along with its role as the first enzyme in glycolysis, HK3 has shown to be involved in the regulation of diverse immune system activities. In this study we studied the role of HK3 in the regulation of macrophage polarization into inflammatory phenotype (termed as M1) and anti-inflammatory and repair phenotype (termed as M2). Using CRISPR-Cas9 technology, we generated mice with deletion of exons 2 and 3 of the Hk3 gene, which are needed for the function of the protein. Bone marrow-derived macrophages (BMDM) isolated from Hk3
-/-
mice displayed higher levels of M2 markers, including Arg1, Ym1, Mrc1, PD-L2, IRF4, Mgl2, and Mgl1 compared to BMDMs from wild-type mice. Also, we found that pSTAT6 protein, was higher in BMDMs from Hk3
-/-
mice. Furthermore, there was a higher oxygen consumption rate and lower extracellular acidification rate in Hk3
-/-
BMDMs. Steady-state metabolomics in BMDMs isolated from WT and Hk3
-/-
mice showed that there was a significant increase in metabolites in the serine biosynthesis pathway (SBP), indicating that glucose metabolism is more shunted into the SBP with Hk3 deletion. The removal of serine/glycine from media resulted in a significant decrease in the M2 markers in wild-type BMDMs; However, higher M2 markers were observed in Hk3
-/-
BMDMs. This result indicated that the SBP is needed for the polarization of macrophages into the M2 subtype, and HK3 mediated M2 polarization is likely through the SBP. We then subjected Hk3
-/-
to LAD and assessed cardiac function after seven days using echocardiography. Result demonstrated that both male and female Hk3
-/-
mice display improved cardiac function, as assessed by measurement of ejection fraction (EF), fractional shortening (FS), and interventricular septum thickness in diastole (IVSd). Our results demonstrate that HK3 is a novel regulator of macrophage polarization into the phenotype with more anti-inflammatory and tissue repair characteristic. This study is clinically important by providing a potent novel approach to the treatment of ischemic heart disease.
Abstract P3090: The Role Of Circular Cdr1as In Macrophage Cardiac Inflammation And Fibrosis
Authors: Gonzalez Carolina , Cimini Maria , Benedict Cindy , Mallaredy Vandana , Trungcao May , Rajan Sudarsan , Garikipati Venkata N , Kishore Raj
Published: October 2023

In response to cardiac injury, macrophages are recruited in high numbers to the infarcted myocardium and undergo phenotypic switching from pro-inflammatory in the early stage to anti-inflammatory in the later stage. Thereby, controlling the initiation, maintenance, and resolution of the inflammatory response. Circular RNAs are newly discovered non-coding RNA generated from protein-coding genes that implicated in the regulation of immune cells. However, there are currently no reports on the role of circRNAs in macrophage biology and function during cardiac ischemia. We aim to determine the specific role of circ-CDR1as in macrophage class switching and how circ-CDR1as modulates function post-cardiac injury. We performed circular RNA microarray analyses to assess circRNA transcriptome changes using RNA isolated from bone marrow derived macrophages (BMDM) polarized to a M1 or M2 phenotype. We validated circRNA expression by RT-qPCR in 3D post-MI hearts and within specific cell types. We generated circCdr1as overexpression plasmid transfected into BMDM, changes in phenotype were determined by FACS and RT-qPCR analysis. We generated circ-CDR1as overexpression associated adenoviral vector plasmid under control of CMV promoter and administered by tail vein injection. LV function was determined by echocardiography. Macrophage phenotype and CD31 was determined by immunohistochemistry and fibrosis by masson trichrome staining. Circ-CDR1as is downregulated in M1 macrophages and upregulated in M2 macrophages. There is a downregulation of cdr1as in cardiomyocytes and macrophages (F4/80+) 3 days post MI, during which pro-inflammatory macrophages are dominant. Administration of AAV9 circCDR1as
in vivo
significantly improved %EF and %FS at 21 and 28D days post-MI. Masson trichrome staining showed significant decrease in fibrotic area. Additionally, there is an increase in CD206+ cells at the border zone/infarcted area. This study suggests that circ-Cdr1as plays a role in macrophage biology by upregulating anti-inflammatory markers to promote an M2 phenotype and exogenous gene delivery of circCDR1as may improve LV function over time. Therefore, circ-Cdr1as may potentially be a therapeutic for the resolution of inflammation after cardiac injury.
Abstract P3093: Early Myocarditis In The Desmoglein 2 Mutant Mouse Model Of Arrhythmogenic Cardiomyopathy
Authors: Valdez Steven , Taylor Lindsey , Rosenzweig Rachel , Bansal Shyam S , Palatinus Joseph
Published: October 2023

