bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2066: Forced Exercise Prevents Reductive Stress Induced Cardiomyocyte Remodeling And Cardiac Dysfunction
Authors: Jyothidasan Arun , Sunny Sini , Litovsky Silvio , Pogwizd Steven M , Namakkal-Soorappan Rajasekaran N
Published: October 2023

Background:
Disruption of the endoplasmic reticulum (ER) function has been recognizedas a clinical signature of congestive heart failure, ischemia, and atherosclerosis. Whilepro-oxidative redox milieu is vital for the ER to regulate protein folding events. Recently,we reported that overabundant antioxidants in the heart cause reductive stress (RS) andimpair the protein folding mechanisms due to unresolved ER stress. Here, we testedwhether forced exercise prevents RS and improves ER function through augmenting ROSsignaling, thereby improving the PQC mechanisms in the heart.

Methods:
Cardiac-specific constitutively active Nrf2 (CaNrf2-TG) and non-transgenic (NTG) mice at 5-6weeks of age were subjected to forced treadmill exercise (FTE) for 20 weeks (15meters/minute with a 5° slope; for 45 minutes/day; for 6 days every week). Cardiacstructure and function were analyzed before and after the exercise. At 22 weeks, micewere sacrificed for analysis to assess the benefits of FTE.

Results:
RNA sequencingrevealed significant downregulation of key protein folding genes and chaperones in TGmice in association with the pathological remodeling at 6 months. Particularly,
Pdi, Atf6,
Calr,
and
Ero1α
were downregulated (>2 fold) at 24 weeks in TG mice, indicating impairedER function in the remodeling hearts. These changes were coupled with increasedcardiomyocyte size and hypertrophy. Upon FTE, we observed significant improvementsin the structure and function of the cardiomyocytes. WGA staining showed the cross-section area of the cardiomyocytes was significantly reduced in the exercise groupcompared to the sedentary-TG mice experiencing cellular hypertrophy (115.7μm
2
NTGvs 428.8μm
2
TG vs 202.5 μm
2
TG-FTE). Echocardiography analysis demonstratedpreservation of cardiac structure and function (EF, FS, IVD, IVS, EDV, and ESV).Specifically, compensatory improvement in diastolic function (MVE/A ratio) was observedin TG-FTE exe group. Additionally, we observed that the ubiquitination of proteins wassignificantly reduced in the exercise group indicating deaccumulation of the misfoldedproteins in RS hearts.

Conclusion:
Together these results demonstrate that chronicexercise could ameliorate RS-induced ER stress and cardiac dysfunction.
Abstract P2073: Hyperbaric Oxygen Therapy Protects The Myocardium From Reductive Stress-induced Proteotoxic Remodeling
Authors: Sunny Sini , Houg jennifer , Navaneeth Shibu , Aniqa Sayed , John Kofi Afortude , Namakkal-Soorappan Rajasekaran N
Published: October 2023

Background:
Oxygen is the prime source of reactive oxygen species (ROS) in cells. We hypothesize that hyperbaric oxygen therapy (HBOT) can be used as an effective intervention to maintain redox homeostasis in the myocardium. Here, we assessed whether HBOT could prevent RS through enhanced ROS signaling, thereby protecting the hearts from proteotoxic stress.

Methods:
Cardiac-specific human Nrf2 transgenic (hNrf2-TG) and their non-transgenic littermates (NTG) were used in this study. Mice at the age of 8 weeks were exposed to hyperbaric oxygen (2.5 Bars, 2 hr/day) for 8 weeks. The TG and NTG (n=6) mice without HBOT served as controls. Pre- and post- echocardiography (Vevo 2100) analysis were carried out to assess the structural and functional changes over time in response to HBOT exposure. The influence of HBOT on matrixins (MMP2/9), redox (GSH & NQO1), and cardiac hypertrophy markers (ANF, BNF) were examined using zymography, immunoblotting, and qPCR analysis. The aggregation of proteins in myocardial tissues was detected by Proteostat staining.

Results:
While the expression of hNrf2 induces RS in the mouse heart, we noticed that HBOT ameliorates the RS through enhanced ROS signaling. A decrease in systemic GSH levels along with the downregulation of ANF and BNF mRNA levels observed in the HBOT group indicates the restoration of the redox homeostasis as well as reduced remodeling of the myocardium. Interestingly, hNRF2-induced RS was suppressed by HBOT as supported by changes in the protein levels for NQO1, GCLC, and GCLM. The degree of protein aggregation in RS hearts was significantly reduced by HBOT. Vevo strain analysis reveals that 8 weeks of HBOT restored the diastolic stretch seen in the RS hearts to a normal diastole-systole velocity equal to NTG mice. A significant decrease in the ejection fraction (80% vs. 55% in TG vs. TG-HBOT), was evident in the TG mice that received HBOT. Repression of matrixins, observed in the hearts of HBOT-TG mice signifies the benefits of HBOT in RS hearts.

