bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1106: Loss Of Function Of The Nuclear Envelope Protein LEMD2 Causes DNA Damage-dependent Cardiomyopathy
Authors: Caravia Xurde M , Ramirez-Martinez Andres , Gan Peiheng , McAnally John R , Bassel-Duby Rhonda , Liu Ning , Olson Eric N
Published: November 2022

Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, which primarily affect the heart and skeletal muscle. A mutation in the NEP LEMD2 causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. As a consequence, there is no specific therapy for this cardiomyopathy. To explore the basis of this pathology, we generated mice carrying the human c.T38>G Lemd2 mutation. These mice represent a new preclinical model that phenocopied the human disease and developed severe dilated cardiomyopathy with extensive cardiac fibrosis leading to premature death. At the cellular level, LEMD2 mutant cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of LEMD2 also died shortly after birth due to heart abnormalities. Both LEMD2 mutant models displayed aberrant cardiac gene expression and extensive DNA damage linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus significantly rescued cardiac function in the c.T38>G mice. Together, our results reveal the essentiality of the nuclear envelope protein LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.
Abstract P1111: A SOX17-PDGFB Signaling Axis Regulates Aortic Root Development
Authors: Lu Pengfei , Bhattachan Punit , Russell Megan , Benkaci Sonia , Cheng Wei , Feng Xuhui , Wu Bingruo , Zhou Bin
Published: November 2022

Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of
Sox17
in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve (BAV) due to the absence of non-coronary leaflet (NCL) and mispositioned left coronary ostium (LCO). The respective defects are associated with reduced proliferation of NCL mesenchyme and aortic root smooth muscle derived from the second heart field cardiomyocytes. Mechanistically, SOX17 occupies a
Pdgfb
transcriptional enhancer to promote its transcription and
Sox17
deletion inhibits the endothelial
Pdgfb
transcription and PDGFB growth signaling to the NCL mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the NCL and LCO defects in
Sox17
nulls. These data support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries.
Abstract P1115: SnoRNAs As Potential Biomarkers For Assessment Of Health Risks In Astronauts
Authors: Rai Amit , Rajan K. Shanmugha , Bisserier Malik , Brojakowska Agnieszka , Sebastian Aimy , Evans Angela , Coleman Matthew , Mills Paul , Arakelyan Arsen A , Uchida Shizuka , Hadri Lahouaria , Goukassian David A , Garikipati Venkata
Published: November 2022

During spaceflight, astronauts are exposed to various physiological and psychological stressors, such as microgravity, sleep deprivation, isolation, confinement, and high ionizing radiation have shown adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts' health. Small nucleolar RNA (snoRNA) is a type of short ncRNAs (60-300 nucleotides) known to guide 2’-O-methylation (Nm) or pseudouridine (ψ) on ribosomal RNA (rRNA), snRNA, or mRNA. Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in plasma extracellular vesicles (EV) and astronaut's peripheral blood mononuclear cells (PBMCs) can be utilized as potential biomarkers. Using unbiased small RNA sequencing (sRNAseq), we evaluated the EV snoRNA changes in peripheral blood (PB) plasma of astronauts (n=5/group) who underwent median 12-day long Shuttle missions between 1998-2001. Using stringent cutoff (> log 2-fold change, FDR < 0.05), we detected 20 down-regulated snoRNAs and 10 upregulated PB-EVs at R+3 compared to L-10. qPCR validation revealed that SNORA74A was significantly down-regulated at R+3 compared to L-10. We next determined snoRNA expression levels in astronauts' PBMCs at R+3 and L-10 (n=6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts' PBMCs at R+3 compared to L-10 Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings unveiled that spaceflight induced changes in EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as novel biomarkers for monitoring astronauts' health.
Abstract P1118: Genetic Validation Of Cardiac Cytoskeleton And Extracellular Matrix Targets For Drug Discovery
Authors: Khomtchouk Bohdan B , Davidson Michael H
Published: November 2022

Currently, cardiovascular disease (CVD) drug discovery has focused primarily on addressing the inflammation and immunopathology aspects inherent to various CVD phenotypes such as cardiac fibrosis and coronary artery disease. However, recent findings suggest new biological pathways for cytoskeletal and extracellular matrix (ECM) regulation across diverse CVDs, such as the roles of matricellular proteins (e.g. tenascin-C) in regulating the cellular microenvironment. The success of anti-inflammatory drugs like colchicine, which targets microtubule polymerization, further suggests that the cardiac cytoskeleton and ECM provide prospective therapeutic opportunities. In this Abstract, we will present genetic validation evidence supporting the efficacy of pharmaceutical modulation of the cytoskeleton and the associated ECM that supports its cellular architecture to find new computationally-derived therapeutics in the treatment of CVD using integrated in-silico, in-vitro, and in-vivo methods. Our focus will be on the dynamic role that the cytoskeleton and ECM play in CVD pathophysiology, highlighting how novel target discovery in cytoskeletal and ECM-related genes enables new therapeutics development to alter the regulation of cellular architecture in plaque formation and rupture, cardiac contractility, and other molecular mechanisms.
Abstract P1130: A Minimally Invasive Injectable Syncsc Adhesive (misa) For Aging Rats With Chemotherapy-induced Heart Failure
Authors: Huang Ke
Published: November 2022

