bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo131: Time-Restricted Feeding Normalizes Dim Light at Night-Induced Disruption of Cardiovascular Rhythms in Mice
Authors: Prabhat Abhilash , Sami Dema , Ehlman Allison , Stalcup Rebecca , Seward Tanya , Su Wen , Gong Ming , Schroder Elizabeth , Delisle Brian
Published: October 2024

Background:
Light input to the suprachiasmatic nucleus entrains circadian rhythms in physiology and behavior to the day-night cycle. Exposure to light at night in people is associated with cardiometabolic disease. Pre-clinical studies show that artificial light at night, including at very low levels, disrupts day-night rhythms in activity, feeding behavior, heart rate, and blood pressure dipping.

Hypothesis:
Dim light at night (dLAN) disrupts day-night rhythms in feeding behavior to blunt day-night rhythms in autonomic input to the heart and blood pressure dipping.

Methods:
Mice (n=5-6/sex) in thermoneutral housing were implanted with telemetry probes to record heart rate, blood pressure, and core body temperature. Autonomic input to the heart was assessed by measuring heart rate and subtracting the temperature-dependent changes in the heart rate after pharmacological inhibition of muscarinic and β-adrenergic receptor activation. Mice were housed in 12 h light: 12 h dark cycles (LD, 200 lux: 0 lux) with ad libitum access to food (LD-ALF), subjected to 12 h light: 12 h dLAN cycles (dLAN-ALF; 200 lux: 5 lux) for two weeks, and then feeding was time-restricted (not calorically restricted) to the dLAN cycle (dLAN-RF). Data were extracted from Ponemah and Clocklab, and statistical analysis was done using GraphPad PRISM software.

Results:
Compared to LD-ALF mice, dLAN-ALF mice showed reduced amplitudes in day-night activity, feeding, heart rate, and blood pressure rhythms, with males more affected than females (p<0.001). dLAN-ALF male and female mice had decreased amplitudes in the day-night rhythms in autonomic input to the heart. In addition, dLAN-ALF male mice had less blood pressure dipping. dLAN-RF normalized autonomic input to the heart and heart rate in male and female mice (p<0.05, p<0.01, respectively). dLAN-RF also improved blood pressure dipping in male mice (p<0.001). dLAN-RF did not normalize activity rhythms.

Conclusion:
dLAN disrupts day-night rhythms in activity, feeding, heart rate, and blood pressure dipping in mice, with males being more impacted. Time-restricted feeding to the dLAN cycle normalizes autonomic input to the heart and blood pressure dipping. These data suggest that time-restricted feeding counteracts the light-at-night-induced circadian disruption of cardiovascular function.
Abstract Or102: Macrophage-Mediated Interleukin-6 Signaling Drives Ryanodine Receptor-2 Calcium Leak in Postoperative Atrial Fibrillation
Authors: Keefe Joshua , Aguilar Yuriana , Navarro-Garcia Jose Alberto , Paasche Amelie , Abu-Taha Issam , Tekook Marcel , Bruns Florian , Kamler Markus , Shen Ying , Dobrev Dobromir , Wehrens Xander
Published: October 2024

Postoperative atrial fibrillation (poAF) is self-limited AF that occurs days after cardiac surgery in one-third of patients. The degree of perioperative increase in systemic inflammation, particularly involving interleukin (IL)-6, correlates with poAF risk. However, no studies to-date have identified the cell types involved nor mechanistically linked IL-6 signaling to fundamental arrhythmia mechanisms.

To study poAF, we developed a mouse model consisting of atrial pericardiectomy and transient aortic cross-clamping followed by intracardiac burst pacing on postoperative day three to assess poAF inducibility. Single-cell RNA sequencing of atrial non-myocytes revealed macrophages to be the most prominently altered atrial cell type – increased 2.4-fold (
P
<0.001) in mice with versus without poAF. Pseudotime trajectory analyses revealed IL-6 to be the top cytokine altered in macrophages, which we confirmed by western blot demonstrating a 1.7-fold (
P
=0.012) increase in atrial IL-6 protein in mice with versus without poAF. Indeed, both macrophage depletion and conditional knockout of IL-6Ra in macrophages decreased poAF inducibility by 5.2-fold (
P
=0.048) and 5.5-fold (
P
=0.027), respectively. Downstream inhibition using an FDA-approved STAT3 inhibitor also rescued poAF (6.0-fold decrease,
P
=0.022). At the single atrial cardiomyocyte (ACM) level, IL-6 challenge led to a 6.3-fold (
P
<0.001) increase in calcium (Ca
2+
) spark frequency after normalization to sarcoplasmic reticulum Ca
2+
load, indicative of ryanodine receptor-2 (RyR2) dysfunction. Indeed, RyR2 phosphorylation at Ser2814, the target of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII), was increased in the atria of mice with poAF, and CaMKII inhibition was sufficient to rescue arrhythmogenic Ca
2+
sparks in ACMs from mice with poAF. We corroborated our findings in human ACMs and atrial tissue to demonstrate translational relevance.