Conclusion:
HBOT directly improves myocardial structure at the tisuue level; maintain the redox status at systemic level, and improves the myocardial performance at functional level. Our study established that HBOT can rescue the myocardium from RS-induced proteotoxic insults.
Abstract P2089: Myocardial Long Non-Coding RNAs Expression Signatures In Heart Failure With Preserved Ejection Fraction
Authors: Guo Alina , Yoo Edwin , Knuttsdottir Hildur , Kass David A , Sharma Kavita , Hahn Virginia S
Published: October 2023

Heart failure with preserved ejection fraction (HFpEF) accounts for greater than half of all HF cases and has 5 year mortality rates on par with HF with reduced ejection fraction (HFrEF). Unlike HFrEF, however, HFpEF patients have limited therapeutic options. Improved understanding of myocardial epigenetics are needed to develop novel therapeutics. We have previously conducted myocardial transcriptomics in HFpEF and found several thousand differentially expressed genes between HFpEF vs Controls. Targeting a single gene or pathway will likely not address the many abnormalities seen in HFpEF, highlighting the need for epigenetic approaches. Long non-coding RNAs (lncRNAs) exert epigenetic control over large numbers of transcripts and represent potential therapeutic targets in HFpEF.We measured lncRNA expression from myocardial tissue from HFpEF (n=41), HFrEF (n=30), and control (CON, n=24). Agnostic principal component and hierarchical gene clustering analyses separated HFpEF, HFrEF and CON with minimal overlap. LncRNA’s differentially expressed in HFpEF were predicted to control pathways related to neurotransmitter release, cytoskeleton, and muscle cell development. Three lncRNA-derived HFpEF subgroups were identified with distinct clinical correlates and clinical outcomes, and a subset of these predicted a poor prognosis. Differential expression analysis using DESeq2 revealed 95 upregulated and 342 downregulated lncRNAs.Our findings suggest that HFpEF has a lncRNA profile distinct from HFrEF and controls, highlighting potential epigenetic therapeutic targets for HFpEF.
Abstract P2104: Branched-chain Amino Acids Are Required For Acquisition Of The Cardiac Myofibroblast Phenotype
Authors: Nguyen Daniel C , Wells Collin , Dassanayaka Sujith , Jones Steven P , Hill Bradford G
Published: October 2023

Introduction:
Branched-chain amino acids (BCAAs) are critical mediators of anabolic signaling and are essential for cell growth. Findings in both animals and humans indicate that intra-cardiac BCAA levels are markedly elevated in the myocardium after infarction. Nevertheless, it remains unclear how BCAAs contribute to processes involved in post-infarction cardiac remodeling, such as fibrosis. The goals of these studies were to evaluate whether BCAAs can regulate collagen production and facilitate myofibroblast transdifferentiation.

Methods and Results:
To determine how profibrogenic stimuli influence genes critical for BCAA uptake and catabolism, we performed bulk RNA sequencing on fibroblasts isolated from sham and post-infarcted hearts as well as from naïve fibroblasts treated with TGFβ. Compared with fibroblasts from sham hearts, fibroblasts from post-infarcted hearts had higher expression of BCAA transporters (
Slc7a8
,
Slc3a2
,
Slc38a1
,
Slc1a5
), lower levels of the inhibitory kinase
Bckdk
, and higher levels of Bckdh complex constituents (
Dbt,
Dld
; n=3–6/group;,q<0.05). Similarly, TGFβ elevated
Slc3a
and
Slc38a1
as well as the BCAA catabolic gene,
Bcat1
(n=3/group; q<0.05). Culture of cardiac fibroblasts in BCAA-free medium completely prevented TGFβ-induced Col1a1 and periostin upregulation and markedly diminished α-smooth muscle actin abundance (n=3/group; p<0.05).

Conclusion:
In cardiac fibroblasts, profibrogenic stimuli increase the expression of genes involved in BCAA uptake and catabolism. BCAA availability significantly impacts fibroblast activation by regulating collagen synthesis and myofibroblast transdifferentiation. Ongoing studies will elucidate whether BCAA catabolism contributes to myofibroblast activation and whether lower dietary BCAA levels influence cardiac remodeling after myocardial infarction.
Abstract P2129: Myocardial Stunning Model In Rats
Authors: Elmahdy Ahmed , Shekka Espinosa Aaron , Hussain Shafaat , Kakei Yalda , Pylova Tetiana , Al-Awar Amin , Krasnikova Maryna , Zulfaj Ermir , Jha Abhishek , Andersson Axel , Sevastianova Valentyna , Nejat Amirali , Redfors Björn
Published: October 2023

Background:
Myocardial stunning is a temporary loss of contractility without irreversible histological damage. In myocardial infarction(MI), stunning could lead to acute ischemic heart failure (AIHF). We aim to establish a model of myocardial stunning in rats.

Methods:
Rats were randomized to either two 5 min cycles of preconditioning(PC) or non-preconditioning (NPC) before ligating LAD for 13.5 min. Echocardiography (ECHO) derived wall motion score index (WMSI). Speckle tracking assesed heart function. TTC staining determined infarction size

Results:
PC-13.5 yielded akinesia at 4h and recovery at 48h by WMSI. Speckle tracking showed a worsening in LVEF, global longitudinal strain (GLS), and time to peak radial strain (TP) in the ischemic segment at 4h which normalized at 48h. In NPC-13.5 at 48h, LVEF, GLS, and TP radial strain didn′t recover. TTC staining showed minimal necrosis in PC and substantial necrosis in NPC.

Conclusion:
The study characterized a reproducible model of myocardial stunning. ECHO showed a human-like course of myocardial stunning. TTC showed absence of irreversible damage.
Abstract P2132: The Raf Kinase Inhibitor Protein Protects Mitochondria From Ischemic Stress
Authors: Brand Theresa , Lukannek Ann-Kathrin , Kautz Christian , Schanbacher Constanze , Gedik Nilgün , Kleinbongard Petra , Lorenz Kristina
Published: October 2023

Background:
The Raf kinase inhibitor protein (RKIP) increases cardiac inotropy without detrimental remodeling and even protects from the development of heart failure in response to chronic pressure overload or catecholamines. These RKIP effects are mediated via simultaneous and persistent activation of cardiac β1- and β2-adrenergic receptors (βAR).

Hypothesis:
Since an intact mitochondrial function is central for the energy supply and cardiomyocyte health, which are parameters that had been proven to be positively influenced by RKIP in the heart, we here aimed to analyze the impact of RKIP and βAR signaling on mitochondria.