Statement of Purpose:
Long-term chemotherapy in cancer treatment may lead to serious myocardiopathy that then prompts heart failure (HF) or even death. Additionally, patients over 65 years of age are likely to have complications in other organs, making the treatment an insurmountable obstacle. In previous studies, regenerative therapy has been reported as a robust method to improve HF. Despite the successful proof of concept, one big challenge for clinical translational is to deliver the therapeutics in a minimally invasive way. To that end, we sought to develop a minimal-invasively injectable synthetic cardiac stromal cells (synCSC) adhesive (MISA) that could be delivered with small incisions on the chest wall, without an open-chest surgery. The MISA should be gelatinized easily right after transplantation to increase the retention of synCSCs in the pericardium cavity. To mimic aging patients with cardiomyopathy caused by HF, we have established an aging rat model with HF induced by doxorubicin. The overarching hypothesis is that minimal-invasively delivery of MISA improves synCSC therapeutic efficacy in the aging rat model of HF.

Methods:
We created an injectable MISA by embedding optimized synCSCs into a myoECM-fibrinogen gel in an adjusted concentration. MISA retains my ECM proteins and peptide fragments while gelatinizing fast after injection. After MISA fabrication, we characterized its 3D morphological features to determine synCSC embedding efficiency. To determine the safety, efficacy, and mechanism of MISA treatment, we employed multiple doxorubicin intraperitoneal injections as an oxidant generator and topoisomerase II inhibitors to create an aging rat model of HF.

Results:
MISA improved therapeutic efficacy in HF treatment after being injected minimal-invasively. Also, MISA polymerized fast after administration and formation of a thin layer of regenerative gel that released regenerative factors to the myocardium in a sustained release manner while degradation. MISA as therapeutic biologics has the potential to revolutionize cardiovascular regenerative medicine.
Abstract P2004: Beta-Hydroxybutyrate Attenuates Heart Failure With Reduced Ejection Fraction Via Epigenetic Regulation Of Pgc1α
Authors: Jankauskas Stanislovas S , Gambardella Jessica , Varzideh Fahimeh , Kansakar Urna , Mone Pasquale , Sidoli Simone , Santulli Gaetano
Published: November 2022

Mitochondrial dysfunction plays a crucial role in the pathogenesis of heart failure with reduced ejection fraction (HFrEF). The ketone body beta-hydroxybutyrate (BHB) was suggested to improve mitochondrial function. However, the mechanisms are not fully understood and no studies investigated BHB effect on mitochondrial dysfunction and cardiac insufficiency in HFrEF. HFrEF was modeled in mice by permanent occlusion of the left anterior descending coronary artery causing myocardial infarction (MI). After surgery, mice were fed with BHB diet or standard chow. Sham operated mice on standard chow served as a control. BHB diet increased BHB plasma level 2.75 times compared to mice on standard chow. Four weeks after MI, BHB diet significantly improved cardiac remodeling, evident by decreased myocardial fibrosis, higher ejection fraction, lower left ventricular diastolic volume, and robust downregulation of atrial natriuretic peptide. Strikingly, survival rate was 25% for mice on normal chow and 60% for mice on BHB-diet. In primary isolated cardiomyocytes, we observed that BHB diet also shortened sarcomere time to peak contraction. Basal and maximal cardiac mitochondria respiration was reduced 4 weeks after MI alongside with downregulation of the master-regulator of mitochondrial biogenesis, PGC1α. BHB-diet ameliorated MI-induced downregulation of PGC1α, restored mitochondrial respiration, and prevented mitochondrial calcium overload. By analyzing cardiac human specimens, we discovered that Histone 3 lysin 26 dimethylation and lysin 36 methylation (H3K26me2/H3K36me) were significantly higher in human heart samples from patients with HFrEF compared to healthy controls; similar findings were observed in MI hearts compared to sham. BHB diet downregulated H3K26me2/H3K36me in murine hearts. Chromatin immunoprecipitation of H3K26me2/H3K36me revealed a PGC1α promoter enrichment. H3K26me2/H3K36me was also upregulated after ischemia
in vitro
and was accompanied by a decrease in PGC1α expression, dampened mitochondrial respiration, and decreased mitochondrial mass. Histone methyltransferase inhibition precluded PGC1α downregulation after ischemia mimicking BHB effect.In this study, we demonstrated for the first time that BHB mitigates HFrEF. This effect is mediated via reversing the epigenetic silencing of PGC1α with the subsequent restoration of mitochondrial function.
Abstract P2011: The Role Of A Novel Alpha-crystallin B Chain Variant In Hypertrophic Cardiomyopathy
Authors: Chou Chun , Martin Gregory , Perera Gayani , Awata Junya , Blanton Robert M , Cao Xuehong , Alissa Abdullah , Madias Christopher , Galper Jonas B , Chin Michael T
Published: November 2022