Taken together, we use an integrated approach incorporating mouse and human biochemical, functional, and single-cell sequencing data to demonstrate that infiltrating atrial macrophages, specifically IL-6Ra from macrophages, are fundamentally required for poAF development (
Fig. 1
). Molecularly, enhanced atrial IL-6 signaling led to arrhythmogenic RyR2 dysfunction through CaMKII. Our findings portend therapeutic utility by providing mechanistic evidence that targeting the IL-6 axis may be used for poAF prevention and treatment in a clinically amenable timeframe.
Abstract Or109: Regulation of UFM1 modification from ER dynamics to cardiac homeostasis
Authors: Tandra Varsha , Singh Nagendra , Li Honglin , Su Huabo , Li Jie
Published: October 2024

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. Understanding the molecular mechanism governing CVD has become a major focus in developing new therapeutics. Ubiquitin (Ub) like protein post-translational modifications (PTMs) have been known to play an important role in the normal functioning of the heart. UFM1 (ubiquitin-fold modifier 1) is a novel Ub-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade, named ufmylation. Ufmylation regulates diverse cellular processes and is implicated in various human diseases. However, its importance in the heart is unknown. For the very first time, we have reported dysregulation of ufmylation in dilated cardiomyopathy, hypertrophic human patients, and animal models. It remains unclear whether ufmylation of cardiac proteins is required for heart function and if so, how ufmylation regulates cardiac function. To determine the role of UFM1 modification in cardiomyocytes we have generated cardiac-specific UFM1-knockout (UFM1CKO) mice. The UFM1CKO mice developed dilated cardiomyopathy at resting condition, as indicated by the left ventricle (LV) wall thinning, chamber dilatation, and impaired contractility at 4 months of age, which were further exacerbated by 6 months. It appears that UFM1 modification constrains pathological cardiac remodeling by maintaining ER homeostasis and regulating ER stress response. To test the hypothesis that UFM1 modification, instead of UFM1 itself is required to maintain cardiac function, we replenished UFM1CKO mice with cardiac-specific WT or conjugation-deficient UFM1 via AAV9. By echocardiography, we revealed that, unlike WT UFM1, overexpression of the conjugation-deficient mutant failed to attenuate the cardiac remodeling and dysfunction of the UFM1CKO heart. Interestingly, the conjugation-deficient mutant further exacerbated the cardiac dysfunction of the UFM1CKO mice. Extensive fibrosis and upregulation of ER stress response protein were also noted in conjugation-deficient mutant UFM1 injected UFM1CKO heart. Overall this study indicates that modification of proteins by UFM1 is critical to cardiac function via maintaining ER homeostasis.
Abstract Or122: Noncoding regulation of epicardial gene expression and epithelial-to-mesenchymal transition during heart development
Authors: Nunes Vieira Joaquim , Roque Silva Carolina , Valenzuela Sanchez Mariana , Scheucher Hannah , Dias Tiago , Kalisch-Smith Jacinta , Sparrow Duncan , Giacca Mauro , Riley Paul
Published: October 2024

During organogenesis, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), contributing essential cell types and paracrine signalling to the growing heart. The epicardium is integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties in development. Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for
Wt1
in heart development is established, the mechanisms underpinning its activation remain elusive. We identified two
e
volutionary
c
onserved
r
egions (ECRs) shared in mouse and human, located within intron 1 of
Wt1
locus. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development.

Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR or a deletion comprising both ECRs. Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation. Mendelian ratios indicated an overall underrepresentation of

Wt1
ΔECR/ΔECR

mutants in adulthood. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular and membranous defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal patterning of coronary artery stems in mutant hearts. Expression of
Wt1
was markedly reduced in

Wt1
ΔECR/ΔECR

hearts but not kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue immunostaining revealed abnormal coronary vessel and innervation patterning in the subepicardium, as well as reduced EMT and myocardial non-compaction/hyper-trabeculation in

Wt1
ΔECR/ΔECR

.

Collectively, we demonstrated a requirement for novel
Wt1
intronic enhancers regulating locus activity and essential epicardial EMT-associated biological processes in normal heart development. Importantly, observation of septum and semilunar valve defects in Δ
ECR
hearts suggests an hitherto unrecognised role for WT1-driven EMT, opening new avenues of research to improve our understanding of congenital heart disease affecting at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.
Abstract Tu003: The angiotensinogen/vasorin ratio in peripheral blood, a biomarker for preeclampsia
Authors: Murugesan Saravanakumar , Tubinis Michelle , Powell Mark , Hussey Hanna , Saravanakumar Lakshmi , Melvin Ryan , Sinkey Rachel , Mobley James , Tita Alan , Jilling Tamas , Berkowitz Dan
Published: October 2024

Background:
Preeclampsia (PE) significantly impacts maternal and perinatal health worldwide. Despite limited treatment options, predictive tests could help in early diagnosis and personalized treatment/prevention strategies. Current biomarker, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placenta growth factor (PIGF) offer reasonable negative predictive values, but their use as positive predictive markers is limited. Using unbiased discovery proteomics on urine extracellular vesicles (EV), followed by verification by ELISA and western blotting we identified a novel biomarker candidate for PE, vasorin (VASN), which is significantly decreased in PE, along with a known mediator of PE, angiotensinogen (AGT), that was significantly increased in PE as biomarker candidates.

Hypothesis:
Levels of AGT, VASN and their ratios in peripheral blood can be utilized as biomarkers for PE.

Goals/Aims: 1)
To determine gestational age (GA)-dependence of AGT, VASN and AGT/VASN plasma levels in human and murine pregnancy. 2) To assess disease association of AGT, VASN and AGT/VASN in a cohort of pregnant women with PE with severe features (sPE) and gestational age-matched normotensive pregnant women (NTP) and in a murine model of PE.

Methods:
Plasma was collected from GA-matched NTP and sPE. Timed pregnant mice were injected with adenoviral vector (AD) encoding sFLT-1 on embryonic day 10.5 (mouse PE), or with AD encoding enhanced green fluorescent protein injection (mouse control). AGT and VASN were measured by ELISA in human samples and were quantified by western blotting in murine samples.