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease caused by mutations in desmosome proteins. Patients with ACM are born with normal hearts but develop arrhythmias, fibrofatty infiltration, and sudden death. The desmoglein 2 (DSG2) mutant mouse is a well-established model of ACM that recapitulates the human phenotype of the disease. This mouse develops severe ventricular arrhythmias, systolic dysfunction, myocardial fibrosis and exercise induced sudden death. Here, we present evidence of early myocardial immune cell infiltration as well as an inverse relationship between cardiac function and adult myocardial cytokine expression in DSG2 mutant mice. We examined 20 week old adult DSG2 homozygote mice and wildtype littermate controls for systolic function using echocardiography, cytokine expression using rtPCR and fibrosis using masons trichrome staining. Consistent with previous studies we found ventricular fibrosis is significantly increased in mutant hearts (p=0.003). We demonstrate that osteopontin, an inflammatory cytokine released by macrophages, is highly expressed in these hearts (p=2.0x10
-4
). We detected an inverse correlation between systolic function and the osteopontin expression levels in these mutant mice (p=0.0015, r=-0.8315, R
2
=0.6914). These results lead us to suspect that immune cell infiltration may contribute to the disease progression. Subsequently, we examined immune cell populations in 3 week old DSG2 hearts, a period of time before the development of fibrosis, systolic dysfunction or arrhythmias. We measured >40-fold higher number of CD45
+
leukocytes (with increased Ly6G
+
neutrophils and Ly6C
+
monocytes) within the heart muscle of DSG2 homozygotes via flow cytometry (p=7.4x10
-5
), consistent with myocarditis. These results suggest myocarditis precedes fibrosis in the DSG2 mutant mouse model. Our ongoing work is focused on further characterizing the immune response in early post-natal life in the DSG2 mouse heart and determining whether early myocarditis contributes to the phenotype in ACM. Our results highlight potentially novel therapeutic targets to prevent disease progression in ACM.
Abstract P3094: Cell-autonomous Immune Signaling In Human Cardiac Fibroblasts Modulates Fibrosis
Authors: Pillai Indulekha C , Latha Anisha , Vijayakumar Gayathri
Published: October 2023

Cardiac fibrosis is the pathophysiological hallmark of injury in an adult heart. Immune response in the injury niche modulates fibrosis. However, how cell-autonomous immune signaling in cardiac fibroblasts modulates fibrosis is unknown. In this report, we demonstrate that cardiac fibroblasts express TLR4 receptors, and inhibition of TLR4 signaling inhibits fibrotic changes such as proliferation and myofibroblast conversion in human cardiac fibroblasts. Using FACS, and single-cell data of cardiac fibroblasts from Colagen1 GFP mouse, we demonstrate that TLR4 is the predominant TLR and approximately 90 % of human cardiac fibroblasts express TLR4 receptors. Using EdU labeling, cardiac fibroblasts taking up EdU have reduced from 91.65 ± 3.89% from control conditions to 71.15 % ± 2.05 % (mean ± SEM, n=3, p< 0.05) in TLR4 inhibited condition. Ki67 staining shows a similar reduction from 93.1 ± 2.34 % to 59.3% ± 3.54%. TLR4 inhibition in TGF-β treated human cardiac fibroblasts reduced the expression of Periostin expression by 2.6 ± 0.58 and Sma1 by 1.46-± 0.18(mean ± SEM, n=3)fold. Further, migration of cardiac fibroblasts demonstrated a 76.3± 10.23 % (mean ± SEM, n=3, p< 0.05) reduction in the presence of TLR4 inhibitor. Transcriptome analysis of TLR4-inhibited cardiac fibroblasts shows downregulation of proliferation-related genes and pathways. Ingenuity pathway analysis (IPA) of differentially expressed genes in TLR4-inhibited TGF-β -treated cells compared to TGF-β alone shows a reduced expression of a subset of target genes of TGF-β signaling. Further, compared to healthy controls, single-cell data of heart failure patients show significant elevation of Histone 3 and Histone 4 in the fibroblast cluster along with chromatin readers, Brd4 and Nox4. Inhibition of TLR4 in human cardiac fibroblasts reduced the expression of Histone 3 and Histone 4, with Nox4 modulating the chromatin architecture required for fibroblast activation. Thus our data demonstrate that cardiac fibroblast act as an immune cell having cell-autonomous immune signaling, regulating fibrotic changes. Ongoing experiments in 3D human cardiac organoid injury models will reveal how TLR4 signaling regulates fibrotic activation of human cardiac fibroblasts in a heterogeneous system.
Abstract P3096: Nitric Oxide Supplementation During Cardiac Arrest Induces Inflammation And Apoptosis In Rat Hearts
Authors: Salama Abou Bakr M , Linardi Daniele , Mani Romel , Tessari Maddalena M , Ou Qinghui , El Sady Bahaael Din , Shaker Ahmed M , Abdulsaboor Ahmed , Gebreil Ahmad , Abouleisa Riham , Luciani Giovanni Battista
Published: October 2023

Background:
Neuroprotection during cardiac and aortic arch surgeries has been a topic of extensive concern. Several protocols were developed in the trial to achieve the best neurological outcome; amongst them is nitric oxide (NO) supplementation through extracorporeal membrane oxygenation (ECMO). While NO was shown to influence the neurological outcome when supplemented during cardiac surgeries, a little is known about the cardiac effects. The current study addresses the molecular effects associated with NO supplementation on the myocardium.

Methods:
Male Sprague Dawley rats (n=20, 450-550gm) were anesthetized and cooled with cardiopulmonary bypass (CPB) to a core temperature of 19±1
o
C and maintained in hypothermic cardiac arrest for 30 minutes. Then, rats were rewarmed to a target temperature of 35
o
C for 60 min using CPB without (control group, CPB), or with NO 20 ppm supplementation (experimental group). By the end of experiment, hearts were harvested and analyzed.

Results:
RNA sequencing analysis showed NO supplementation results in the differential expression of more the genes related to lipid metabolism, oxidative phosphorylation, inflammatory signaling, proteasome formation, autophagy, calcium handling, smooth muscle relaxation, and apoptotic response compared to the control group. RT-PCR confirmed the RNA sequencing analysis. The experimental group showed a six-fold increase in inflammatory markers IL-1 and NF-κβ. The control group showed increased expression of α1 and β1 adrenergic receptors, NO treatment has led to normalization of expression and a three-fold increase in β2 receptors expression compared to shams. NO supplementation induced apoptosis in the myocardium, as indicated by a 50% increase in TUNEL-positive nuclei compared to the control group.