Methods and Results:
Electron microscopy, respiration measurements and confocal analysis of the mitochondrial membrane potential (mtMP) revealed that the mitochondria of RKIP knockout mice are of smaller size and abnormal shape, show a reduced ADP-stimulated respiration and a hyperpolarized mtMP compared to WT mitochondria. And, expression levels of several respiratory proteins were reduced. In contrast, mitochondrial content in RKIP-tg mouse hearts was enhanced, mitochondrial function maintained, and expression of proteins involved in respiratory complexes and mitochondria-related transcription (e.g. Tfam, NRF-1) was enhanced. To evaluate the stress resistance of RKIP-tg hearts, we subjected isolated cardiomyocytes or hearts of RKIP-tg or WT, respectively, to ischemia/reperfusion (I/R) stress. I/R effectively decreased the mtMP in WT cardiomyocytes and impaired ADP-stimulated respiration of isolated WT mitochondria after ex vivo I/R. In contrast, RKIP-tg mitochondria were protected from mtMP collapse and impaired respiration after I/R. To understand this protective RKIP-mediated effect and whether βAR are involved, we analyzed mtMP in neonatal cardiomyocytes of β2AR knockout mice, upon application of different βAR ligands, pertussis toxin and inhibitors of the PI3K or AKT. These experiments suggest a RKIP-mediated mitochondrial protection via β2AR/Gi/PI3Kα/pAKT signaling.

Discussion:
Our study shows that RKIP-βAR signaling protects mitochondria from I/R stress suggesting βAR modulation as a potential therapeutic strategy to protect the myocardium in ischemic conditions.
Abstract P2140: Orally Delivered Modified Citrus Pectin Attenuates Aortic Dissection Development By Regulating Macrophage Pyroptosis Via Ierruption Of Galectin-3 And Toll-like Receptor 4 Interaction
Authors: Lu Hsin-Ying , Shih Chun-Che
Published: October 2023

Background:
Aortic dissection (AD) is a lethal vascular disease caused by the formation of hematoma in the middle layer of the aortic wall. Inflammation and oxidative stress play important roles in the development of AD. Galectin-3 (Gal-3) is a 26-kDa lectin recognized to regulate many aspects of inflammatory cell behavior. Modified citrus pectin (MCP) is a pH-modified form of the dietary soluble citrus peel fiber known as pectin. It is abundant in β-galactose, which allows it to bind to Gal-3, and is recommended as a therapeutic agent for immune support, cancer, toxicity, and fibrotic diseases. The aim of this study was to explore the effect of MCP on AD and its potential mechanism involved.

Methods and Results:
MCP (100mg/kg) was orally administrated once every two days to retard the development of 3-aminopropionitrile fumarate/angiotensin II-induced AD animal models. In vivo, AD aorta presented an increase in Gal-3 levels that were accompanied by an increase in macrophage. Treatment with MCP decreased the incidence and mortality of AD by reducing inflammatory cell infiltration to the aorta; it downregulated the gene expression of inflammation and pyroptosis, such as IL-6, TNF-α, NLRP3, caspase-1, and IL-1β. In vitro, MCP could inhibit macrophage pyroptosis in H
2
O
2
-stimulated Raw264.7 cells. Additionally, Gal-3 is directly bound to TLR4 in H
2
O
2
-induced macrophages but MCP almost completely disrupted their interaction and suppressed activation of NF-KB by co-immunoprecipitation assays.

Conclusions:
Gal-3 could be a new molecular mechanism associated with AD disease, implying the use of MCP as a promising therapeutic strategy to prevent AD development.
Abstract P2141: Effect Of Anakinra On Peak Oxygen Consumption In Patients With Heart Failure With Reduced Ejection Fraction: A Meta-analysis
Authors: Jain Manisha , Meenashi Sundaram Dakshin , Mahfooz Kamran , Gaddipati Sai Pranathi , Ramnani Subhav , Baig Kaunain , Kaur Gurmehak , Gudi Thulasi Ram , Vasavada Advait
Published: October 2023

Background:
Peak oxygen consumption(VO2) evaluates an individual's aerobic fitness level, and provides information on overall health and fitness. Prior studies on anakinra in heart failure have not evaluated VO2 specifically in the subgroup of heart failure with reduced ejection fraction(HFrEF). Our aim was to evaluate the effect of this drug in this population.

Methods:
We searched five databases: Embase, MEDLINE, Cochrane, Scopus, and PubMed, for RCTs evaluating the effects of Anakinra against a placebo in HFrEF. Meta-analysis was performed using RevMan 5.4 and a random-effects model was used for the analysis. Three RCTs were identified

Results:
The analysis of the Anakinra effect on VO2 in patients with HFrEF was determined using 3 RCTs. Most studied its effect after 2 weeks. Compared to placebo/standard treatment, anakinra was associated with a greater change in peak VO2 from baseline VO2. However, the difference between Anakinra and placebo in peak VO2 changes was not statistically significant (MD 0.07, 95% CI -0.41 to 0.56; I2 = 43%) with an overall test effect of Z = 0.29 at p = 0.77. The VE/VCO2 slope determined the effect of Anakinra on ventilatory efficiency in HF patients. Among the 3 RCTs that evaluated VE/VCO2 slope, no difference was depicted between the Anakinra treatment relative to placebo. Thus, the statistical difference was insignificant between the two groups (MD 0.70, 95% CI -0.06 to 1.46; I2 = 0%) at p = 0.07.

Conclusion:
Anakinra was ineffective in improving VO2 in HFrEF. Future prospective studies with longer duration of treatment and measuring performance monitoring outcomes are needed to understand the effect of this drug for such outcomes.
Abstract P2145: A Review Of Trends In Cardiovascular Medicine Clinical Trials; 2012-2021
Authors: Rawlley Bharat , Bansal Kannu , Dayal Utkarsh , Julka Dhawani , Salooja Ishita , Cordova Sanchez Andres , Kumar Sandeep , Chaudhuri Debanik
Published: October 2023

Introduction:
We examine cardiovascular medicine clinical trials (CTs) from the United States over the past decade focusing on characteristics that are important for assessing the reliability of evidence derived from them.