Results:
AGT and AGT/VASN showed negative correlations with GA, while VASN levels showed positive correlation with GA in NTP and in mouse controls. PlGF and sFLT-1 levels had weak/no correlations with GA. AGT and AGT/VASN were significantly higher in sPE and mouse PE, while VASN was significantly decreased in sPE and mouse PE as compared to NTP and mouse controls, respectively. To differentiate between sPE vs NTP with receiver operating characteristic curve (ROC) for AGT/VASN: AUC (0.90), Sensitivity =92%, Specificity = 85% at AGT/VASN>81.6.

Conclusion:
AGT and AGT/VASN ratio increase, and VASN decrease can serve as biomarkers for PE.
Abstract Tu005: Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43
Authors: Amin Md Ruhul , Marsh Spencer , Beard Claire , Jourdan Jane , Gourdie Robert
Published: October 2024

Background:
Connexin 43 (Cx43) is the body's most widely expressed gap junctional connexin. Researchers have shown evidence of non-canonical functions of Cx43 and the therapeutic promise of Cx43 mimetic peptides in cardiac pathologies. However, some critical barriers, including in vivo stability and potential immunotoxicity of Cx43 mimetic peptides, hinder clinical translation of these pro-drugs. To address these challenges, we are pioneering bovine milk-derived small extracellular vesicles (mEVs) as delivery vehicles for peptidic drugs. While exploring mEVs as drug delivery tools for αCT1 – a peptide mimicking Cx43 carboxyl terminus (CT) undergoing Phase III testing and known for promoting wound healing and cardioprotection - mEVs without loaded drug cargo exhibited similar bioactivity. This study aimed to test the hypothesis that Cx43, particularly CT fragments of Cx43, are naturally present in mEVs.

Methods:
mEVs were isolated from bovine milk using a novel protocol developed by our lab based on Tangential Flow Filtration and characterized by Western Blotting (WB), Nanoparticle Tracking Analysis, and Transmission Electron Microscopy. To determine the presence of Cx43 in mEVs, we used WB, immunoprecipitation and Single-Molecule Localization Microscopy (SMLM).

Results:
Characterization of mEVs confirmed relatively pure and ultrastructurally definitive mEVs isolated from milk at high density. WB for Cx43 in mEVs lysates based on two antibodies - one against the cytoplasmic loop domain and the other against CT-most 20 amino acids of Cx43, showed that full-length Cx43 was present in mEVs. Interestingly, the Cx43 CT antibody also detected a band at around 16 KDa, and this was confirmed by WB by using two more Cx43 CT antibodies. We also immunoprecipitated this Cx43 CT polypeptide from mEVs lysates using the CT antibodies. SMLM suggested that a subpopulation of mEVs contain Cx43 and demonstrated vesicle-to-vesicle heterogeneity to Cx43 immunolabeling.

Conclusions:
We show for the first time that an endogenous Cx43 CT polypeptide is present in mEVs. Our results provide new insight into the role of Cx43 in mEV bioactivity and potentially offer a path to safe, effective, and orally available therapeutic options based on mEVs.
Abstract Tu007: The Impact of Shear-Thinning Hydrogel Delivery on Extracellular Vesicle Cardiac Retention
Authors: Tarvirdizadeh Touba , Alaa Mohamed Mohamed , Andreadis Stelios , Lang Jennifer
Published: October 2024

Background:
Extracellular vesicles (EVs) are essential mediators of intercellular communication. EVs secreted by cardiosphere-derived cells (CDC-EVs) hold promise as vectors in the context of cardiac repair and remodeling through modulation of cardiomyocyte apoptosis and the immune response following injury. Cardiac retention of CDC-EVs is a critical factor influencing their therapeutic efficacy as first-pass uptake and washout from the myocardium remains a variable. We hypothesized that loading CDC-EVs in a shear-thinning, hybrid hydrogel would provide sustained myocardial delivery and reduced EV extraction by the lungs.

Methods:
The hydrogel system was constructed via a two-step approach utilizing Michael-addition reaction and host-guest complexation. This system incorporates four components: (1) an adamantane-functionalized multi-arm polyethylene glycol, (2) monosubstituted β-cyclodextrin-maleimide, (3) a thiolated hyaluronic acid, and (4) a heparin molecule containing thiol moieties. Following LAD ligation, intramyocardial injections of DiR-labeled EVs or hydrogel-encapsulated DiR-labeled EVs were performed on mice. Animals were euthanized after 24 hours and organs harvested. Whole organ biofluorescence was performed using IVIS Spectrum imaging.

Results:
Hydrogel-encapsulated EVs exhibited enhanced cardiac retention 24 hours post-MI when compared with EVs without hydrogel (17 ± 4.4 vs. 3.4 ± 0.9, mean radiant efficiency as a % of dose ± SEM, p<0.01 using unpaired two-tailed t-test). Mice treated with hydrogel encapsulated EVs showed reduced EV retention in the lungs compared to controls treated with EVs alone (5.7 ± 0.9 vs. 10.6 ± 1.4, mean radiant efficiency as a % of dose ± SEM, p<0.05 using unpaired two-tailed t-test).