Conclusions:
NO supplementation during cardiac arrest boosts the inflammatory response in the myocardium and is associated with increased apoptosis.
Abstract P3101: Dietary Nitrates Negate Beneficial Cardiac Adaptations To Aerobic Exercise In A Sex-specific Manner
Authors: Bisset Elise S , Howlett Susan E
Published: October 2023

Dietary supplements such as nitrates are commonly combined with aerobic exercise with the idea that this will improve performance and cardiac function. Whether this combination leads to beneficial effects on cardiac function at the cellular level is unclear, and few studies have used females. We determined whether dietary nitrates and/or aerobic exercise improves fitness and cardiovascular health in adult mice (both sexes) and explored underlying mechanisms. C57Bl/6 mice (9-mos) were given 1mM sodium nitrate (drinking water) and/or access to a running wheel for 3 mos. Cardiac function (echocardiography), blood pressure (tail cuff) and fitness (treadmill) were measured in mice at endpoint; calcium transients (Fura-2) and calcium currents were measured in isolated ventricular myocytes at endpoint. Exercised mice had better fitness and improved cardiac function when compared to sedentary mice, regardless of sex. Nitrates had no effect on blood pressure, total voluntary wheel running, or endpoint fitness measured as distance to exhaustion (242 ± 28 vs 294 ± 37 m, p=0.29; 122 ± 29 vs 136 ± 16 m, p=0.69 in females and males, respectively) compared to exercised controls. However, nitrates combined with exercise impaired cardiac function in a sex-specific manner. Nitrate-treated male mice who exercised had lower E/A ratios (1.8 ± 0.3 vs 1.3 ± 0.1, p=0.01) and thus worse diastolic function than control exercised male mice. By contrast, nitrate-treated female mice who exercised had smaller global longitudinal strain (-19.5 ± 1.5 vs -11.8 ± 3.0%, p=0.01) than control exercised mice, indicative of worse systolic function. Corresponding changes were seen at the cellular level. Male mice given nitrates had slower times to 50% calcium transient decay than controls (44.8 ± 4 vs 73.8 ± 6 ms, p=0.01), while females given nitrates had slower velocity to peak calcium (0.031 ± 0.004 vs 0.018 ± 0.003 μm/ms, p=0.005). These changes in calcium transients were attributable, at least in part, to corresponding changes in calcium currents. Thus, exercise improves fitness regardless of nitrates and promotes beneficial cardiac adaptations in systolic and diastolic function, but these benefits are negated when exercise is combined with dietary nitrates in a sex-specific fashion.
Abstract P3105: Stress Exacerbates Ischemia/reperfusion Injury In The Female Heart Through The Glucocorticoid Receptor Signaling In Cardiomyocytes
Authors: Kamberov Lilly , Dhaibar Hemangini , Patel Sonya , Pruiett Jaylen , Shen Xinggui , Orr Wayne W , Bailey Steven R , Kevil Christopher , Oakley Robert H , Cidlowski John A , Cruz-Topete Diana
Published: October 2023

It is estimated that over 30,000 women younger than 55-years-old die of acute myocardial infarction (AMI) in the United States annually. Clinical data suggest that exposure to mental stress exacerbates the outcomes of an AMI in women. However, a knowledge gap exists regarding the molecular mechanisms whereby stress contributes to AMI in women and how they differ in men. The present project aims to define the mechanisms underlying the link between stress, glucocorticoids (stress hormones), the glucocorticoid receptor (GR) signaling, and the worse outcomes of AMI in females than in their male counterparts. We challenged male and female transgenic mice lacking GR in cardiomyocytes (cardioGRKO) and their respective wild-type controls in a murine model of "mental" stress followed by ischemia/reperfusion (AMI) injury. Our data show that stress exacerbated the response to AMI injury in control females compared to their male counterparts. Females without cardiomyocyte GR were protected and presented significantly reduced infarct areas compared to cardioGRKO males and control males and females with intact GR signaling. These results demonstrated that stress directly affected the heart and that these effects are sexually dimorphic. The severity of the infarct and cardiomyocyte death was further exacerbated during the highest estrogen activity phase (proestrus) of the female cycle. Our
in vitro
studies additionally supported these data, showing that glucocorticoids block estrogen's pro-survival effects on cardiomyocytes in hypoxia/re-oxygenation conditions in a mechanism dependent upon GR inhibition of estrogen regulation of antioxidant pathways, including decreased levels of the master regulator of the antioxidant response, nuclear factor-erythroid factor 2-related factor 2 (Nrf2), and increased superoxide and lipid peroxides production. Our
in vivo
models displayed similar findings. Our study is the first to test the impact of mental stress on activating glucocorticoid signaling in the heart in the setting of AMI. This study is relevant to human health as a proof of concept that exposure to high stress levels aggravates AMI in premenopausal hearts by cardiomyocyte GR-regulated mechanisms that may be targeted to develop therapies tailored to women.
Abstract P3114: Sex Differences In Ventricular Function And Mortality In Sema3a Deficient Mice With Dilated Cardiomyopathy
Authors: PENNINO MARISSA , Dajani Alhassan , Contreras Jaime , Arreola Nigel M , Jay Patrick Y , Zhu Ching , Shivkumar Kalyanam , Ajijola Olujimi A
Published: October 2023

Introduction:
Clinical studies consistently report sex differences in heart failure (HF) outcomes. Disrupted cardiac sympathetic innervation patterns have not been investigated as a potential mechanism.