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, cardiac fibrosis and increased risk of sudden cardiac death, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in the sarcomere gene can account for a substantial proportion of familial cases of HCM, up to 70% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYAB
R123W
) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB
R123W
promotes HCM phenotype remains unclear. Here, we generated mice carrying the
Cryab
R123W
-knockin allele and demonstrated that hearts from these animals exhibit increased elastance, reduced end diastolic volume and increased E/E’ consistent with increased systolic and reduced diastolic function. Upon transverse aortic constriction, mice carrying the
Cryab
R123W
allele developed pathological left ventricular hypertrophy with substantial cardiac fibrosis and progressively reduced ejection fraction. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB
R123W
despite its potent effect on driving cellular hypertrophy. Unexpectedly, CRYAB
R123W
appears to enhance calcium signaling by promoting nuclear localization of NFAT through direct interaction with calcineurin. Mice also were more susceptible to ventricular tachycardia with programmed stimulation. Single nuclei RNA-sequencing of mouse hearts is underway and preliminarily reveals reduction in oxidative phosphorylation, consistent with known effects of
Cryab
on mitochondrial function. Thus, our data establishes the
Cryab
R123W
allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiomyocyte hypertrophy.
Abstract P2018: Tmem65 Is Critical For The Structure And Function Of The Intercalated Discs In Mouse Hearts
Authors: Teng Allen , Gu Liyang , Di Paola Michelle , Lakin Robert , Williams Zachary , Au Aaron , CHEN WENLIANG , Callaghan Neal I , Hakem Zadeh Farigol , ZHOU YU-QING , Fatah Meena , Chatterjee Diptendu , Jourdan Jane , Jack Liu , Simmons Craig A , Kislinger Thomas , Yip Christopher , Backx Peter , Gourdie Robert G , Hamilton Robert M , Gramolini Anthony
Published: November 2022

The intercalated disc (ICD) is unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin 43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigated the role of Tmem65 in ICD organization
in vivo
. A mouse model was established by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA which resulted in a 90% reduction of Tmem65 expression in mouse ventricles compared to mice injected with scrambled shRNA. Tmem65 knockdown (KD) resulted in increased mortality which was accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection, progressing to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts displayed depressed hemodynamics, measured echocardiographically, accompanied by electrocardiogram changes (prolonged PR intervals and QRS duration) consistent with impaired conduction, which was confirmed with optical mapping of isolated hearts. Immunoprecipitation and super-resolution microscopy demonstrated a physical interaction between Tmem65 and sodium channel β subunit (β1) in mouse hearts and this interaction appeared to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at the ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na
+
currents but did show reduced Ca
2+
and K
+
currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.
Abstract P2030: Defining Transcription Factor Dependent Regulatory Networks In Cardiac Rhythm And Disease
Authors: Lazarevic Sonja , Wang Zhezhen , Shen Kaitlyn , Gadek Margaret , Park David , Moskowitz Ivan
Published: November 2022

The transcriptional basis of homeostasis and disease is implied by the non-coding nature of genetic variation identified by GWAS. Functional genomic approaches to unveil the gene regulatory networks (GRNs) relevant to disease is therefore a high priority. Most models for transcriptional dysregulation presume that perturbation of a wild-type GRN causes disease risk. For example, the T-box transcription factor, TBX5, is essential for atrial rhythm homeostasis and directly drives a physiologically relevant GRN composed of cardiac channel genes. Alternatively, examination of the upregulated genes after the deletion of
Tbx5
implicates the activation of a disease-specific GRN. Using TF-dependent noncoding RNA (ncRNA) profiling, through the differential deep ncRNA sequencing from atria of wild-type and
Tbx5
mutant mice, we focused on the ncRNAs that were only active following the deletion of TBX5. Therefore, we identified putative TBX5-dependent enhancers, based on knowledge that ncRNAs can identify transcription at regulatory elements. We hypothesized that these regulatory elements may reveal disease-response enhancers, essential for coping with atrial dysfunction. We sought to identify the cell-specificity of these enhancers and therefore generated cell-type specific ATAC-seq datasets for left atrial tissue, cardiac fibroblasts, and cardiomyocytes, then overlapped each dataset with the ncRNAs. These candidate regulatory elements included a putative enhancer upstream of Sox9, a known modulator of cardiac fibrosis, along with other cardiac stress-response pathways, including mediators of TGF-β signaling. The ncRNA candidate is expressed in isolated cardiac fibroblasts treated with TGF-β, and the enhancer demonstrated luciferase activation in fibroblasts. To examine the hypothesis that the disease-acquired GRN in TBX5 mutant atria may be generalizable to other cardiac insults, we examined the transcriptional changes occurring in the left atria of a Transverse Aortic Constriction (TAC) mouse model. We revealed remarkable correlation between the differentially expressed genes between these disparate disease models. We also performed differential ncRNA profiling from this pressure overload mouse model, and identified the conservation of acquired ncRNAs when compared to the cardiac arrhythmia mouse model. The conservation of the transcriptional analysis and ncRNA candidates supports the paradigm of a common disease-specific GRN that mediates and may participate in the physiologic consequences of disease.
Abstract P2043: Interleukin-1 Blockade Restores Exercise Capacity After Non-reperfused Acute Myocardial Infarction In The Mouse.
Authors: Vohra Habeebah , Narayan Pratyush , MEZZAROMA Eleonora , Van Tassell Benjamin W , Abbate Antonio , Toldo Stefano
Published: November 2022

Background:
Acute myocardial infarction (AMI) is associated with an intense inflammatory response promoting progression to heart failure (HF), characterized by dyspnea, fatigue, exercise intolerance. Interleukin-1 (IL-1) is the prototypical inflammatory cytokine increased in AMI. IL-1 injections reduce exercise capacity in healthy mice. Whether IL-1 reduces exercise capacity after AMI is unknown.