Methods:
The Clinicaltrials.gov API interface was queried with a comprehensive search strategy to identify trials in the field of cardiovascular medicine from the US registered from 1/1/2012 till the 12/31/2021. We extracted data on the presence of data monitoring committees (DMC), masking status and allocation methods used in the CTs.

Results:
Our initial query identified 1299 CTs. After reviewing and excluding unrelated trials, 536 were found to be cardiovascular medicine CTs. Of these, 507 (95%) reported if they had a DMC or not, 261 (49%) reported having a DMC while 246 (46%) reported not having one. 471 (88%) CTs reported on their design allocation method, most common being randomized as reported by 312 (58%) of the trials, 43 (8%) were non-randomized and 116 (22%) reported that a design allocation was not applicable to them. 471 (88%) CTs reported on their masking status, most common being open label as seen in 247 (46%) of the trials followed by double masking seen in 71 (13%) of the trials. On performing a Cox and Stuart trend test, no trend was seen in the annual proportion of trials with randomization, a DMC or masking over the past decade [P > 0.05 for all three].

Conclusions:
In these analyses of 536 CTs, we found that > 50% did not have a DMC or any masking and no favorable trend was seen in the annual proportion of same over the past decade. Additionally, majority of the trials reported randomization as the design allocation method.
Abstract P2148: Beyond Traditional Pharmacology: A Critical Appraisal Of Enhanced External Counterpulsation (EECP) As A Promising Alternative Treatment For Hypertension
Authors: Kapoor Abhay A , Vaja HariOm S , Kaur Gurleen , Rangrej Vraj J , Karri Juhu Kiran Krushna , Aneef Asiff Nathi
Published: October 2023

Introduction:
Hypertension is a significant global public health concern and a leading cause of cardiovascular disease. Despite the availability of numerous pharmacological therapies, many hypertensive patients experience inadequate blood pressure control, medication side effects, or resistance. Thus, non-pharmacological interventions such as Enhanced External Counter pulsation (EECP) have garnered interest as a promising alternative treatment for hypertension.This systematic review aims to evaluate the existing clinical evidence on the efficacy, safety, and underlying mechanisms of EECP as a non-invasive therapy for hypertension. Eligible studies included randomized controlled trials, non-randomized controlled trials, and observational studies.

Material and Methods:
A comprehensive search of the MEDLINE database was done from March 2002 to March 2023 to identify studies evaluating the use of “EECP in hypertensive patients”. Initially, 31 articles were screened for eligibility, and subsequently, 9 articles met the inclusion criteria, & were thereby included in the review. The inclusion criteria being relevance to the research question, thereby excluding the rest.

Results:
The review finds that EECP therapy can have beneficial effects on blood pressure control, endothelial function, arterial stiffness, and inflammation in patients with hypertension. The underlying mechanisms of EECP involve enhancing arterial blood flow, improving endothelial function, and reducing peripheral vascular resistance. Although the evidence base for EECP is limited, the available studies suggest that this therapy is safe and well-tolerated, with few adverse effects reported.

Conclusions:
EECP represents a promising alternative treatment option for hypertension beyond traditional pharmacotherapy. However, further high-quality studies are needed to confirm its efficacy and to identify optimal treatment protocols for different patient populations. Nevertheless, the current evidence suggests that EECP is a valuable addition to the armamentarium of hypertension management strategies.
Abstract P2149: Modulation Of Cardiac Fibrosis By SGLT-2 Inhibitor Canagliflozin And DPP-4 Inhibitor Sitagliptin In A Swine Model For Chronic Myocardial Ischemia: A Comparative Analysis
Authors: Harris Dwight D , Sabe Sharif A , Broadwin Mark , Sabra Mohamed , Xu Cynthia , Abid Ruhul , Sellke Frank W
Published: October 2023

Objective:
Clinical data suggests cardiovascular benefits from anti-diabetes drugs including SGLT-2 and DPP-4 inhibitors, but the mechanisms remain unclear. This study evaluates the effect of DPP-4 inhibitor sitagliptin and SGLT-2 inhibitor canagliflozin on myocardial fibrosis in a swine model for chronic myocardial ischemia.

Methods:
Twenty-one Yorkshire swine underwent ameroid constrictor placement on the left coronary circumflex to model chronic myocardial ischemia. Two weeks post-op, swine were assigned to one of three groups: control (CON, n=8), canagliflozin 300mg daily (CAN, n=8), or Sitagliptin 100mg daily (SIT, n=5). After 5 weeks of treatment, swine underwent terminal harvest and tissue collection. Fibrosis was quantified using Masson's trichrome. Immunoblotting was used to quantify protein expression.

Results:
Trichrome stain showed a significant decrease in perivascular and interstitial fibrosis in the CAN and SIT groups compared to CON (all p<0.05) but no difference between the SIT and CAN groups (Figure 1). Immunoblot showed a decrease in Jak2, pSTAT3 to STAT 3 Ratio, pSMAD 2/3, and SMAD 2/3 (all p<0.05) in the SIT group compared to CON, and decreased pSTAT3 to STAT 3 Ratio, pSMAD 2/3, and SMAD 2/3 (all p<0.05) in the CAN group compared to CON. Jak2 was decreased in the SIT compared to CAN group (p=0.04), and MMP-13 was decreased in the CAN compared to SIT group (p<0.001).