Hypothesis:
We aimed to explore whether cardiac sympathetic innervation patterns disrupted by deletion of the neurorepellent, Semaphorin3a (Sema3a), in murine dilated cardiomyopathy (DCM), has a deleterious effect in male compared to female mice.

Methods:
Utilizing the established Tg9 model of nonischemic HF (DCM+), we compared serial left ventricular ejection fraction (LVEF), ventricular arrhythmogenesis, mortality, and innervation patterns using immunohistochemistry in male vs. female DCM+Sema3a
KO
mice (with disrupted innervation) to male and female DCM+Sema3a
WT
animals.

Results:
Immunohistochemistry revealed greater disruption in sympathetic innervation amongst Sema3a deficient (DCM+Sema3a
KO
) mice (n=11) compared to DCM+Sema3a
WT
(n=10) and WT mice (n=7) ( ****p<0.0001). There was no significant difference in arrhythmogenesis between DCM+Sema3a
WT
versus DCM+Sema3a
KO
mice. Mortality was significantly greater in DCM+Sema3a
KO
mice compared to DCM+Sema3a
WT
and WT mice (*p=0.04). This was attributable to pump failure, as LVEF was lower in DCM+Sema3a
KO
mice that died compared to DCM+Sema3a
KO
mice who did not (*p=0.03). When outcomes were compared for male versus female mice, we observed sex differences in mortality, with female DCM+Sema3a
KO
mice exhibiting higher mortality and greater LV dysfunction than males (*p=0.03). This difference was not seen in male vs female DCM+Sema3a
WT
mice, suggesting that the sex differences are related to Sema3a gene deletion, and thus, disrupted innervation patterns, not DCM alone.

Conclusion:
Our data suggests that there are sex-based differences in DCM outcomes that may involve Sema3a signaling. Further studies validating this finding and identifying underlying mechanisms are warranted.
Abstract P3116: Age-Related Cardiac Changes In S-Nitrosoglutathione Reductase Knockout Mice
Authors: Ebenebe Obialunanma V , Kabir Raihan , Garbus Haley , Jani Vivek , Adamo Luigi , Kohr Mark J
Published: October 2023

Age associated cardiac aging differs between males and females, yet these age-related sex-dimorphic pathways remain poorly explored. We have previously shown that inhibition of S-nitrosoglutathione reductase (GSNOR) was cardioprotective during ischemic injury in male but not female mice. We therefore, hypothesized that GSNOR activity might play a role in sex-related differences in cardiac aging, and GSNOR deletion (KO) might exacerbate age-related cardiac dysfunction in females but not males. WT and KO mice underwent longitudinal 2D-echocardiography at 4, 7, 10 and 14 months of age, and echocardiographic data were analyzed with mixed-effects analysis. We saw a significant decrease in GSNOR activity in old (18 months) WT mice relative to young (4 months) in both sexes but more pronounced in males (p<0.0001) vs. females (p=0.02). Left ventricular (LV) internal diameter at end-diastole significantly increased with age in WT males (2.65 ± 0.02 to 3.60 ± 0.13) and females (2.61 ± 0.02 to 3.32 ± 0.04) with a significant effect of sex across age (p<0.0001). LV Mass (indexed to group body weight at 18 months) increased with age in both males (82 ± 2 to 127 ± 3) and females (85 ± 3 to 113 ± 3) with a significant age and sex interaction (p<0.0001). Similar trends were observed for other echocardiographic parameters. Contrary to our hypothesis, the age- and sex-related differences observed in WT were absent in KO mice. LV internal diameter at end-diastole and LV Mass did not significantly change in male or female KO mice from 4 to 14 months of age neither did the majority of echocardiographic parameters tested. No differences were observed between male and female KO mice. Taken together, our data show that GSNOR deletion attenuates aging-induced cardiac dysfunction and abrogates age-related sex differences in the mouse heart.
Abstract P3122:
Rap1gap2
Fine-mapping For Causal Variants Of Prevalent Ischemic Heart Disease Among Diverse Postmenopausal Women From The Women’s Health Initiative
Authors: Dungan Jennifer R , Qin Huaizhen , Franco-Sazaqui Ayumi , Eynon Matthew , Gregory Simon G , Cooper-Dehoff Rhonda M , Pepine Carl J
Published: October 2023

Background:
RAP1GAP2
is a GTP-ase activating protein that regulates secretion of dense granules from platelets at sites of endothelial damage, eliciting maximal platelet aggregation, a hallmark of ischemic events. Our published sex-stratified GWAS significantly associated
RAP1GAP2
(Chr 17p13.3) variants with ischemic heart disease (IHD) case status and increased mortality risk among women but not men. We hypothesize that
RAP1GAP2
genetic variation may demonstrate causal effects for the increased risk of IHD case status among postmenopausal women.

Purpose:
To fine-map
RAP1GAP2
to identify causal markers for risk of IHD case status among racially and ethnically diverse postmenopausal women.

Methods:
We performed secondary analysis of 8,699 postmenopausal women from the Women’s Health Initiative (Observational cohort) who have publicly available genome-wide and densely imputed genotype data via NIH/dbGap (Affymetrix Affy 6.0). IHD case status was defined as prevalence of myocardial infarction, stroke, angina, congestive heart failure, peripheral arterial disease, or atherosclerotic interventional procedures at or prior to baseline; non-IHD controls were absent of all IHD criteria. PCA and Efficient Local Ancestry Inference (ELAI) were used to infer global and SNP-wise local ancestries. For each SNP, we performed SNP-wise logistic regression modeling, calibrating for the confounding effects of local and global ancestries and controlling for major cardiac risk factors, hormone replacement and cardiac medication exposure, IHD type (obstructive, nonobstructive), eGFR, education, income, and occupation.