Conclusion:
Usage of shear-thinning hydrogel as an EV delivery vehicle enhances EV retention in the heart and decreases off-target uptake of EVs following acute MI. This finding suggests that hydrogel delivery may be a promising strategy to improve the therapeutic efficacy of CDC-EVs for cardiac repair.
Abstract Tu008: Efficient Intramyocardial Delivery of EV-Encapsulated AAVs to Target Cardiomyocytes in a Pre-Clinical Swine Model
Authors: Gallinat Alex , La Salvia Sabrina , Li Xisheng , Raisinghani Nikhil , zhang shihong , Shao Alan , Phan Anh , Mavropoulos Spyros , Ishikawa Kiyotake , Sahoo Susmita
Published: October 2024

Background:
Adeno-associated viruses (AAV) have been widely used to deliver therapeutic genes to the heart due to their safety and efficacy. However, a major obstacle to their successful gene delivery is the presence of neutralizing antibody (NAb) which form during natural exposure to AAV or following AAV administration. Our recent publication has shown that EV-encapsulated AAVs (EV-AAVs) shields AAVs from NAb neutralization, and overcoming this key roadblock associated with AAV-mediated gene therapy.

Hypothesis:
In this study, we addressed whether EV-AAVs can target cardiomyocytes
in vivo
.

Aims:
To test whether cardiomyocytes effectively uptake EV-AAVs
in vivo
, in a pre-clinical swine model.

Methods and Results:
Adult pigs prescreened for a lower NAb titer were randomly assigned to receive intramyocardial administration of either vehicle, AAV6-Luciferase or EV-AAV6-Luciferase. A total of six injections across three injection sites within the left ventricle (LV) were administered per animal. All animals were sacrificed at four weeks and liver and the LV tissues were collected. Luciferase activity assay, flow cytometry analysis of isolated cardiomyocytes (CM) and non-myocytes (NM), and immunofluorescence microscopy were performed to evaluate transgene expression and cellular tropism. Echocardiography was performed at baseline and at 4W to monitor cardiac function. At 4W, EV-AAV6s resulted in a higher luciferase expression in LV compared to nude AAV6 particles. Expression in the LV was higher compared to the liver. Flow cytometry analysis revealed a higher luciferase expression in CM compared to NM, which was corroborated by Immunofluorescence. No differences were detected in the global heart function across groups. Biodistribution of EV-AAV uptake and transgene expression are being studied with intramyocardial and intracoronary delivery mechanisms.

Conclusion:
EV-AAVs deliver higher amounts of genes than nude AAV6 via intramyocardial administration in a pre-clinical swine model. CMs preferentially uptake EV-AAVs compared to NMs. Our studies suggest that EV-AAVs are a superior cardiac gene delivery vector for future clinical studies.
Abstract Tu018: Integrin alpha 1 plays a critical role in angiotensin II-induced abdominal aortic aneurysm rupture
Authors: Amioka Naofumi , Miyoshi Toru , Iida Toshihiro , Saito Yukihiro , Kondo Megumi , Akazawa Kaoru , Akagi Satoshi , Nakamura Kazufumi , Yuasa Shinsuke
Published: October 2024

Background:
Abdominal aortic aneurysm (AAA) is a life-threatening condition without an established specific therapy. This study aims to identify a new therapeutic target for preventing AAA rupture.

Methods and Results:
Angiotensin II (AngII)-infused ApoE-/- mice develop AAA, an established model of AAA. To identify molecular pathways contributing to AAA development, we conducted single-cell RNA sequence analysis using a dataset of aortic tissues harvested from mice infused with AngII for 4 weeks (GSE186865). Gene set enrichment analysis revealed upregulation of integrin- and TGFB-related pathways in AngII-stimulated smooth muscle cells (SMCs). Integrin α1 (Itga1) constitutes half of the α1β1 integrin duplex and serves as a major collagen receptor in SMCs. Male wild-type (n=37) and Itga1 knockout (n=36) mice with an ApoE-/- background received a continuous infusion of AngII for 4 weeks to develop AAA. Kaplan-Meier analysis indicated that ablating Itga1 significantly suppressed AAA rupture (p=0.009), without affecting the maximum diameter of the abdominal aorta. Considering extracellular matrix degradation as a critical hallmark of AAA rupture, we examined the abundance of major matrix metalloproteinases (MMPs) in the aortic wall, specifically MMP2 and MMP9, two major MMPs in the aorta. Gelatin zymography revealed that ablation of Itga1 suppressed AngII-induced MMP9 increase, without affecting MMP2 abundance. The expression of MMP9 is controlled by inflammation and proliferation signals; hence, we focused on these signals. qPCR analysis (n=8 per group) demonstrated that AngII-induced upregulation of Tnfa (p<0.001) and Tgfb1 (p=0.026) was mitigated by Itga1 ablation in aortic tissues.

Conclusion:
The integrin pathway is activated in the tissue of AAA tissue and deletion of Itga1 suppresses AAA rupture. Here we showed that the Itga1-Tgfb1-MMP9 axis would be a novel molecular target to inhibit AAA rupture.
Abstract Tu022: Gut-derived bacterial metabolites promote cardiac inflammation, leading to heart failure in murine model of DSS-induced colitis
Authors: Ranjan Prabhat , Dutta Roshan , Colin Karen , Prasad Ram , Maynard Craig , Verma Suresh
Published: October 2024

Background:
Inflammatory Bowel Disease (IBD) affects millions of people all over the world. This has been known to contribute to several other comorbidities. Recently, impaired gut-barrier permeability has been known to contribute to cardiovascular diseases in IBD patients. However, the precise mechanisms remain unclear. In this study, we propose that bacterial metabolites, like peptidoglycan, may play a role in promoting cardiac inflammation and ultimately leading to heart failure in mice following colitis.

Methods:
We induced colitis in C57 black mice with DSS (dextran sodium sulfate) treatment. Further, we collected fecal sample for 16s rRNA analysis to evaluate microbial flora. Additionally, heart and intestine tissues were collected to determine inflammatory genes expression (qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify the circulating peptidoglycan (PGN) level. The effect of PGN on NFkB inflammatory pathways was evaluated in macrophages.

