bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1111: Dysfunctional SCARNAs And Failed Pseudouridylation Result In Irregulation Of Cardiac Genes In Noonan Syndrome Patient-derived Induced Cardiomyocytes
Authors: Sigamani Vinoth , Perales Selene G , RAJASINGH SHEEJA , Vembuli Hemanathan , Pushparaj Rahul , Rajasingh Johnson
Published: October 2023

Background:
Small Cajal body associated RNAs (scaRNAs) can control alternative splicing and the maturation of mRNAs by regulating the pseudouridylation (PsU) of small nuclear RNAs (snRNAs), which are involved in the formation of the spliceosome complex, via RNA-dependent mechanism. Failure of snRNA PsU results in dysregulated mRNA splicing and maturation which causes congenital heart defects. In this study, we aim to find the importance of snRNAs PsU in Noonan syndrome (NS) patients-derived induced pluripotent stem cell-derived cardiomyocytes (NS-iCM) when compared to normal skin fibroblast iCM (SF-iCM).

Hypothesis:
We hypothesize that snRNAs PsU by scaRNAs will have a significant role in the development of cardiomyocytes in NS patients.

Methods and Results:
We generated iCMs from SF-iPSCs and NS patient-derived NS-iPSCs. The qRT-PCR analysis showed a significantly lower expression of scaRNA6, scaRNA20, and scaRNA28 in NS-iCMs when compared with control SF-iCMs
(Fig. A).
The PsU of snRNA U12 was quantified by RNA Immunoprecipitation (RIP) using the Pseudouridine (Ψ)-specific antibody and mouse IgGas a negative control. The data showed that snRNA U12 was significantly reduced in the NS-iCM when compared with negative IgG
(Fig. B).
Furthermore, the PsU modification was quantified by using the 1-cyclohexyl- (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) method. Our data showed that relative Ψ units were significantly reduced in the NS-iCMs when compared with normal SF-iCMs
(Fig. C).

Conclusion:
Our results indicate that snRNAs PsU by scaRNAs were dysfunctional in NS-iCMCs, which may result in cardiac defects in NS patients.
Abstract P1115: Epicardial Signaling Regulates Myocardial Contractility Via Mirna
Authors: Nebigil Canan G
Published: October 2023

Background:
A congenital left ventricular aneurysm is a rare cardiac malformation that maycause heartfailure, ventricular wall rupture, ventricular arrythmia or sudden cardiac death. However, mechanisms of congenital left ventricular aneurysm are not known. Paracrinesignals from cardiomyocytes, such as a cardiokine, prokineticin-2, can activate EPDCsdifferentiation in adult heart to promote neovascularization. How the reciprocal interaction viaprokineticin and miRs from epicardium to cardiomyocytes affect heart growth and function,and how EMT-related events contributecardiomyocyte proliferation and function areunknown. MicroRNA (miR124) has an important role in the adult heart, however, its role incardiac development is unknown.

Results:
Here in this study, by deletion of a prokineticin-2 receptor PKR1 through epicardialcell linage tracing at the different developmental stages of mice embryos, we found that thesemice exhibited ventricular rupture and embryonic lethality. Indeed, miR-124 was significantlyupregulated in
PKR1
-deficient epicardial cells both
in vitro
and
in vivo
, whereas ectopicexpression of these miRs in epicardial cells resulted in the reduced expression of SNAI2 andsubsequently led to impaired EMT and EMT-related events such impaired migration andincreased apoptosis. Wild type mice cardiomyocytes that were cultured in condition mediumderived from PKR1-deficient epicardial cells had a lower beating rate and contractility. Treatment of the cardiomyocytes with miR-124mimic exhibited the same phenotype, that wasrestored by miR-124 inhibitor, indicating involvement of mir124 in epicardium-cardiomyocyte communication and regulation of cardiomyocyte contractility.