Results:
Mean age 64.08 (±7.74) for a sample of 3,125 (36%) non-Hispanic Whites, 3,841 (44%) African Americans and 1,733 (20%) Hispanic Americans. We observed 1,932 IHD cases and 6,767 non-IHD controls. Novel PCA and ELAI uncovered strong spatial global population structure, varying ancestries, and ancestry-genotype correlations across genomic regions. Fine mapping analyses are underway and results will be available at time of presentation.

Conclusions:
Identifying sex- and ancestry-related causal genetic factors can serve as future targets for precision prevention of IHD among postmenopausal women.
Abstract P3141: Age-associated Alterations In Sialylation Affect Nav1.5 Channel Function And Contribute To Cardiac Dysfunction
Authors: Tarantino Adriana , Coviello Simona , Ghiroldi Andrea , Melgari Dario , Frosio Anthony , Calamaio Serena , Piccoli Marco , Ciconte Giuseppe , Pappone Carlo , Anastasia Luigi
Published: October 2023

Voltage-gated sodium channel Nav1.5 plays a pivotal role in the proper functioning of cardiac muscle cells by mediating their depolarization. Sialylation, a post-translational modification involving the addition of negatively charged sialic acid moieties, is essential for Nav1.5 functionality. Altered sialylation has been observed in various cardiovascular diseases, including Chagas disease, and congenital disorders of glycosylation affecting the heart. Furthermore, sialylation status has been shown to change during human adult life, impacting brain and muscle structural properties. Cardiac arrhythmias increase in prevalence with age, contributing to higher morbidity and mortality in the elderly. We aimed to investigate the impact of age-related changes in sialylation on Nav1.5 channels in cardiac cells. The Nav1.5 protein and sialic acid (α-2,3 and α-2,6) levels were quantified using SNA and MAA lectins in heart tissue from an aging mouse model. To investigate the role of sialic acid on Nav1.5 channel regulation, in vitro studies were conducted on HEK293 cells expressing Nav1.5 channels and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Treatment with a mix of sialidase enzymes was performed for monitoring Nav1.5 protein biosynthesis, trafficking, degradation, and recycling and patch clamp analysis was used to examine changes in sodium ion current. To investigate the impact of exogenous sialic acid production on Nav1.5 protein expression, cells were fed with modified mannosamine. Our results revealed a reduction in both α-2,3 and α-2,6 sialic acid levels in aged mice, which positively correlated with Nav1.5 protein levels. In vitro experiments demonstrated that decreased external sialic acid upon treatment induced Nav1.5 channel internalization and activated endocytic pathways, leading to proteasome-mediated degradation. Conversely, exogenous sialic acid restored Nav1.5 protein levels on the cell membrane. In conclusion, our findings suggest that age-related reductions in sialylation status in cardiac tissue induce Nav1.5 channel internalization. Modulating sialic acid levels could potentially serve as a promising therapeutic strategy to prevent cardiac dysfunction in the elderly population.
Abstract P3144: A Deep Neural Network Algorithm For Accurate Detection Of Brugada Syndrome Features In Electrocardiograms
Authors: Melo Luke , Ciconte Giuseppe , Christy Ashton , Vicedomini Gabriele , Anastasia Luigi , Grant Edward , Pappone Carlo
Published: October 2023

Brugada Syndrome (BrS) is a rare but potentially fatal cardiac condition that is difficult to diagnose due to the elusive nature of its characteristic ECG pattern. The diagnosis of BrS requires identification of a specific electrocardiographic (ECG) pattern, which often appears only upon administration of sodium-channel blockers. Here, we present a deep neural network (DNN) algorithm that accurately detects BrS features in ECGs without the need for a drug challenge. We trained and validated the DNN on two independent cohorts of patients who were enrolled in electrophysiological studies for BrS diagnosis. The internal prospective validation cohort included 370 subjects, and the multicenter external validation cohort included 110 subjects from three other medical institutions in Italy. All subjects who did not present a spontaneous type 1 ECG underwent an ajmaline drug challenge to aid in confirming a BrS diagnosis. We found that the DNN approach classified ECGs for BrS with a sensitivity of 79.6%, specificity of 93.6%, accuracy of 88.4%, and area under the curve (AUC) of 0.934±0.027 in the validation cohorts. The DNN model also detected BrS with 100% accuracy in all cases in which a patient presented the spontaneous type 1 pattern. Our results demonstrate that a multivariate machine-learning algorithm can accurately detect the features of BrS in a conventional ECG without the challenge of a sodium-channel blocker. The computer-assisted analysis of digital ECG traces from current electrocardiographs might help physicians recognize patients affected by this disease. In conclusion, we have developed an advanced deep learning algorithm that can accurately detect BrS features in ECGs. Our results demonstrate that the DNN approach could be an effective tool to improve the diagnosis of BrS and potentially prevent sudden cardiac death in affected individuals.
Abstract P3150: Extracellular Vesicles From Endothelial Progenitor Cells Limit The Development Of Ventricular Dysfunction In A Mouse Model Of Myocardial Infarction
Authors: Dantas Ana Paula V , Bobi Joaquim , LAZARO IOLANDA , Van Beusekom Heleen M , Francisco Alarcon , Francisco Alarcon , Ortiz José Tomas , Roque Merce , Sabate Manel , Brugaletta Salvatore
Published: October 2023