Results:
Echo-data showed cardiac dysfunction in DSS mice. Masson’s trichrome staining showed deteriorated intestinal villi, while western blot data indicated decreased Occludin and increased PV-1 level, suggesting a leaky gut in DSS treated mice. Comparative analysis of 16s rRNA sequencing data showed significantly altered Firmicutes/Bacteroidetes ratio in DSS treated mice as compared to control mice. Moreover, plasma PGN level was significantly increased 3 weeks following DSS treatment. Real-time gene expression data of pro-inflammatory cytokines (TNFα) and fibrosis associated genes (col1α1, fibronectin) were increased in DSS mice heart. Increased CD68 expression in the immunostaining data of DSS mice heart suggests the recruitment of inflammatory macrophages. At the molecular level, CDK9 expression was significantly increased in macrophage following PGN treatment, which ultimately leads to an increase in NFkB signaling. Finally, CDK9 inhibition attenuated PGN-induced NFkB signaling.

Conclusion:
Together, our study suggests that colitis-induced gut leakage leads to CDK9-mediated NFκB upregulation, contributing to cardiac inflammation. Ongoing research will shed light on the underlying molecular mechanisms of cardiovascular dysfunction.
Abstract Tu027: Impact of Atherosclerosis-Related Small Cerebral Vessel Damages on Tau Pathology in Atherosclerosis Mouse Model
Authors: Liu Xinyuan , Wang Yanru , Han Jingyan
Published: October 2024

Background:
The linkage between atherosclerosis and vascular dementia, including its ties to Alzheimer’s disease (AD), is well-established. This research explores how small cerebral vessel damages, a less recognized consequence of atherosclerosis, contribute to the development of tau pathology, a key AD hallmark.

Methods and Results:
Through the ApoE-/- mouse model, we evaluated the impact of hypercholesterolemia and AngII infusion on tau pathology. The regimen included an atherogenic diet for three months and subsequent AngII infusion (1mg/kg/min) for 28 days in three-month-old mice. Key outcomes measured were phosphorylated tau (AT8), cerebral microbleeds, blood component deposition, and oxidative stress markers. Results indicated a synergistic effect of diet and AngII on increasing microbleeds, vascular leakage, and oxidative stress, which coincided with heightened p-tau in affected brain regions. A specific focus on endothelial glutaredoxin-1 (Glrx) deficiency revealed its critical role in moderating these pathological changes, linking endothelial oxidative stress directly with tau pathology.

Conclusion:
Our research highlights the significant role of small cerebral vessel damage in advancing tau pathology in atherosclerosis, with endothelial oxidative stress as a key mediator. This underscores novel pathways through which vascular health influences AD pathology.
Abstract Tu031: Immunosuppression Drug Modulation of Endothelial Cell Function
Authors: Dexheimer Ryan , Wu David , Elezaby Aly , Sayed Nazish , Chen Ian , Sallam Karim
Published: October 2024

Heart transplantation is a life-saving therapy for patients with end-stage heart failure with over 4,000 transplants occurring per year. The use of immunosuppression drugs is necessary in post transplantation to prevent graft rejection. Proliferation signal inhibitors have previously been shown to effectively reduce the incidence of cardiac allograft vasculopathy (CAV) compared to calcineurin inhibitors but the differential mechanisms that drive this benefit and differences in vascular profile of immunosuppression drugs remain incompletely understood. In this study, we examined the direct effect of immunosuppressive agents on endothelial function to define cellular mechanisms that may contribute to differential vascular phenotypes. We used endothelial cells derived from human induced pluripotent stem cells (iPSCs) and treated them with calcineurin inhibitor (tacrolimus), mTOR inhibitor (sirolimus), or vehicle for 5 days with daily media changes. We examined endothelial function using scratch wound assay, tube formation assay, and LDL uptake. We also examined metabolic changes using Seahorse assay owing to metabolic changes induced by immunosuppression drugs in other organs. Our results indicate mTOR inhibitor (sirolimus) resulted in a reduction in endothelial cell proliferation and migration using the scratch wound assay and tube formation assay compared to vehicle or calcineurin inhibitor (tacrolimus)-treated cells. Sirolimus also reduced LDL uptake in iPSC-derived endothelial cells compared to vehicle or tacrolimus-treated cells. Tacrolimus was associated with a trend towards altered endothelial energetics. Tacrolimus and sirolimus exert differential endothelial cell functional profiles that do not fully explain the clinical outcomes observed suggesting a need to explore the drug endothelial profiles in the context of vascular injury and inflammation.
Abstract Tu033: CXCL5: The Next Guardian of the Galaxy to challenge Atherosclerosis
Authors: Sanchez-Hodge Rebekah , Bahnson Edward , Schisler Jonathan
Published: October 2024