Conclusion:
These finding reveals a key signaling pathway in which a cardiokine,prokineticin-2 promotes EMT to form EPDCs and their migration in an autocrine manner, andsupports myocardial growth and contractility through repressing miR124. Our study mayprovide novel insight in etiology of congenital defects and new strategies to promotemyocardial regeneration.
Abstract P1119: Deep Learning-based Cardiac Microphysiological Systems For Studying Reentry Arrhythmia
Authors: Son Young Hoon , Park Hyeong Kyun , Sheng Christina , Jang Jeongin , Park Junbeom , Park Sung-Jin
Published: October 2023

Arrhythmias can be caused by reentry and focal activities, leading to a disruption of the normal sinus rhythm. Understanding these mechanisms and their contributions to arrhythmias in individual patients is crucial for developing effective treatments. To achieve this, in vitro human models based on induced pluripotent stem cell-derived cardiomyocytes have been developed to replicate arrhythmogenic activities and identify pathophysiological mechanisms specific to patients. However, the current methods for analyzing electrophysiology (EP) videos are limited and subjective, making the interpretation of clinical EP videos challenging. To address these limitations, we have developed a deep cardiac EP system called cEP-Net. The system comprises a cardiac microphysiological system designed for reproducing various forms of reentry and focal activities in vitro and a deep learning-based EP video classifier with a spatial and temporal streams architecture. The deep EP classifier analyzes cardiac EP videos, identifies arrhythmogenic activities, and categorizes videos into four categories based on their characteristics: (1) localized activities, (2) normal linear propagation, (3) focal activity, and (4) reentrant activity. We pre-trained cEP-Net using 111,110 annotated in vitro EP videos produced by a cardiac microphysiological system and evaluated its performance for in vitro and in vivo clinical settings. Our pre-trained cEP-Net demonstrated high Kappa (91.0%) and concordance rate (94.7%) for all categories. cEP-Net visualized in vitro EP videos in UMAP domains and successfully clustered them into four categories. Additionally, cEP-Net successfully captured the development and evolution of reentry during early in vitro cardiac tissue formation and the initiation of focal activities in the HCN4 (a pacemaker protein)-expressing tissues. We also used cEP-Net to quantify the arrhythmogenic risks of cardiac agents and found that cEP-Net could capture Flecainide-induced vulnerability to reentry. Lastly, we applied cEP-Net to analyze electroanatomical mapping videos in vivo. The cEP-Net demonstrated its capability to identify phase singularities in hearts with atrial fibrillation (AF) and capture the EP patterns of persistent AF hearts.
Abstract P1127: Induced Pluripotent Stem Cell-derived Macrophages Modulate Human Engineered Cardiac Tissue Function
Authors: Lock Roberta I , Graney Pamela L , Tavakol Naveed , Nash Trevor , Sanchez Eloy , Vunjak-Novakovic Gordana
Published: October 2023

In the native human myocardium, cardiac resident macrophages are a critical mediator for normal cardiac function, performing a variety of biological roles in electrical conduction, regeneration, and phagocytosis and efferocytosis. However, most models of human engineered cardiac tissues (hECT), which aim to recapitulate the native adult myocardium for disease modeling and drug screening applications, do not include a macrophage population. Thus, in this study we introduced an iPSC-derived macrophage (iM0) population into an all iPSC-derived tissue engineered model of the human myocardium in order to assess its impact on tissue phenotype and function. CD14
+
macrophages were differentiated from iPSCs and added as 7% of the total cell composition (7% iM0, 69.75% iCM, 23.25% iCF) in the hECT model. The hECTs were then cultured for 4 weeks and subjected to a ramped electrical stimulation protocol for matured function. The resulting tissues showed that the presence of a CD68
+
macrophage population was maintained over this extended period of culture and significantly impacted hECT function, increasing contractile force production and modulating contraction and relaxation velocity. Interrogation of calcium signaling via isoproterenol revealed that iM0 presence in the tissues may be causing these functional differences via modulation of the cAMP signaling pathway. These observations demonstrate that macrophages significantly contribute to cardiac function in iPSC-derived hECT models and emphasize the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.
Abstract P1128: CRISPR-mediated Activation Of DLK1 Induces A Regenerative State In Engineered Human Myocardium
Authors: Berecic Branimir , Bader Niklas , Rika Anastasia , Schoger Eric , Buchholz Eric , von Haehling Stephan , Zelarayan Laura C , Dieterich Christoph , Toischer Karl , Zimmermann Wolfram H , Tiburcy Malte
Published: October 2023

Background:
The human heart displays limited potential to recover from injury. Delta-like non-canonical Notch ligand-1 (
DLK1
) is an imprinted gene strongly expressed during embryo-fetal development. Expression in postnatal organs is low but re-expression commonly occurs in regenerative organs after injury. We hypothesized that enhancing
DLK1
expression in engineered human myocardium (EHM) may induce a regenerative response.