After myocardial infarction (MI), angiogenesis in the ischemic myocardium is essential for an optimal reparative process to limit the development of ventricular dysfunction. Cell-derived extracellular vehicles (EVs) are emerging as the next-generation strategy in tissue engineering. In this study, we aimed to determine the role of EVs derived from endothelial progenitor cells (EPC) in cardiac function post-MI. EVs were purified from the supernatant of EPC by density gradient centrifugation. MI was induced in C57BL/6 mice (n=23) by left coronary artery ligation and confirmed by EKG. Mice were randomized to receive purified EVs (n=10) or saline (n=10) in two zones of the myocardium, peri-ischemic and central ischemia zones, followed by weekly I.P. injection of EVs or saline for six weeks. Three mice underwent sham surgical procedures without coronary artery ligation. Cardiac magnetic resonance at the end of the follow-up showed significant impairment of left ventricle (LV) performance in the saline group, which was better preserved in mice treated with EVs
(Fig 1)
. Histological analysis in the LV showed increased collagen deposition in both MI groups
(Fig 2A)
. However, capillary density decreased more pronouncedly in the saline than in the EV group
(Fig 2B)
. Quantitative mRNA array revealed a differential relative mRNA expression in the EV group compared to saline
(Fig 3)
. The administration of EPC-derived EVs has a modest effect on preventing the development of LV adverse remodeling after ischemia, as it limits LV dilation at the long term in a mouse model of chronic MI.Funding: ISCIII (PI19/00264, SEC/FECINV- BAS 20/012)
Abstract P3152: Diabetic Upregulation Of Grk2 Impairs Adipocyte Exosome Surface Adiponectin-mediated Cardioprotection And Exacerbates Ischemic Heart Failure
Authors: Zhang Zhen , Liu Caihong , Zhu Di , Meng Zhijun , Zhang John , Lau Wayne , Lopez Bernard L , Christopher Theodore , Gao Erhe , Koch Walter J , Zhao Jianli , Ma Xinliang (Xin) , Wang Yajing
Published: October 2023

Background:
Type 2 diabetes mellitus (DM) significantly exacerbates ischemic heart failure (IHF) by incompletely understood mechanisms. We recently reported that DM switches adipocyte-derived exosome (Exo) from cardioprotective signaling activators in nondiabetes (ND) to vehicles carrying cytotoxic molecules from DM adipocyte to heart. However, it remains unclear how DM triggers this switch and whether blocking it can protect against DM exacerbation of IHF.

Methods and Results:
ND or DM (12 weeks of high-fat diet) mice were subjected to MI/R (90 minutes/4 weeks) and treated with Exo (isolated from ND epidydimal fat pads and intramyocardially injected immediately before reperfusion). Exo administration significantly attenuated IHF in ND but not in DM mice. In vitro mechanistic and in vivo concept-prove experiments were conducted to determine how Exo protects against IHF and how DM blocks this protection. In cardiomyocytes (CM) from ND mice, Exo rapidly activated multiple injury salvage kinases (ISKs, including ERK, AMPK, and ACC) and attenuated cell death, suggesting that Exo-surface molecules mediate Exo cardioprotection. Exo-Flow (a method specifically detects Exo surface molecules) experiments demonstrated that adipocyte-derived Exo carries high levels of adiponectin (APN) on their surface. APN antibody-neutralized Exo or APNKO mice-derived Exo neither activated ISKs nor attenuated IHF. Exo failed to activate ISKs and protect IHF in AdipoR1KO mice. These results demonstrated that the adipocyte Exo surface-localized APN and CM-expressed AdipoR1 are essential in protective communication between adipocyte and heart. GRK2, a kinase that phosphorylates AdipoR1 at S205 and blocks APN cardioprotection, was dramatically upregulated in DM hearts. GRK2 overexpression blocked Exo-induced ISKs activation and cellular protection in WT CM but not in AdipoR1
S205A
knock-in CM. Finally, Exo administration in AAV9-mediated CM-specific AdipoR1
S205A
knock-in mice significantly attenuated diabetic IHF, while it failed to protect in AdipoR1
S205E
mice.

Conclusions:
This study sheds light on the mechanisms underlying DM exacerbation of IHF and highlights the potential of targeting the APN/AdipoR1/GRK2 axis to protect against this condition.
Abstract P3154: Characterized Expression Of MicroRNAs In Plasma Exosome In Patients With Coronary Chronic Total Occlusion
Authors: Suh Jon , Son Ji-Hye , Moon Inki , Kong Min Gyu , Kim Dongeon , Park Jeong Kyu , Choi Hyung Oh , Seo Hye-Sun , Cho Yoon Haeng , Chang Hun Soo
Published: October 2023

Backgrounds:
Even with similar coronary artery occlusion, chronic total occlusion (CTO) has different clinical features from acute myocardial infarction (AMI). MicroRNAs (miRNA; miR) have been regarded as biomarker to identify heterogenous patients, as well as new therapeutic targets in cardiovascular disease. We aimed to identify exosomal miRNA important for CTO.

Methods:
Patients with percutaneous coronary intervention (PCI) of CTO (n=30) and AMI (n=30) were performed miRNA expression profiling via exosomal small RNA sequencing in the plasma for these groups. Receiver operating characteristic (ROC) analysis, gene ontology, and functional pathway were analyzed in the miRNA exhibiting >4 absolute fold changes with FDR < 0.05.