Atherosclerosis is a persistent inflammatory disease characterized by the buildup of lipid-laden macrophages (foam cells) in arterial walls and has been linked to several cardiovascular ailments. Despite advances in understanding its pathogenesis, the underlying mechanisms of atherogenesis, particularly regarding chemokine influence, has yet to be investigated. Our laboratory has previously identified CXCL5, a chemokine known for its involvement in inflammatory responses, as a potential mediator of atherosclerosis. We found that patients with elevated CXCL5 levels were less likely to have experienced a previous heart attack or develop coronary artery disease, suggesting that CXCL5 may be atheroprotective. To further investigate the role of CXCL5 in atherosclerosis, we conducted a study using mice on an apolipoprotein E (apoE) knockout background, to promote the formation of plaque, with varying alleles (0, 1, or 2) of CXCL5. Mice were placed on a high-fat diet regimen starting at approximately 10 weeks of age and continued until 26 weeks of age. Aortas, echocardiograms, and blood plasma were obtained to assess the development of atherosclerosis and its impact on cardiac function and metabolic parameters. Our
en face
oil red o staining results showed that the aortas of mice lacking one (p=.0314) or both alleles (p=.0783) of CXCL5 exhibited elevated plaque formation compared to that of wild-type mice. Echocardiographic data revealed that left ventricular mass (corrected) was highest in CXCL5 knockout mice and increased significantly over time (p=.0170) in these animals. Additionally, a significant increase in heart rate (p=.0095) and glucose (p=.0274) levels was only observed in CXCL5 heterozygous mice over the 16-week period, while triglyceride levels increased in both CXCL5 heterozygous (p=.0010) and knockout (p=.0241) mice. Interestingly, high-density lipoprotein (HDL) levels, which is often considered as "good cholesterol," increased significantly only in wild-type (p=.0031) mice, where CXCL5 was present, over the 16-week period. These findings suggest that CXCL5 may play a protective role against atherosclerosis and its associated cardiac complications. In conclusion, our study provides novel insights into the complex interplay between inflammation, lipid metabolism, and cardiovascular disease, highlighting the potential therapeutic value of CXCL5 in the prevention and treatment of atherosclerosis.
Abstract Tu040: A Novel Anti-Thrombotic and Anti-AF Drug without an Adverse Bleeding Problem
Authors: Ricchiuti Nikola , Niebrzydowski Maxwell , Richardson Abigail , Yan Jiajie , Ai Xun
Published: October 2024

Introduction:
Aging is an independent risk factor of atrial fibrillation (AF), which leads to a high mortality due to thromboembolic stroke. But available antithrombotic therapies are limited for the aged AF patients due to aging-associated bleeding risk. We recently revealed critical roles of activated cardiac stress kinase JNK2 in both enhanced AF risk and thrombogenesis in aging through a mechanism of heart-platelet crosstalk. Here, we assessed anti-thrombotic potentials of JNK2 inhibition.

Methods:
Young (Yg) and aged (2 vs 24 mo) wildtype (WT) mice with and without a JNK2-specific inhibitor (JNK2I, 5 mg/kg i.p.; every other day/4 doses) or meloxicam (0.5 mg/kg/day, i.p.; five days) treatment were used for
in vivo
FeCl
3
-induced carotid artery occlusion (thrombus formation) and tail bleeding assays. The effects of cardiac JNK2-specific inhibition in thrombogenesis and bleeding were assessed in our unique cardiac-specific mice of MKK7D (inducible overexpression of active MKK7D, a JNK upstream activator) and MKK7D-JNK2dn (overexpressed inactive dominant negative (dn) JNK2 to competitively inhibit endogenous JNK2 in MKK7D mice).

Results:
Aged mice had a shortened artery occlusion time compared to Yg (5.8±0.2 min vs. 7.1±0.2 min, p<0.001, n=12,7). JNK2I reversed the occlusion time in aged mice to that seen in Yg (p=NS, n=8,7). Yg MKK7D mice also showed a shortened occlusion time compared to WT-controls (p<0.001, n=9,8) that was rescued by cardiac-specific JNK2 inhibition in MKK-JNK2dn mice (p=NS, n=5,8), suggesting a key role of cardiac JNK2 in thrombus formation. Next, we found that 40% of aged WT mice had continuous bleeding compared to its absence in Yg WT. However, meloxicam (an anti-platelet NSAID drug) increased continuous bleeding risk in both aged (83% had it) and Yg (20%) and prolonged the initial clotting time in aged mice (385±56s vs 84±18s in aged controls; p<0.001, n=8,6). In contrast, JNK2I eliminated this bleeding issue, evidenced from the absence of continuous bleeding and shortened initial clotting time compared to aged controls (p=NS, n=8,8).

Conclusion:
Heart JNK2 enhances thrombotic risk in the blood. JNK2 inhibition could be a promising anti-thrombotic and anti-AF therapeutic approach without adverse bleeding for aged AF patients.
Abstract Tu049: Stabilization of RyR2 with dantrolene treatment ameliorates left ventricular remodeling and ventricular tachycardia after myocardial infarction
Authors: Fujii Shohei , Suetomi Takeshi , Kobayashi Shigeki , Nakamura Yoshihide , Uchinoumi Hitoshi , Yamamoto Takeshi , Yano Masafumi , Sano Motoaki
Published: October 2024

Background: Dantrolene has been reported to suppress diastolic Ca
2+
leakage through the cardiac ryanodine receptor (RyR2) by restoring defective inter-subunit interactions within RyR2 and enhancing the binding affinity of calmodulin (CaM) to RyR2. Here, we investigated whether chronic administration of dantrolene which was started after myocardial infarction (MI) inhibits ventricular tachycardia (VT) and alleviates left ventricular remodeling.

Methods: We developed MI model by left anterior descending coronary artery (LAD) ligation in mice. Wild type mice were divided into 4 groups; sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN, 20mg/kg/day, i.p.), MI mice (WT-MI) and MI mice treated with dantrolene (WT-MI-DAN). Then, they were followed up for 12 weeks.