Methods and Results:
RNA sequencing of muscle biopsies from non-failing (n=14) and failing (n=12) human hearts demonstrated a reduced expression of
DLK1
in heart failure. In addition, serum levels of soluble DLK1 were reduced in patients with heart failure and correlated to left ventricular ejection fraction. Similar to patient samples,
DLK1
expression was reduced in EHM with simulated heart failure. To test if
DLK1
restoration prevents contractile dysfunction, we made use of a CRISPRa transgenic iPSC cell line, which stably expresses an enzymatic inactive dCas9 complex to specifically increase and modulate
DLK1
expression. Cardiomyocyte-specific
DLK1
activation (
DLK1
a) in EHM made with primary cardiac fibroblasts in a collagen hydrogel enhanced global
DLK1
expression levels to 12±5 fold (n=5) of non-target (NT) control in simulated heart failure. While
DLK1
a EHM showed comparable baseline function, contractile dysfunction by simulated heart failure was attenuated in
DLK1
a EHM (Force of contraction: -13±5 % vs -45±5 % in
DLK1
a vs NT EHM, n=8, p<0.05). RNA sequencing identified a regenerative transcriptome pattern in
DLK1
a EHM with differential expression of cell motility, extracellular matrix, cell cycle and metabolism-related processes.

Conclusion:
Reactivation of the developmental gene DLK1 induces a regenerative state and may protect from heart failure.
Abstract P1142: Environmental Regulation Of LncRNAs In Human Congenital Heart Defects
Authors: Hwang John , Xuedong Kang , Touma Marlin
Published: October 2023

Introduction:
Long non-coding RNA (lncRNA)-mediated transcriptional regulation is emerging as an important regulatory mechanism during development and diseases affecting all aspects of transcriptome programming. However, how systemic hypoxia alters lncRNA regulation in congenital heart defects (CHDs) remains poorly understood.

Objectives:
To examine the impact of systemic hypoxia on lncRNA-mediated regulatory networks in CHDs.

Methods:
Deep bulk RNA-seq datasets were derived from specimens of ventricular septum defects (VSD), tetralogy of fallot (TOF), and control hearts. TOF stratification (cyanotic vs noncyanotic) was based on average O2 saturation levels. Correlation statistics served to identify the lncRNA-mRNA pairs. The lncRNA-mRNA networks were generated using weighted gene co-expression network analysis (WGCNA).

Results:
Principal component analysis showed that lncRNAs from CHDs and control samples formed distinct clusters. Compared to control, 385 lncRNAs were differentially expressed in CHDs (adj
P
≤ 0.05) with 15% exceeded two-fold change in TOF. Subgroup analysis of the TOF cohort, revealed 111 differentially expression lncRNAs in cyanotic compared to noncyanotic TOF, after normalization to control. Out of 850 lncRNAs located within 50 kb of a protein coding genes, 65% exhibited significant correlation with the expression of their partner genes at Pearson’s correlation coefficient -0.7≤ and ≥0.7 and B-H corrected
P
value ≤0.05. The partner genes were enrichment with cardiac development, RNA processing, and chromatin remodeling. WGCNA identified nine lncRNA-mRNA modules specifically associated with clinical diagnosis or hypoxia status. Furthermore,
NONHSAT05860
and
NONHSAT049671
were identified as the top hypoxia-associated hub lncRNAs, potentially driving the molecular network of cyanotic TOF.