Hypothesis:
IL-1 blockade preserves exercise capacity in mice with experimental AMI.

Methods:
Ten-week-old male CD1 mice (N=45) were trained to run on a treadmill. Baseline exercise capacity was assessed. Based on the distance run, the mice were divided into tertiles. Mice in the middle tertile underwent surgical ligation of the coronary artery. Ten mice survived and were randomly assigned to treatment with daily intraperitoneal injections with anakinra, a recombinant human IL-1 receptor antagonist (10 mg/kg, 0.2 ml, N=4), or 0.9% NaCl vehicle (N=6). We measured exercise capacity at 1, 2, and 4 weeks. Data were analyzed using T-test for unpaired groups and are presented as mean and standard error of mean.

Results:
Baseline exercise capacity was 217±16 and 230±16 m in the anakinra and vehicle groups, respectively. Exercise capacity was significantly reduced 1 week after surgery in both groups (-57±9% vs -60±7%, P=0.80; P<0.01 vs respective baseline). By week 4, however, anakinra treatment significantly improved exercise capacity compared to the vehicle (+42±4% vs +8±7%, P<0.001) (Figure).

Conclusion:
Non-reperfused AMI leads to a severe reduction in exercise capacity that is reversed by IL-1 blockade. These data support IL-1 as a therapeutic target for preventing HF after AMI.
Abstract P2047: Identification Of Selective Small-molecule ERBB4 Agonists With Cardioprotective Effects
Authors: Feyen Eline , Cools Julie , Van fraeyenhove Jens , Tubeeckx Michiel , De Winter Hans , Audenaert Dominique , Bruyns Tine , De Keulenaer Gilles , Segers Vincent
Published: November 2022

Introduction:
Morbidity and mortality of heart failure remain high, mandating new therapeutic approaches. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and an attractive target for treatment. Clinical trials with recombinant NRG1 are ongoing, but require intravenous administration, limiting applicability and efficacy.

Purpose:
To develop selective small-molecule ERBB4 agonists with cardioprotective effects.

Methods:
A high-throughput screening of 10,240 compounds (cpds) was performed on a ERBB4/ERBB4 dimerization assay. Hit cpds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, toxicity and metabolic stability were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization and adenylate kinase assays or LC-MS/MS. Apoptotic and hypertrophic effects of cpds (4-32μM) on cultured cardiomyocytes were studied after exposure to 100μM H
2
O
2
and 100nM angiotensin II (AngII). Antifibrotic effects (4-32μM) were studied on TGF-β-induced collagen synthesis in cultured human fibroblasts, and in mice (n=9-10/group) treated with AngII (1000 ng/kg/min) or cpd (83 μg/kg/h) using osmotic pumps.

Results:
We identified 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 dimerization (Emax 9-33% relative to NRG1, EC50 6E-6 to 2E-7M). Competition assays indicated allosteric receptor binding and potentiation of NRG1-induced ERBB4 receptor dimerization, up to 2.7 fold (P<0.0001). Six were non-toxic and induced ERBB4 phosphorylation, but ERBB1, ERBB2 or ERBB3 phosphorylation remained unaffected and cpds did not induce ERBB2/ERBB3 dimerization, showing ERBB4 selectivity. Cpds showed a t1/2 of 170min in human liver microsomes. Cpds attenuated hypertrophic and apoptotic effects of AngII or H
2
O
2
(P<0.05), and decreased collagen upregulation
in vitro
(P<0.05).
In vivo
, a selected cpd attenuated AngII-induced myocardial interstitial fibrosis (-76±26%, P<0.01), and cardiac
Col1a1, Col3a1
(-64±22%; -71±25%, P<0.01) and
Nppa
(-77±22%, P<0.01) mRNA expression.

Conclusion:
We identified selective novel pyrimidine derivative small-molecule ERBB4 agonists with cardioprotective effects
in vitro
and
in vivo
.
Abstract P2056: A Micropeptide Encoded By An Endogenous Retroviral-derived Lncrna Regulates Cardiomyocyte Multinucleation
Authors: Anderson Kelly M , Poosala Pornthida , Lindley Sean , Anderson Douglas M
Published: November 2022