Conclusions:
Sitagliptin and canagliflozin result in a similar reduction in perivascular and interstitial fibrosis in a swine model for chronic myocardial ischemia. This reduction in fibrosis likely contributes to the improved cardiovascular outcomes seen with DPP-4 and SGLT-2 inhibitors.
Abstract P2152: Asperuloside Ameliorates Obesity-associated Endothelial Dysfunction Via Activating Nrf2/HO-1 Signaling Pathway
Authors: He Chufeng
Published: October 2023

Objectives:
Asperuloside (ASP) is the major bioactive components in Eucommia species but its effects on vascular health and the underlying mechanism remain unknown. In this study we aim to investigate the protective vascular effect of ASP and we hypothesize that ASP ameliorates obesity associated endothelial dysfunction.

Methods:
After induction of obesity in C57BL/6 male mice fed with high fat diet for twelve weeks, they were treated daily with ASP (50 mg/kg) via oral gavage for seven weeks in
vivo
study. C57BL/6 mice aortae were used for
ex vivo
study and endothelial cells were used for in
vitro
study. Vascular function was determined by wire myograph. DHE and MitoSOX staining were used to detect ROS levels. QPCR, aortic en-face immunofluorescence staining and Western blotting were used to analyze mRNA and protein expressions. Small interfering (si)RNA of Nrf2 and HO-1, inhibitors of Nrf2 (ML385) and HO-1 (OB-24) were used to elucidate the mechanistic pathway of ASP ’s action.

Findings:
ASP significantly improved endothelial-dependent relaxations, suppressed oxidative stress, reduced VCAM-1 (~2 fold) and ICAM-1 (~2 fold) expression, increased both Nrf2 (~1.5 fold) and HO-1 (~1.5 fold) expression in obese mice aortae, IL-1β-treated aortae
ex vivo
and endothelial cells. However, ASP failed to prevent endothelial dysfunction in HO-1 inhibited aortae and HO-1-siRNA transfected endothelial cells, indicating that ASP improved endothelial function through upregulating HO-1. Nrf2 knockdown attenuated HO-1 upregulation by ASP, showing that ASP regulated Nrf2/HO-1 pathway. ASP increased nuclear Nrf2 and dual luciferase reporter assay illustrated that ASP increased ARE binding activity. Moreover, inhibition of Nrf2/ARE binding by ML385 blocked vascular protective effects and HO-1 upregulation by ASP, showing that ASP activated Nrf2/HO-1 signaling by stimulating Nrf2/ARE binding.

Conclusions:
This study for the first time shows that ASP rescues endothelial dysfunction and suppresses vascular oxidative stress associated with obesity and IL-1β via activating Nrf2/HO-1 pathway. These findings explore the pharmacological effects of ASP and contribute to the development of a novel treatment for endothelial dysfunction in cardiovascular diseases.
Abstract P2153: A Long Noncoding RNA Plentiful In Cardiosphere-derived Cell Extracellular Vesicles (CDC-EVs), BCYRN1, And Its Short Synthetic Derivatives Enhance Regulatory T Cell (Treg) Proliferation, Migration, And Activation
Authors: liao ke , Mohammadigoldar Zahra , Akhmerov Akbarshakh , Ibrahim Ahmed G , Marban Eduardo
Published: October 2023

Background:
By modulating inflammation, Treg cells influence outcomes in myocardial infarction (MI). Here, we test the hypothesis that BCYRN1 (Brain Cytoplasmic RNA 1) and BCYRN1-derived binding small sequences (BDSSs) are cardioprotective by activating Tregs.

Methods:
RNA sequencing identified BCYRN1 as plentiful in cardiosphere-derived cell extracellular vesicles (CDC-EVs), validated by qPCR. RNA pulldown and luciferase assays probed BCYRN1's potential function as a miRNA sponge. We assessed BCYRN1 and BDSS effects on Treg proliferation (Cell Counting Kit-8), migration (trans-well migration), and IL-10 (qPCR and ELISA).
In vivo
, MI was induced in C57BL/6 mouse by left coronary artery ligation for 45 minutes followed by 15 minutes reperfusion. Afterwards, mice received (via tail vein) CDC-EV-BCYRN1 (CDC-EVs overexpressing BCYRN1), BDSSs, mut-BDSSs (scrambled inactive versions of BDSS) or vehicle (PBS). MI size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post-MI.

Findings:
BCYRN1 was found to contain putative binding sites for miR-138, miR-150 and miR-98; these miRs target ATG7, CCR6 and IL-10 mRNAs, respectively. BCYRN1 serves as a miRNA sponge, leading to Treg cell ATG7/autophagy dependent proliferation (3.0± 0.1 fold increase, N=5), CCR-6 dependent migration (1.8 ± 0.1 fold increase, N=5) and increased IL-10 production (fold increase, qPCR: 2.6 ± 0.4, N=6 and ELISA: 6.1 ± 0.8, N=5). Lipid nanoparticles containing a cocktail of 3BDSSs likewise induce Treg cell proliferation (1.9± 0.6 fold increase, N=6), migration (1.3± 0.2 fold increase, N=6), and IL-10 induction (6.8± 0.5 fold increase, N=6). In vivo (N=5/group), CDC-EV-BCYRN1 (but not vehicle) and BDSS cocktail (but not mut-BDSS) induced Treg infiltration, proliferation and IL10 production in the heart with a concomitant decrease of infarct size.