Results:
We detected 10 miRNAs -including miR-21-5p, miR-206, miR-215-5p, and miR-1-3p upregulated in CTO and 3 miRNAs -including miR-9-5p, miR-3529-3p, and miR-127-3p downregulated in CTO as compared to AMI (FDR q < 0.05). Gene enrichment and pathway analysis showed that the target genes of the 10 upregulated miRNAs were involved in regulation of osteoblast proliferation and interleukin-4 and interleukin-13 signaling. In contrast, the target genes of the 3 down-regulated miRNAs were enriched in heart valve development and blood vessel development. We selected 7 miRNAs -including miR-21-5p, miR-206, miR-215-5p, miR-1-3p, miR-9-5p, miR-3529-3p, and miR-127-3p which were related to major pathway term in gene ontology and validated by quantitative real-time polymerase chain reaction (qRT-PCR). In qRT-PCR analysis, the expression levels of miR-9-5p and miR-127-3p of plasma exosome were both lower in CTO than those of AMI, similar to the results of exosomal small RNA sequencing. The ROC curve were analyzed in the 2 miRNAs and for the integrative miRNA expression score showed high accuracy in distinguishing between the CTO and AMI (AUC = 0.948) with 79% specificity and 97% sensitivity.

Conclusions:
We identified significantly different miRNA expression patterns between the CTO and AMI. The levels of these miRNAs in plasma exosomes may be promising markers that distinguish CTO from AMI.
Abstract P3158: Extracellular Vesicles From Ischemic Myocardium Mediate Regulatory T Cell Dysfunction Through MicroRNA-1-mediated Suppression Of VEGF-A
Authors: Mohammadigoldar Zahra , Liao Ke , Ibrahim Ahmed G , Li Liang , Liu Weixin , Marban Eduardo
Published: October 2023

Motivation:
Myocardial infarction (MI) triggers an efflux of pro-inflammatory extracellular vesicles (EVs) from the heart into the circulation. Here, we test the hypothesis that circulating ischemic heart EVs (iEVs) attenuate regulatory T cell (Treg) proliferation and function post-MI.

Methods:
Hearts were explanted from mice and Langendorff-perfused with Tyrode buffer either continuously for two hours (nonischemic), or subjected to no-flow for 30 min, and reperfused for 1.5h. Heart effluent (~700ml) was collected for EV isolation by ultracentrifugation. EV size and concentration were measured by nanoparticle tracking analysis; EV markers were characterized by western blot. EVs from nonischemic heart effluent (nEVs) served as controls. RNA sequencing (RNAseq) followed by qPCR validation was used to identify microRNAs in EVs. Assessments of human Tregs included cell proliferation (Cell Counting Kit-8 assay), IL-10 production (qPCR of Tregs, and ELISA of Treg-conditioned media), and RNAseq after exposure to EVs. In vivo, an antagomir was administered retro-orbitally immediately post-MI, and splenic Tregs were measured by flow cytometry 7 days post-MI.

Findings:
MiR-1-3p was highly enriched in iEVs compared to nEVs. Exposure of human Tregs to iEVs or a miR-1 mimic resulted in decreased proliferation (
P
<0.0001, N=8) and production of IL-10 (qPCR: P<0.0001, n=9; ELISA:
P
<0.0001, N=6) compared to nEVs. Antagomir-1 reversed iEV- and miR-1-induced inhibition of Treg proliferation. RNAseq on human Tregs revealed that VEGF-A, a miR-1 target gene, was suppressed after exposure to iEVs compared to nEVs. In vivo, the number of splenic Tregs decreased on day 7 post-MI, with a concomitant decrease in IL-10
+
and VEGF-A
+
Tregs. Intriguingly, administration of antagomiR-1 restored numbers of splenic IL10
+
and VEGF-A
+
Treg cells.

Conclusions:
iEVs cause Treg dysfunction by transferring miR-1. In turn, miR-1 suppresses VEGF-A, a known modulator of Treg function. Our findings reveal novel paracrine modulation of immune cells by ischemic myocardium. Targeting EV signaling post-MI represents a potential new strategy to augment Treg-mediated tissue repair.
Abstract P3161: Engineering Mesenchymal Stem Cell-derived Extracellular Vesicles To Enhance Their Therapeutic Efficacy In Cardiovascular Disease
Authors: Choudhury Sarah , Lang Jennifer K
Published: October 2023

Background:
Extracellular vesicles derived from cardiosphere-derived cells (CDC-EVs) have demonstrated the ability to regulate cardiomyocyte apoptosis and induce cardiomyocyte proliferation in pre-clinical models of acute MI. While CDC-EVs exhibit cardioprotective effects, they require invasive intramyocardial injections to achieve therapeutic efficacy, detracting from their translatability. Mesenchymal stem cells (MSC) also generate cardioprotective EVs (MSC-EVs), which have demonstrated therapeutic efficacy in models of cardiac disease following intravenous administration. However, MSC-EVs lack several of the cardioprotective miRs expressed by CDC-EVs, including miR146a, which has been shown to modulate cardiomyocyte apoptosis. The objective of our current study was to determine the effect of enriching MSC-EVs with cardioprotective CDC-EV miRNA on cardiomyocyte apoptosis.

Methods:
MSCs were transfected with miR-146a-5p mimics using Trans-IT TKO in serum free growth media. EVs were isolated using a combination of ultrafiltration and size exclusion chromatography (SEC) and characterized by size, morphology and expression of surface markers. RT-PCR was performed on EV samples to quantify miR-146a expression. Primary neonatal mouse cardiomyocytes were treated with MSC-EVs, miR-146a loaded MSC-EVs, CDC-EVs or matched volume vehicle control. Cardiomyocyte apoptosis was assessed by a TUNEL assay.