Endogenous retroelements (REs) are pervasive in mammalian genomes, and consist mostly of silenced mobile genetic sequences long considered to be ‘junk DNA.’ In a few known instances, mammalian genomes have captured and repurposed endogenous retroviral sequences to play important roles in regulating mammalian cell functions, notably cell fusion. Our previous work has shown that some mammalian transcripts misannotated as lncRNAs can be translated into small functional peptides, called micropeptides. Here we report the discovery of a cardiac-specific micropeptide encoded by a misannotated lncRNA transcribed from a murine endogenous retroviral element. We named this transcript CARDI, and the encoded micropeptide CARDI-B, since translation of the micropeptide occurs from the second ATG start codon. We show that CARDI-B shares homology with a singular domain of a retroviral protein and localizes to the nucleus. Genetic deletion of CARDI-B in mice from an isogenic background significantly increased the frequency of binucleated cardiomyocytes, at the expense of mononucleated cardiomyocytes. In line with previous reports that the frequency of mononucleated cardiomyocytes is correlated with cardiac regeneration, we show that CARDI-B-deficient hearts perform worse following myocardial infarction injury. These data reveal that functional micropeptides can be concealed within mammalian retroelements, and suggests that RE-derived micropeptides may contribute to the natural variation in heart development and regeneration observed across species.
Abstract P2058: LncRNA PCAT19 Limits Angiogenesis And Safeguards Quiescent Endothelial DNA By Preventing Uncontrolled Phosphorylation Of Replication Protein A2
Authors: Oo James A , Pálfi Katalin , Warwick Timothy , Wittig Ilka , Boos Frederike , Leisegang Matthias , Brandes Ralf P
Published: November 2022

Background:
Long noncoding RNAs (lncRNA) provide an additional layer of molecular control in pathways crucial for vascular development and cardiovascular disease. We set out to identify novel endothelial lncRNAs that could be exploited to treat vascular disease.

Methods and Results:
We identified the endothelial cell-specific long noncoding RNA (lncRNA) PCAT19, which contributes to the endothelial proliferation-quiescence switch and acts as a safeguard for the endothelial genome during quiescence. PCAT19 was upregulated with endothelial cell confluence and quiescence. Knockdown of PCAT19 promoted endothelial proliferation and angiogenic sprouting. Conversely, PCAT19 overexpression reduced endothelial proliferative capacity and angiogenic sprouting. Using a cardiac organoid model system, we observed a markedly denser vascular network after the removal of PCAT19. RNA-sequencing after PCAT19 knockdown revealed multiple differentially regulated cell cycle genes. An antisense-oligonucleotide pulldown of PCAT19 followed by mass spectrometry identified multiple DNA damage response and cell cycle-related proteins as PCAT19 interaction partners; among them was the full DNA replication protein A complex. PCAT19 knockdown was found to sensitise DNA to damage, as measured by heightened p53 and γH2AX levels as well as a positive TUNEL signal and longer comet tail olive moments. Mechanistically, PCAT19 limited the phosphorylation of RPA2 on the serine 33 (S33) residue by inhibiting RPA2 interaction with the ATR kinase.

Conclusions:
In healthy vessels, endothelial cells maintain a stable, differentiated and growth-arrested phenotype. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. PCAT19 was identified as a highly enriched endothelial lncRNA that acts as a dynamic guardian of the endothelial genome, promotes cellular longevity and facilitates rapid switching to proliferation. Therapeutic targeting of PCAT19 could potentially alter the course of vascular disease development and progression by modulating endothelial growth and angiogenic sprouting.
Abstract P2064: High Resolution Single Cell Analysis Of The Developing Human Heart
Authors: Bayraktar Semih , Colzani Maria , Davaapil Hongorzul , Knight-Schrijver Vincent , Rericha Patrick , Lee Jonathan , He Xiaoling , Gambardella Laure , Sinha Sanjay
Published: November 2022

The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression.
Abstract P2072: Complex Remodeling Of Transcriptomes And Chromatin Accessibility Epigenomes In Acute Post-infarct Myocardium Revealed By Massive Parallel Single-nucleus Sequencing Analyses
Authors: Nguyen Ba Thong , Abdi Lovina , MacLellan Robb , Zhenhe Zhang , Zhang Yiqiang
Published: November 2022

Background:
Adverse remodeling in post-infarct myocardium includes weakened contractility due to substantial cardiomyocyte (CM) loss, augmented inflammatory and fibrotic responses, and re-organization of vasculatures. Current mechanistic understanding of cellular interaction within and between cardiac cell populations in post-injured myocardium remained limited.

Aims:
To identify molecular regulations converged in post-infarct heart cells.

Methods and Results:
We simultaneously analyzed total heart cells in post-infarct myocardium by using the latest single-nucleus (sn) dual transcriptomics-epigenomics approaches (snRNA-seq and snATAC-seq) in our new triple-transgenic multi-reporter mouse model featuring CM lineage tracking and maturity visualization. The compositions of cell types clustered based on transcriptomic profiles are highly comparable to those identified by open chromatin accessibility. There were significant reductions of CMs and endothelial cells, along with remarkable increases of immuno/inflammatory cells and cardiac fibroblasts in post-infarct hearts compared to controls, as revealed by comparable cell populations clustered based on transcriptomic profiles versus epigenomics chromatin accessibility profiles. We characterized the dual-omic molecular signatures of all heart cell populations from acute post-infarct murine hearts in contrast to controls. We identified the features as various molecular states in pseudotime trajectory, distinguishing each cell population and sub-cluster presented in diseased and normal hearts. Heart cells in post-infarct myocardium were clustered along the newer pseudotime series while those in control hearts were mostly located in earlier phases of the pseudotime trajectory. Lastly, we identified the signaling pathways and TFs enriched by differentially-expressed genes (DEGs) and the differentially active regions (DARs) of the chromatin in dedifferentiated CMs as well as other heart cell types and their relationships in post-MI and normal hearts.