Conclusions:
A single LncRNA (BCYRN1) and shorter synthetic derivatives mimic the effects of CDC-EV on Treg
in vitro
and
in vivo
. Our study provides a novel approach, targeting Treg, to treat MI. This approach could be useful for other diseases where increased Treg activity is a therapeutic goal (e.g. autoimmune disorders).
Abstract P2169: Identification Of Pro-adhesive Drugs By A Novel High-throughput Approach
Authors: Hanns Pauline , Castellanos-Martínez Ramón , Spindler Volker , Schinner Camilla
Published: October 2023

Defective cell-cell adhesion contributes to the patho-mechanism of various diseases. In line with this, a mouse model recently developed by our group demonstrate that loss of cell-cell adhesion is an important initial step leading to Arrhythmogenic Cardiomyopathy (ACM), a disease which presents with severe arrhythmia, ventricular fibrosis and impaired cardiac function. Derived from the central role of defective cell-cell adhesion for the patho-mechanism, we here aim to identify new compounds, which restore intercellular adhesion and could serve as potential therapeutics. To screen a high number of agents, we developed an adhesion-based high-throughput approach by adapting a standard cell-cell dissociation assay to 96-well plate format including establishment of an automated acquisition and analysis pipeline. To model loss of intercellular adhesion, cells deficient for the desmosomal adhesion molecule desmoglein-2 were used. After establishing settings to sensitively detect changes in cell-cell adhesion, this method was applied to screen a library of 1946 FDA-approved drugs in three concentrations. Several new compounds strengthening intercellular adhesion were identified in addition to known pro-adhesive drugs, confirming the robustness of our assay. The increased adhesiveness of selected top hits was confirmed in cells expressing ACM patient mutations. In conclusion, we here developed an adhesion-based high-throughput approach capable of identifying adhesion modulators which have the potential to be used as therapeutics for diseases with defective desmosomal adhesion such as ACM.
Abstract P2194: Investigating The Role Of Angiotensin II In The Pathogenesis Of Atrial Fibrillation In Covid-19 Patients Through Blocking The Renin-Angiotensin-Aldosterone System
Authors: Khidhir Angela , Drago Thomas , Najam Usman , Goyal Sourabh , Beshara Michael , Palatnic Leonard , Chatterton Brian
Published: October 2023

Background:
COVID-19 was found to be associated with a greater risk of developing atrial fibrillation (AF). It is thought that COVID-19 enters cells using its surface spike protein, which binds to the ACE-2 receptor and downregulates it. One of the functions of ACE-2 is converting the pro-inflammatory, anti-fibrinolytic angiotensin II (Ang II) to angiotensin I (Ang I). Therefore, when ACE-2 is downregulated, more Ang II is available to trigger its downstream detrimental effects. Thus, we hypothesized that blocking the effects of Ang II using ACE inhibitors (ACEis) or ARBs would confer a protective effect against the risk of AF.

Objective:
The purpose of this project was to conduct a retrospective cohort study to determine whether treatment with an ACEi/ARB would prevent the development of AF in patients hospitalized for COVID-19. This would help us better understand the pathogenesis of AF in COVID-19 patients. It would also help us decide whether prophylactic treatment with ACEi/ARB would help reduce the risk.

Methods:
We reviewed the records of all patients admitted for at least seven days for COVID-19 between January 2021 and June 2021. Of 5430 charts, 188 met the inclusion criteria.

Results:
Approximately 4.8% of patients hospitalized for COVID-19 developed AF. About 6.74% of patients that were not on an ACEi/ARB at any point during their hospitalization developed AF, whereas none of the patients on an ACEi/ARB developed AF, making the relative risk (RR) 0.1871 (95% CI 0.0108 to 3.2377, p-value 0.2492), and the number needed to treat 14.8. Interestingly, in the patients with a previous diagnosis of AF, there was also no statistically significant difference in the need to escalate rate or rhythm control strategy between patients on an ACEi/ARB compared to those who were not (6.67% vs 14.29%, respectively), with a RR of 0.4667 (95% CI: 0.0546 - 4.0629, p-value 0.4900).

Conclusion:
Our data to date show that the use of an ACEi/ARB is not associated with a statistically significant decrease in the risk of AF in patients hospitalized with COVID-19, which suggests that there may be another cellular mechanism by which COVID-19 causes AF. Therefore, prophylactically treating patients with an ACEi/ARB or holding them on admission may have no influence on the risk of AF.
Abstract P3032: 15(
R
)-lipoxin A
4
Contributes To Splenic Erythrophagocytosis Via Activation Of ALX/FPR2
Authors: Sansbury Brian , Asplund Haley , Zheng Jing-Juan , Boyd Nolan , Hellmann Jason
Published: October 2023

Red blood cells (RBCs) are the most abundant cell type in the body and are traditionally appreciated for their role in respiration and gas exchange. Recently, however, RBCs have garnered new attention for their immunomodulatory effects which is primarily achieved via their interaction with resident macrophages in the spleen, known as red pulp macrophages. As RBCs enter the spleen, red pulp macrophages probe the cells for signs of mechanical and oxidative damage, as well as inflammatory stimuli bound to cell surface receptors (e.g., foreign pathogens, endogenous chemokines). Once recognized, red pulp macrophages prune damaged portions of RBC membranes or phagocytose the cell entirely, efficiently processing and recycling the cellular cargo of the RBC. In a healthy human, this process, termed erythrophagocytosis, accounts for the daily turnover of roughly 150-200 billion RBCs. Consequently, defects in the system can have wide-ranging health effects including anemia, impaired host defense, and even sepsis. Recent data suggest that arachidonic acid (AA)-derived prostaglandins promote erythrophagocytosis, yet the impact of other eicosanoid species on the process has not been well described. We performed targeted lipidomics analyses focused on the AA metabolome in two distinct models of erythrophagocytosis. First, mouse bone marrow-derived macrophages were incubated with freshly isolated RBCs; then, spleens from mice were collected following exogenous delivery of damaged RBCs. In both models, we found increased production of the 15(
R
)-epimer of lipoxin A
4
(15(
R
)-LXA
4
), as well as its biosynthetic intermediate, 15-HETE. Similar to LXA
4
, 15(
R
)-LXA
4
has been shown to robustly promote the active resolution of inflammation by regulating leukocyte function via its interaction with the G protein-coupled receptor, ALX/FPR2. In mice genetically deficient in this receptor, we measured a significant decrease in splenic clearance of damaged RBCs. Together, these data suggest that 15(
R
)-LXA
4
activation of ALX/FPR2 on red pulp macrophages may be a critical aspect regulating erythrophagocytosis and maintaining RBC homeostasis.
Abstract P3036: Circular RNA Decoys Targeting MiR-146a As A Promising Therapeutic Strategy To Improve Vascular Regeneration
Authors: Boettcher Solveig , Kalies Katrin , Knoepp Kai , Dutzmann Jochen , Huettelmaier Stefan , Sedding Daniel
Published: October 2023