Results:
Treatment with miR-146a loaded MSC EVs reduced cardiomyocyte apoptosis compared with cardiomyocytes treated with unmodified MSC exosomes (14±1% vs. 31±3%) (mean ± SEM, p=0.001, one-way ANOVA). miR-146a loaded MSC-EVs and CDC-EVs demonstrated a similar level of reduction in cardiomyocyte apoptosis (14±1% vs. 15±1%). There was no difference in the level of apoptosis between cardiomyocytes treated with unmodified MSC exosomes and cardiomyocytes treated with vehicle control (31±3% vs. 27±1%) (mean ± SEM, p=0.366, one-way ANOVA).

Conclusion:
Engineering MSC-EVs to express miR-146a enhances their ability to reduce cardiomyocyte apoptosis
in vitro
. This highlights the potential of cargo engineering as a means to improve the therapeutic efficacy of EVs for cardiovascular disease.
Abstract P3166: Circulating Extracellular Vesicles In Human Cardiorenal Syndrome Promote Renal Injury In Kidney On A Chip System
Authors: Chatterjee Emeli , Rodosthenous Rodosthenis , Kujala Ville , karalis Katia , Helge Lehmann , Spanos Michail , Oliveira Getulio , Miller Tyne , Shi Mingijan , Gokulnath Priyanka , Li Guoping , Ghiran Ionita , Lindenfeld Joann , Mosley Jonathan , Lau Emily , Ho Jennifer E , Sheng Quanhu , Shah Ravi V , Das Saumya
Published: October 2023

Objective:
Cardiorenal syndrome (CRS)—renal injury during heart failure (HF)—is linked to higher morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.

Methods:
We investigated the transcriptional effects of circulating EVs from patients with CRS on renal epithelial/endothelial cells, using a microfluidic kidney chip model (KC). We performed small RNA-seq and regression against serum creatinine to prioritize subsets of EV miRNAs associated with renal function. Long RNA-seq was performed on the same EVs to identify the source organs via deconvolution analysis.
In silico
pathway analysis, human genetics, and interrogation of expression of miRNA target genes in the KC model and in a separate cohort of individuals post-renal transplant with microarray-based gene expression were performed for validation. To enhance the rigor of our observation, kidney epithelial cells were treated with corresponding anti-miRs and ago-miRs under various experimental conditions.

Results:
Renal epithelial and endothelial cells in the KC model exhibited uptake of EVs. EVs from patients with CRS led to higher expression of renal injury markers (
IL18
,
NGAL
,
KIM-1
), a greater cystatin C secretion relative to non-CRS EVs. Small RNA-seq and regression identified 15 miRNAs related to creatinine, targeting 1143 gene targets specifying pathways relevant to renal injury, including TGF-b and AMPK signaling. Deconvolution analysis identified 6 major organs as the main source of the EVs. Finally, we observed directionally consistent changes in expression of TGF-b pathway members (BMP6, FST, TIMP3) in KC model exposed to CRS EVs, as well as in the cells treated with corresponding inhibitors and mimics of miRNAs. Similar trend was observed in renal tissue after transplant rejection. Mendelian randomization suggested a role for FST in renal function.

Conclusion:
EVs from patients with CRS directly elicit adverse transcriptional and phenotypic responses in a KC model by regulating biologically relevant pathways, suggesting a novel role for EVs in CRS.
Abstract P3182: Promoting Mitophagy By Enhanced Fatty Acid Oxidation Improves Phenotypes Of Heart Failure With Preserved Ejection Fraction
Authors: Yoshii Akira , McMillen Timothy , Tian Rong
Published: October 2023

Although metabolic remodeling is known to contribute to the development of heart failure with reduced ejection fraction (HFrEF), the role of energy metabolism in HF with preserved ejection fraction (HFpEF) is poorly understood. Here we assessed cardiac energy metabolism in both HFpEF and HFrEF mice by multi-nuclear NMR spectroscopy. HFpEF was induced by a 2-hit of high fat diet (HFD) and Nω-nitrol-arginine-methyl-ester (L-NAME, in drinking water) for 10 weeks. Mice fed with chow diet, HFD or L-NAME administration alone were used as controls. HFpEF phenotypes were assessed as the exercise intolerance, pulmonary congestion, high E/e’ by echocardiography and left shift of pressure-volume relationship obtained from isolated hearts. High energy phosphate content, assessed by 31P NMR spectroscopy of isolated perfused hearts, showed a lower phosphocreatine (PCr) content in HFpEF hearts while ATP level was unaltered compared to all the controls, resulting a reduced PCr/ATP ratio similar to that in HFrEF induced by TAC. Decreased respiratory function and increased ROS production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile, assessed by 13C-isotopmer analysis, showed a high dependency on fatty acid oxidation (FAO) in HFpEF hearts, which is opposite of HFrEF but similar to that in HFD hearts. However, PCr/ATP ratio and mitochondrial function were sustained in the HFD hearts with the same duration of treatment. We found that mitophagy was activated in HFD heart but did not change in HFpEF hearts despite similar extent of obesity suggesting that HFpEF heart is maladaptive to the metabolic stress. Promoting mitophagy by TAT-Beclin1 treatment improved mitochondrial function in HFpEF hearts and partially rescued cardiac diastolic dysfunction and lung congestion. Furthermore, enhancing FAO in HFpEF hearts by deleting acetyl-CoA carboxylase 2 stimulated mitophagy and improved HFpEF phenotypes. In summary, our results show that mitochondrial dysfunction is an important pathogenic mechanism for cardiac dysfunction in HFpEF. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control which can be improved by stimulating FAO.