Conclusions:
The results demonstrated the complex cellular and molecular remodeling at single-cell (nucleus) level in acute post-infarct hearts in which diverse and converging signaling networks may be targeted for heart failure therapeutics.
Abstract P2079: Rbl2 Regulates Cardiac Sensitivity To AnthracyclineChemotherapy
Authors: Chen Jingrui , Xia Peng , Sapkota Yadav , Scott Erika N , Liu Yuening A , Hudson Melissa , Rassekh Shahrad R , Carleton Bruce C , Ross Colin , Chow Eric , Cheng Zhaokang
Published: November 2022

Anthracycline chemotherapies cause heart failure in a subset of cancer patients. Emerging evidence suggests that genetic factors might contribute to the interindividual variations in cardiac sensitivity to anthracyclines. We previously reported that the anthracycline doxorubicin (DOX) induces cardiotoxicity through activation of cyclin-dependent kinase 2 (CDK2). The aim of this study was to determine whether retinoblastoma-like 2 (RBL2/p130), an emerging CDK2 inhibitor, regulates CDK2 activity and anthracycline sensitivity in the heart. Here, we showed that loss of endogenous Rbl2 increased basal cardiac CDK2 activity. Mice lacking Rbl2 were more sensitive to DOX-induced cardiotoxicity, as evidenced by rapid deterioration of heart function and loss of heart mass. Disruption of Rbl2 exacerbated DOX-induced mitochondrial damage and cardiomyocyte apoptosis. Mechanistically, Rbl2 deficiency enhanced CDK2-dependent activation of forkhead box O1 (FOXO1), leading to upregulation of the pro-apoptotic protein Bim. Inhibition of CDK2 desensitized Rbl2-depleted cardiomyocytes to DOX. In wild-type cardiomyocytes, DOX exposure induced Rbl2 expression in a FOXO1-dependent manner. Importantly, human RBL2 gene single nucleotide polymorphism (rs17800727) was also associated with anthracycline cardiotoxicity in childhood cancer survivors. In conclusion, loss of Rbl2 provokes cardiac CDK2 activation, resulting in increased sensitivity to DOX-induced cardiotoxicity. Rbl2 is an endogenous CDK2 inhibitor in the heart and represses FOXO1-mediated pro-apoptotic gene expression. Our findings suggest that RBL2 could be used as a biomarker to predict the risk of cardiotoxicity in individual patient prior to initiation of anthracycline-based chemotherapy.
Abstract P2082: Igfbp-3 Activates Pro-survival Pathways To Mitigate Anthracycline-induced Cardiomyocyte Injury
Authors: Chen Junjie , Awada Jerome , Jong Jeremy , Packard Rene R
Published: November 2022

Rationale:
Anthracyclines such as doxorubicin are among the most effective chemotherapeutic agents; however, their use leads to frequent cardiovascular complications, including heart failure, due to the activation of multiple cardiomyocyte injury pathways. Insulin-like growth factor binding protein-3 (IGFBP3) is strongly upregulated in cardiomyocytes after administration of doxorubicin. However, its role in doxorubicin-induced cardiomyocyte apoptosis and survival remains controversial.

Methods and Results:
We demonstrate that IGFBP3 is strongly induced by doxorubicin in neonatal ventricular myocytes (NRVMs) 12 hours after doxorubicin treatment by RT-PCR (4.2 fold vs. control, n=3, P<0.05) and Western blot. Adenoviral overexpression of IGFBP3 in NRVMs leads to a significant reduction of phosphorylated H2A histone (γH2AX) after doxorubicin treatment, a hallmark of DNA double-strand breaks (DSBs), as well as topoisomerase IIβ, a key mediator of doxorubicin induced DNA DSBs. Conversely, silencing of IGFBP3 with siRNA leads to a significant increase of γH2AX expression in NRVMs. Adenoviral shRNA silencing of IGFBP3 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) further demonstrates a 44% and 52% decrease in contraction magnitude and velocity in response to doxorubicin compared with scramble shRNA (scramble shRNA, n=5, shIGFBP3, n=3, P<0.05). Immunofluorescent imaging and cell fractionation assays conducted in NRVMs indicate that doxorubicin initiates IGFBP3 nuclear translocation, suggesting an active role in the cardiomyocyte response to doxorubicin-induced cell injury. Next generation sequencing of hiPSC-CMs treated with doxorubicin reveals that overexpression of IGFBP3 leads to upregulation of cardiomyocyte survival pathways, including TNF receptors without cytoplasmic death domain (TNFRSF10C and D), BCL2 family gene BCL2L1, the p53 target ACER2, the p53 co-factor FBXW7, the β-catenin mediator TBX3 and the ataxia telangiectasia mutated (ATM) activator ZNF423.