Results: The 12-week survival rate was significantly higher in MI-DAN group (71 %) than in WT-MI (40%). Serial echocardiography showed that adverse LV remodeling was ameliorated in WT-MI-DAN group compared to WT-MI group (WT-MI 6.3 ± 0.25 mm, WT-MI-DAN 5.5 ± 0.14 mm, P<0.01). VT test by epinephrine (2mg/kg, i.p.) showed that VT was induced in all WT-MI group, although it was significantly inhibited in WT-MI-DAN group.

An increase in diastolic Ca
2+
spark frequency and a decrease in sarcoplasmic reticulum Ca
2+
content were observed in isolated cardiomyocytes from WT-MI group whereas both were significantly ameliorated in WT-MI-DAN group.

Conclusions: Dantrolene improves the diastolic Ca
2+
leakage from sarcoplasmic reticulum of damaged cardiomyocytes. Chronic administration of dantrolene after MI inhibits VT and ameliorates LV remodeling, leading to improved prognosis after MI. RyR2-targeting therapy could be a novel strategy for the treatment of post MI.
Abstract Tu054: Deficiency or targeting inhibition of histone lysine demethylase KDM5B
in vivo
for potential therapeutic effects on both HFrEF and HFpEF
Authors: Zhao Wen , Qiao Hui , Wang Ziwei , Wang Meifei , Wang Haopeng , Wang Wenlong , Wang Qiqi , Tan Shixuan , Jiao Lemin , Xu Jiale , Li Yalan , Gao Erhe , Yuan Ziqiao , Liu Hong-Min , Dong jianzeng , Yu Bin
Published: October 2024

Heart failures with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF) have been recognized as the leading cause of morbidity and mortality worldwide. Histone lysine demethylase KDM5B, an important anti-tumor drug target, has been involved in several cardiovascular diseases. However, its role in heart failure remains largely unknown. In the current study, we found that the expression of KDM5B were significantly enhanced and KDM5B’s downstream substrate: the methylation level of H3K4me3 was decreased in the mouse hearts of both trans-aortic constriction (TAC)-induced HFrEF and high fat diet (HFD) with Nω-nitro-L-arginine methyl ester (L-NAME) (two-hit)-induced HFpEF. By generating a tamoxifen-induced cardiomyocyte- (CM-KO) and a myofibroblast- (MF-KO) specific KDM5B Cre-loxP conditional knockout mice, specifically, we further found that either KDM5B CM-KO or MF-KO mouse significantly ameliorated pathological ventricular remodeling, fibrosis and rescued the cardiac dysfunction phenotypes induced by TAC or the two-hit models, specifically. Furthermore, pretreatment of a KDM5B specific inhibitor TK-129, which was previously identified by our group, also significantly attenuated myocardial hypertrophy, fibrosis and improved cardiac dysfunction in the two mouse heart failure models. Our findings demonstrated, for the first time, that genetic deficiency of KDM5B in either cardiomyocytes or myofibroblasts
in vivo
or pharmacological inhibition of KDM5B display a similar therapeutic effect in the pathological cardiac remodeling and heart failure in both pressure overload-induced HFrEF and HFpEF mouse models, suggesting that KDM5B is a potential therapeutic target for these two different HFs.
Abstract Tu066: Loss of Sigmar1 Aggravated Cardiac Proteotoxicity and Cardiac Dysfunction in Mutant αB-Crystallin Mouse
Authors: Aishwarya Richa , Abdullah Chowdhury S. , Remex Naznin Sultana , Bhuiyan Mohammad , Orr Wayne , Bhuiyan Md Shenuarin
Published: October 2024

Background:
Deposition of misfolded protein aggregates observed in diverse cardiac disease models leads to proteotoxic cardiomyopathy. In earlier studies, we confirmed mutations in α-B-Crystallin, specifically Arg120Gly (CryAB
R120G
), develop proteotoxic cardiomyopathy. Cardiomyocyte-specific CryAB
R120G
overexpression leads to cardiac dysfunction resulting from the accumulation of toxic pre-amyloid oligomers and protein aggregation. In the event of proteotoxic insult, the chaperone proteins function to prevent the accumulation of protein aggregates. One of the multitasking chaperone proteins in the heart is Sigmar1, whose role in proteotoxic cardiomyopathy and protein aggregation remains unknown.

Hypothesis:
We hypothesize that Sigmar1 plays an essential role in protein aggregate clearance, and the absence of Sigmar1 in CryAB
R120G
mice aggravates pathological cardiac remodeling.

Methods and Result:
We measured Sigmar1 expression level in CryAB
R120G
hearts and found a significant reduction in endogenous Sigmar1 protein and transcript levels in CryAB
R120G
hearts. Co-immunoprecipitation and yeast-two-hybrid assays demonstrated a direct interaction between Sigmar1 and CryAB. Therefore, we evaluated Sigmar1’s function in the proteotoxic model of heart failure. We ablated Sigmar1 in CryAB
R120G
, expressing myocytes both
in vitro
by using Sigmar1 siRNA and
in vivo
by crossing Sigmar1 knockout with CryAB
R120G
(CryAB
R120G
xSigmar1
-/-
)
.
Both i
n vitro
and
in vivo
studies displayed a dramatic increase in protein aggregate size and content in CryAB
R120G
xSigmar1
-/-
heart, assessed by immunostaining and electron microscopy. Echocardiography showed age-dependent aggravation of cardiac contractile dysfunction, and Masson’s Trichrome staining showed increased cardiac fibrosis in the CryAB
R120G
xSigmar1
-/-
hearts compared to CryAB
R120G
hearts.