Heart failure and cardiac remodeling following myocardial infarction (MI) depend on capillary density and neovascularization. MicroRNAs (miRNAs), which are small non-coding RNAs that modulate gene expression, play a major role in the pathogenesis of these conditions. Inhibition of these pathways through miRNA sponges, such as antagomiRs or circular decoys (circRNAs), might represent a therapeutic approach for MI. Here, we evaluate the role of miR-146a and present a promising treatment strategy using synthetically designed circular decoys.

miRNA-146a expression was analyzed in human vascular cells and mice tissue following either myocardial infarction or hind limb ischemia. CircRNAs were synthetically designed and evaluated. Methods to characterize the effect of inhibition of miR-146a included the quantification of the target gene and protein expression and analysis of cellular functions.miR-146a was significantly upregulated
in vitro
in senescent cells (
P
<0.05) and
in vivo
in the ischemic myocardium following ligation of the LAD (
P
<0.001) in C57BL/6 mice. A circular RNA decoy targeting miR-146a was successfully designed and transfected into vascular cells
in vitro
. The following analysis showed a significant target upregulation for NOX4, KLF4, and TRAF6 (
P
<0.001) after circRNA transfection, also compared to antagomiR transfected cells. Functional analysis revealed improved migration and proliferation capacity in vascular endothelial cells (
P
<0.05). First results indicate that inhibition of the upregulation of endothelial miR-146a in mice results in significantly enhanced angiogenesis and revascularization (
P
<0.05) accompanied by a significantly reduced myocardial infarct size (
P
<0.01) and preserved cardiac function post-MI (
P
<0.01).

In summary, we could identify miR-146a as a critical regulator of angiogenesis and vascular regeneration, and present a first strategy for angiogenic therapy of ischemic diseases. However,
in vitro
analysis showed a higher efficiency of circRNAs in the context of inhibiting miR-146a functions in vascular cells. CircRNAs are more stable and more efficient when transfected, compared to antagomiRs, and might be a better alternative and a realistic approach for future clinical studies.
Abstract P3045: MicroRNA-31-5p Is Upregulated During Neointima Formation And Promotes Smooth Muscle Cell Proliferation And Migration By Regulating Krüppel-like Factor 4 (KLF4)
Authors: Gürlach Simon , Kalies Katrin , Daniel Fiene , Köster Jan Michael , Boettcher Solveig , Hehl Laura , Knoepp Kai , Dutzmann Jochen , Daniel Jan-Marcus , Sedding Daniel
Published: October 2023

In the context of CVDs, microRNAs (miRs) have emerged as potential therapeutic targets for preventing adverse vascular remodeling and restenosis, given their ability to modulate multiple signaling pathways and cellular processes. Here, we propose that targeting miR-31-5p might be an effective approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications.

A screening of murine neointimal tissue for differentially regulated miRs revealed robustly increased expression levels of miR-31-5p over time. Subsequently, expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (EC) and -smooth muscle cells (SMC). Further, the effect of miR-31-5p on cellular function was investigated and possible targets were identified and confirmed on mRNA and protein level.

Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in C57BL/6J mice following wire-induced injury of the femoral artery at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in SMC (p<0,05
)
following serum stimulation, whereas only a slightly enhanced expression was observed in EC. On a functional level, miR-31-5p revealed dissenting effects on EC and SMC: Migration and proliferation were not altered in EC (p>0.05). In contrast, knockdown of miR-31-5p significantly reduced the migration (p<0,05) and proliferation in SMC (p<0,01) without detectable apoptotic effects. Systematic
in silico
target screens suggested KLF4 as potential target. Knock down of miR-31-5p resulted in a significant upregulation of KLF4 in SMC (p<0,01), whereas overexpression resulted in a significant downregulation of KLF4 in SMC (p<0,05) on mRNA- and protein level.

Here, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional role in SMC rather than EC since knockdown of miR-31-5p was able to prevent SMC proliferation and migration. Mechanistically, KLF4 identified as a direct target of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate SMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.
Abstract P3051: Master Splicing Regulator Rbpms2 Is Not Required For Murine Cardiac Development Or Contractile Function
Authors: McLendon Jared M , Zhang Xiaoming , Boudreau Ryan L
Published: October 2023