Conclusion:
Our findings indicate that IGFBP3 is strongly induced in cardiomyocytes by doxorubicin treatment. Overexpression of IGFBP3 attenuates doxorubicin induced DNA DSBs and promotes pro-survival signaling pathways.
Abstract P2093: Immunodominant Human Leukocyte Antigen-ii-restricted Epitopes In Human Apolipoprotein B
Authors: Roy Payel , Sidney John , McNamara Coleen A , Sette Alessandro , Ley Klaus
Published: November 2022

Introduction:
CD4

+

T cell responses to apolipoprotein B are well characterized in atherosclerotic mice and also detectable in humans. CD4
+
T cells recognize peptides displayed on highly polymorphic Human Leukocyte Antigen-II molecules. Immunogenicity of individual APOB peptides is largely unknown in humans. Only one HLA-II-restricted epitope was validated using a DRB1*07:01-APOB
3036-3050
tetramer. We reasoned that the T cell epitope repertoire of APOB is broader and may contain dominant antigenic regions.

Hypothesis:
Human APOB harbors discrete immunodominant sequences that are targeted by autoreactive CD4
+
T cells in majority of donors. These trigger atherosclerosis-related autoimmune responses in humans.

Objective:
To optimize a restimulation-based workflow to examine antigenicity of multiple candidate peptides in HLA-typed donors, to chart TCR specificities and phenotypes of APOB-reactive cells and to profile T helper cytokine responses to APOB epitopes.

Methods and Results:
We selected twenty APOB peptides (APOB
20
) from an
in silico
screen and validated peptide binding to the most commonly occurring HLA-II alleles. Using stringent controls, we confirmed specificity of expansion-based protocols to detect CD4
+
T cytokine responses to APOB
20
pool.
Ex vivo
assessment of activation-induced markers revealed statistically significant autoimmune response to APOB
20
, but not to a ubiquitously-expressed negative control protein, actin. CD4
+
T responses to the APOB
20
pool were resolved to the level of individual peptides using an IFNγ enzyme-linked immune-absorbent spot (ELISpot) assay. This led to the discovery of six immunodominant epitopes (APOB
6
) that triggered robust T cell responses in most donors. High-throughput sequencing identified unique APOB
6
-specific expanded TCR clonotypes. Responding CD4
+
T cells were enriched in antigen-experienced memory phenotypes. APOB
6
induced secretion of both pro-inflammatory and regulatory cytokines. In matched clinical samples, APOB
6
stimulation detected heightened expression of activation and memory markers and augmented secretion of proinflammatory cytokines in patients with more severe coronary artery disease.

Conclusions:
Using >60 donors expressing diverse HLA-II alleles and distributed over a screening and a validation cohort of healthy subjects and a clinical cohort of CAD patients, we discovered six immunodominant APOB epitopes that allowed interrogation of dynamic antigen-specific CD4
+
T responses associated with human atherosclerosis.
Abstract P2098: A Novel Measure Of Myocardial Collagen Structure Using Ultrasonic Backscatter
Authors: Pittman Lindsay A , Chadha Arjun , Milne Michelle L , Chung Charles S
Published: November 2022

Aging and disease are associated with dysregulation of collagen deposition and collagen degradation and crosslinking. These modifications in collagen structure are key determinants of cardiac integrity and function, making it advantageous to monitor collagen’s state throughout disease. Although myocardial fibrosis often occurs alongside both cardiac and systemic disease, monitoring myocardial collagen typically requires expensive, invasive, and/or contraindicated methods. However, collagen content has been correlated to ultrasonic backscatter, a low-cost, non-invasive index. Preliminary studies in our lab suggested collagen also dominates the anisotropy of backscatter(variation in the brightness) in a left ventricular short-axis ultrasound. The purpose of the present study was to determine a relationship between myocardial collagen structure and anisotropy of ultrasonic backscatter while using collagenase to degrade myocardial collagen networks. Hearts were excised from Sprague Dawley rats and perfused with a collagenase-containing solution for either 10 (n=7) or 30 minutes (n=7) or control solution for 30 minutes (control n=8). Serial ultrasound images were acquired throughout collagenase digestion and ultrasonic backscatter was assessed where the collagen is primarily aligned perpendicular to the angle of insonification (bright on ultrasound image), and where collagen is primarily aligned parallel to the angle of insonification (darker on image). Our data suggested that collagenase digestion reduced backscatter anisotropy within the myocardium (p<0.001) with the lateral and septal walls (collagen parallel to ultrasound) showing the greatest change in backscatter intensity. Histology (Trichrome staining) and biochemistry (hydroxyproline assay) suggests that collagen remains present but its crosslinking is altered within 10 minutes (p<0.047). These data suggest that myocardial collagen fiber orientation and crosslinking of collagen correlates with the anisotropy of ultrasonic backscatter. Thus, anisotropy of ultrasonic backscatter could potentially be used to assess collagen crosslinking and deposition.
Abstract P2103: Cannabidiol Protects Cardiac Function
In Vivo
And Inhibits Endothelial-to-mesenchymal Transition
In Vitro
: A Potential Anti-fibrotic Therapy For Heart Failure.
Authors: Krishnamoorthi Muthu Kumar , Sanaee Forughalsadat , Lavasanifar Afsaneh , Youker Keith , Bhimaraj Arvind
Published: November 2022

Introduction:
Cannabidiol (CBD) has been investigated for application in various diseases including cardiovascular ailments. Endothelial-to-Mesenchymal transition (EndoMT) has been reported to contribute to cardiac fibrosis and eventually heart failure (HF). Here we demonstrate that both free (f) and nanoparticle encapsulated (n) CBD preserves cardiac function in a non-ischemic mouse model of HF. Further, we show that CBD inhibits EndoMT
in vitro
.