Conclusion:
Overall, we found that Sigmar1 directly interacts with CryAB and plays a vital role in the degradation of protein aggregates where the absence of Sigmar1 causes increased protein aggregate size and content in CryAB
R120G
mice, leading to aggravated pathology with ventricular dysfunction, increased interstitial fibrosis, and cardiac hypertrophy.
Abstract Tu072: O-GlcNAcylation underlies the activation of sodium-glucose cotransporter 1 in diabetic hearts
Authors: Pandey Vivek , Velmurugan Sathya , Liu Miao , Despa Sanda
Published: October 2024

Rationale:
Type-2 diabetes (T2D) greatly increases the risk for developing heart failure. Recent evidence indicates that the cardiac expression and function of the sodium-glucose cotransporter 1 (SGLT1) is elevated in disease states, including T2D, leading to structural and functional remodeling of the heart. The mechanisms underlying SGLT1 activation in the diabetic heart are currently unknown.

Objective:
To investigate if SGLT1 undergoes the O-linked attachment of β-
N
-acetylglucosamine (O-GlcNAcylation), a post-translational modification that is exacerbated in T2D, and its effect on SGLT1 activity in diabetic hearts.

Methods/Results:
SGLT1 co-immunoprecipitates with O-GlcNAc in heart homogenates and HL-1 cardiomyocyte lysates. Enhancing global protein O-GlcNAcylation in HL-1 cells via si-RNA-mediated silencing of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, resulted in increased amounts of O-GlcNAc that precipitate with SGLT1. SGLT1 function, measured as the SGLT1-mediated Na
+
influx, was significantly increased in HL-1 cells with silenced OGA. These data suggest that SGLT1 undergoes O-GlcNAcylation and this modification results in SGLT1 activation. Using rats that express human amylin in the pancreatic β-cells (HIP rats) as a model of late-onset T2D and their wild-type littermates as controls, we found that a larger fraction of SGLT1 co-immunoprecipitates with O-GlcNAc in hearts from T2D rats compared to controls. The SGLT1-mediated Na
+
influx was larger in myocytes from T2D
vs
. control rats. Increasing total O-GlcNAcylation with Thiamet-G in control myocytes resulted in larger Na
+
influx. In reverse experiments, inhibition of O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine crystalline reduced SGLT1-mediated Na
+
uptake in myocytes from T2D rats. Inhibition of O-GlcNAcylation in T2D myocytes reduced Ca
2+
spark frequency and this effect was significantly less pronounced with SGLT1 blocked. These results suggest that O-GlcNAcylation affects myocyte Na
+
regulation by activating SGLT1, which contributes to myocyte Na
+
overload and its downstream effects on sarcoplasmic reticulum Ca
2+
leak in T2D.

Conclusion:
Exacerbated O-GlcNAcylation underlies SGLT1 activation in diabetic hearts. Preventing O-GlcNAcylation may limit myocyte Na
+
overload and the frequency of pro-arrhythmogenic Ca
2+
sparks in myocytes from T2D rats.
Abstract Tu086: Repairing nuclear envelope ruptures to ameliorate Lamin-related cardiomyopathy
Authors: En Atsuki , Bogireddi Hanumakumar , Thomas Briana , Stutzman Alexis , Ikegami Sachie , Laforest Brigitte , Almakki Omar , Pytel Peter , Moskowitz Ivan , Ikegami Kohta
Published: October 2024

Heterozygous loss-of-function mutations in
LMNA
, encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A prevailing hypothesis posits that
LMNA
insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, the mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted
Lmna
in cardiomyocytes in adult mice (

Lmna
CKO

). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in

Lmna
CKO

cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in

Lmna
CKO

mice, we conducted time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from

Lmna
CKO

cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in

Lmna
CKO

mice. We found that the envelope ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repair. We further found that overexpression of DNA-binding protein BANF1 promoted ESCRT-III recruitment to the ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in

Lmna
CKO

mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for
LMNA
-related dilated cardiomyopathy.
Abstract Tu088: Fibroblast Activation, Collagen Secretion, and Migration Occurs Independently of
Has2 Expression
Authors: Little Danielle , Nguyen Daniel , Brittian Kenneth , Brainard Robert , Jones Steven
Published: October 2024

Background:
Hyaluronan (HA) accumulates after tissue injury and may regulate the wound-healing process. We found that HA is increased in and around the scar following myocardial infarction (MI). Fibroblasts are the main producers of the collagen that comprises the scar, and we have shown that they are responsible for HA accumulation. Given that these two extraordinarily high-volume synthetic processes occur in the same place and simultaneously, we queried whether alterations in
Has2
-dependent HA synthesis impacted collagen secretion and other fibroblast functions.

Goal:
To determine the impact of HA synthesis on fibroblast function.

Methods and Results:
We isolated cardiac fibroblasts from

Has2
fl/fl

mice and treated with Cre adenovirus to delete
Has2
expression or
Has2
adenovirus to overexpress
Has2.
We assessed cell number and found no differences when
Has2
was deleted or overexpressed in both naïve fibroblasts (n=6); activation with TGF-b did not change this response
.
We used a scratch assay to assess migration and found no change between treatments in naïve fibroblasts (n=6). Hydroxyproline assessment of deposited collagen and immunoblotting for soluble collagen secretion in the media indicated no differences between treatments in naïve fibroblasts (n=3).

Conclusion:
Genetic manipulation of HA production did not impact proliferation, migration, or collagen secretion in fibroblasts. Considering the enormous metabolic requirement of fibroblasts to produce HA and collagen, these results suggest that the regulation of these processes may be distinct or that the substrate is not limiting in our experimental conditions. In the intact heart, substrate could become limiting and a relationship between these two biosynthetic processes may emerge