Post-transcriptional gene regulation (PTGR) through RNA-binding proteins (RBP) is an understudied area of cardiac biology. Emerging research describes important roles for Rbpms and Rbpms2 in RNA splicing, cardiomyocyte diploidy and proliferation, cytoskeletal reorganization and translation initiation during cardiomyocyte differentiation and cardiac development. However, the role of Rbpms2 in adult heart is largely unknown. We identified Rbpms2 as a cardiovascular-enriched RBP that is consistently downregulated in failing hearts from humans and mice. Though prior research has focused on roles in splicing, a large portion Rbpms2 protein is extranuclear, and >35% of empirically determined Rbpms binding sites localize in 3’UTRs, suggesting alternative PTGR roles. To assess Rbpms2’s role in cardiomyocyte PTGR, we assessed mRNA-seq changes in neonatal rat cardiomyocytes after overexpression and knockdown of Rbpms2. Pathway analyses revealed significant upregulation of genes involved in cell cycle control, and downregulation of genes involved in viral host defense pathway after Rbpms2 knockdown. We next characterized Rbpms2 knockout (KO) mice, which are viable without overt phenotypes, to provide initial descriptions of adult mouse cardiac function after Rbpms2 deletion. Baseline echocardiography data of male mice at 6 months of age shows normal cardiac size, structure, and function without significant difference between KO and wildtype (WT) mice (n=10). To assess the role of Rbpms2 in cardiac hypertrophy, we induced pressure overload via transverse aortic constriction (TAC) on mix-sex WT and Rbpms2-KO mice, which resulted in robust hypertrophic responses and reduced ejection fraction in both groups, but no significant differences between WT and KO mice (n=11 female, n=7-10 male). Overall, our findings are surprising given recently published data showing that Rbpms2 is a master regulator of RNA splicing affecting sarcomere organization and cardiomyocyte contractile function in zebrafish and stem cell-derived cardiomyocytes, supporting the need for follow-up studies on cardiac Rbpms2. It is possible that Rbpms compensates for the loss of Rbpms2 in the mouse heart and would require double KOs to assess synergistic phenotypes.
Abstract P3054: Further Delineation And Exploration Of The Cardiac MicroRNA-Target Interactome
Authors: Peck Bailey , McLendon Jared M , Boudreau Ryan L
Published: October 2023

MicroRNAs (miRs) have emerged as key players in cardiac biology and disease. These non-coding RNAs are loaded into Argonaute (Ago) proteins to direct suppression of target mRNAs via base-pairing. Despite a mass of research on cardiac miRs in model systems and humans, the translational impacts have been limited since miR-mRNA target interactions in heart tissues have not been adequately defined at the “-omics” level. Researchers heavily rely on predicted miR targets and are often misled by false predictions, since imperfect base-pairing is sufficient for miR binding. Our goal is to comprehensively define miR targeting events and their biological relevance in the heart and to understand the clinical significance of genetic variants (e.g. SNPs) that modulate cardiac miR functions. We previously used high-throughput biochemical means (Ago2 CLIP-seq) to generate the first transcriptome-wide map of miR binding sites in non-failing human hearts, yielding ~4000 sites across >2000 genes. While exploring this dataset, we discovered translationally relevant miR-SNP interactions that associate with heart failure patient outcomes, most notably in
SCN5A
. We have since identified multiple common missense variants in
TTN
that disrupt miR binding. These associate with heart structure/function measures in the general population and could have significant implications for cardiomyopathy patients with
TTN
mutations. Beyond SNPs, we made the surprising discovery that
TTN
mRNA harbors >20 miR-133 binding sites and has the potential to bind up to 20% of the miR-133 pool in cardiomyocytes, perhaps serving as an endogenous miR “sponge”. We experimentally validated the potential for this using a
TTN
mini-gene harboring 18 of the most strongly bound miR-133 target sites; we found that this construct was capable of inhibiting miR-133 activity in cells. Finally, to further expand these studies, we optimized our protocol by adapting state-of-the-art CLIP methods, which recently yielded our 2
nd
generation map of human cardiac miR binding sites (now in failing hearts), leading to the identification of >12,000 bindings sites across >6000 genes. We are now interrogating these data and processing additional samples to investigate how miR binding profiles rewire in failing hearts.
Abstract P3068: Hematopoietic Loss Of Y-chromosome In Circulating Monocytes Derived From Heart Failure Patients Associates With Cardiac Fibrotic Signaling
Authors: Abplanalp Wesley T , Cremer Sebastian , John David , Merten Maximilian , Schuhmacher Bianca , Macinkovic Igor , Dimmeler Stefanie , Zeiher Andreas M
Published: October 2023

Background:
Hematopoietic mosaic loss of Y chromosome (LOY) correlates with age and associates with increased cardiovascular disease incidence. Moreover, new murine studies propose that LOY could promote cardiac fibrosis by stimulating cardiac infiltration and activation of circulating macrophages. Yet, specific profiles of hematopoietic LOY cells in patients with heart failure (HF) are missing.

Methods and Results:
To decipher the genetic profile of human circulating immune cells and assess the effects of LOY, we performed single-cell RNA-sequencing (scRNA-seq) with immune cells of 10 male patients having chronic ischemic HF (mean age 67.3 years, ejection fraction 34.4%). We bioinformatically sorted immune cells by presence or absence of Y-chromosome-associated genes. Relative distribution of LOY was assessed, with monocytes having the greatest absolute count of LOY cells with no significant change in distribution among cell types. Given the abundance of monocytic LOY cells, we examined LOY monocytic cells transcriptional profiles (29810 Y-carrier monocytes, 2205 LOY monocytes) in an unbiased manner. Using a patient-specific, paired analysis, LOY monocytes showed significantly diminished expression of TGF-ß inhibiting genes (SMAD7, TGIF2) in LOY cells versus Y-carrier cells. Opposingly, from 193 upregulated genes, LOY cells showed elevated markers associated with monocyte/macrophage-mediated tissue damage and pro-fibrotic cardiac remodelling (S100A8, CLEC4D, TLR2). Gene ontology terms derived from upregulated genes in LOY cells further supported a mechanistic link to cardiac fibrosis by calling terms associated with TLR signaling, which are known to enhance cardiac fibrosis.

Conclusion:
This is the first study to study the genetic signature of LOY monocytes in HF patients. Our results considerably extend very recent experimental results in mice that LOY sensitizes macrophages to enhanced signaling associated with cardiac fibrosis. Reduced TGF-ß inhibitory molecule expression and enhanced TLR signaling may contribute to the aggravation of heart failure by LOY in patients with HF.