Hypothesis:
CBD administration will improve cardiac function by targeting anti-fibrotic mechanisms.

Methods:
HF was induced in a 3-month old C57BL/6J mice (n=3) with 1% NaCl, 0.3 mg/mL L-NAME and an osmotic pump infusion of angiotensin II (Ang II). Both fCBD and nCBD were administered twice per week subcutaneously and echocardiography and histology evaluated at 5 weeks. We used an
in vitro
EndoMT model with L-NAME+ Ang II as EndoMT inducers in HUVECs. CBD at 0.1, 1 and 2 μM was used to study the dose impact.

Results:
HF mice treated with fCBD, and nCBD exhibited significantly higher EF when compared to untreated HF mice (Fig. 1 A). Histological evaluation showed that nCBD treated group displayed a higher anti-fibrotic activity compared to fCBD (both with less fibrosis compared to HF mice) (Fig. 1 B).
In vitro
, when CBD was added during EndoMT induction, the transition process was inhibited. Immunofluorescence studies exhibited a dose dependent effect of CBD in inhibiting Vimentin (mesenchymal marker) expression during the EndoMT process (Fig. 1 C).

Conclusion:
In conclusion, CBD protects cardiac function and exhibits an anti-fibrotic effect, possibly mediated by EndoMT inhibition. A nano formulation of CBD might have a more potent impact.
Abstract P2112: Human Genetics And Engineered Heart Tissues Link Frail
MYH7
Alleles To Delayed Maturation And Ventricular Noncompaction
Authors: Monroe Tanner O , Fullenkamp Dominic , Puckelwartz Megan , McNally Elizabeth M
Published: November 2022

Background:
Up to 1% of humans are born with a congenital heart defect. Among those defects is a condition known as left ventricular noncompaction (LVNC), which is defined as the ratio of trabeculated myocardium to compact myocardium exceeding 2.3:1. The process of compaction occurs when the layer of compact cardiomyocytes (CMs) proliferates, while CMs in the trabecular layer quiesce. This differential expansion of the CM layers reduces the trabecular invaginations into the cardiac lumen. Deficient compact CM proliferation or excessive trabecular CM proliferation converge on a similar presentation, collectively referred to as noncompaction. Family and population genetics studies have implicated deleterious variants in sarcomere genes as highly correlated with LVNC (PMID: 18506004, PMID: 3350056); specifically,
MYH7
truncations and missense variants that fall within the first 600 amino acids of the beta myosin heavy chain head region.

Hypothesis:
Reduced contractility in certain
MYH7
head domain missense or truncating variants disrupts cardiomyocyte terminal maturation, leading to excessive trabeculation.

Results:
We identified individuals and families with
MYH7
Lys234Asn and Arg243His. Variant carriers had LVNC often in the setting of dilated cardiomyopathy, similar to what has been described with
MYH7
truncations. We engineered
MYH7
truncations in human induced pluripotent stem cells (hiPSCs) which were differentiated into engineered heart tissues (EHTs) to model the developing heart. In EHTs with
MYH7
truncations contractility was reduced by 50%, EHT size increased by 17%, and CM proliferation increased by 40% in EHTs heterozygous for
MYH7
truncations (N=6 each).

Conclusions and Future Directions:
Our observations of impaired differentiation, reduced contractility, and decreased compaction are consistent with LVNC phenotypes. We propose a general model where diminished contractility delays sarcomere maturation, extending the proliferation time of trabecular cardiomyocytes and resulting in excessive trabeculae.
Abstract P2117: TRF2 Rescues Pathogenic Phenotypes In Duchenne Muscular Dystrophy Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells
Authors: Eguchi Asuka , Torres-Bigio Sofia I , Gonzalez Adriana Fernanda G , Koleckar Kassie , Birnbaum Foster , Blau Helen M
Published: November 2022

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart; however, these treatments do not address the root cause of dilated cardiomyopathy: loss of cardiomyocytes. Previously, we showed that longer telomeres are protective against dilated cardiomyopathy. Here we investigated the role of telomeres as a target for therapy in DMD cardiomyocytes using human induced pluripotent stem cells (iPSCs) to model the disease. Compared to healthy controls, DMD cardiomyocytes exhibited reduced telomere lengths, cell size, nuclear size, and sarcomere density. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends. TRF2 levels are reduced relative to healthy controls in DMD cardiomyocytes. We hypothesized that decreased TRF2 drives telomere attrition in the progression of dilated cardiomyopathy. Our data show that TRF2 overexpression prevented telomere attrition and also rescued deficits in cell size, nuclear size, sarcomere density, and calcium handling. These data highlight the benefits of TRF2 upregulation as a potential gene therapy to delay the onset of dilated cardiomyopathy.