Conclusions:
The study revealed a high-resolution landscape of lncRNA contribution to transcriptome regulation in CHDs and uncovered hypoxia-associated lncRNA-mRNA regulatory circuits in cyanotic TOF.
NONHSAT05860
and
NONHSAT049671
were significantly influenced by systemic hypoxia providing conceptional basis for future investigations of their specific roles in transcriptome programming in cyanotic TOF.
Abstract P1150: Integrative Transcriptomics And Cell Systems Analyses Reveal Protective Pathways Controlled By Igfbp-3 In Anthracycline-induced Cardiotoxicity
Authors: Chen Junjie , Awada Jerome , Hassanieh Mackram , Packard Rene R
Published: October 2023

Rationale:
Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents. Their use however is hindered by the risk of anthracycline-induced cardiotoxicity (AIC). Our understanding of protective pathways activated by cardiomyocytes following AIC remains incomplete. Insulin-like growth factor binding protein (Igfbp)-3, the most abundant Igfbp family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cell types. Whereas Igfbp-3 is significantly induced by Dox in the heart, its role in AIC remains ill-defined.

Methods and Results:
We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 overexpression leads to a significant reduction of γ-H2AX, a marker of DNA damage (56% reduction vs. control, n=3, P<0.05), and impedes topoisomerase (Top)-2β expression which forms a Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (44% reduction vs. control, n=3, P<0.01). Next generation sequencing and gene network analyses of iPSC-CMs treated with Dox reveal that Igfbp-3 perturbation modulates genes implicated in microtubule function and cardiomyocyte contraction. Stimulated Emission Depletion (STED) microscopy and immunoblotting further indicate that Igfbp-3 knockdown exacerbates detyrosinated microtubule accumulation (STED, 21% increase vs. control, n=16-19, P<0.05; immunoblotting, 62% increase vs. control, n=6, P<0.01) – a hallmark of increased cardiomyocyte stiffness and heart failure – and favorably affects contractility following Dox treatment.

Conclusion:
Our delineation of the protective role of Igfbp-3 in AIC provides novel insight into pathways induced by cardiomyocytes to mitigate DNA damage and apoptosis, and improve contractile function.
Abstract P1157: Perinuclear Damage From Nuclear Envelope Deterioration Elicits Stress Responses That Contribute To
Lmna
Cardiomyopathy
Authors: Sikder Kunal , Phillips Elizabeth , Saunders Jasmine , Mothy David , Choi Jason C
Published: October 2023

Mutations in the
LMNA
gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating from
LMNA
mutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Nuclear envelope damage resulting from
LMNA
mutations is widely observed and accepted to be involved in the disease pathogenesis but how it contributes is not well understood. To this end, we performed a detailed kinetic study using a temporally controlled, cardiomyocyte-specific deletion model of
Lmna
to elucidate the progression of the disease. We found that cardiomyocyte-specific
Lmna
deletion in 12 weeks-old adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration and extensive damage to the perinuclear space. Perinuclear mitochondria and golgi are particularly affected, leading to mitochondrial loss as well as golgi dilation/fragmentation and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development of
LMNA
cardiomyopathy.
Abstract P1158: Glucolipotoxicity Curtails Mitophagy In Cardiomyocytes
Authors: Hahn Younghee , Chen Hongling , Banker Nidhi , Gadula Srinidhi , Vathappallil Pravin , Silverstein Brett , Van Jennifer , Kobayashi Tamayo , Zhang Ziying , Huang Yuan , Liang Qiangrong , Kobayashi Satoru
Published: October 2023

Diabetic patients suffer from complications of hyperglycemia and hyperlipidemia, which can lead to cardiovascular problems. The precise mechanism behind this phenomenon is unclear, but it is often associated with cellular damage and metabolic dysfunction. Mitochondria are responsible for energy production within cells, and abnormal metabolic conditions like diabetes can cause mitochondrial dysfunction. Mitophagy, the selective lysosomal degradation of damaged mitochondria, is a critical process for maintaining mitochondrial quality. However, the effects of hyperglycemia and hyperlipidemia on mitophagy in cardiomyocytes are not well characterized. We hypothesized that mitophagy is compromised in cardiac muscle cells by hyperglycemic and hyperlipidemic stresses. In this study, we investigated the effects of high-glucose (HG) and high-fat (HF) conditions on mitophagy in cardiomyocytes. We cultured H9C2 cardiac cells in media containing high levels of glucose (30 mM) and/or palmitate (25uM). The mitochondrial fractions were collected and the mitophagy flux was measured. Western blot analysis showed that the protein levels of microtubule-associated protein light chain 3 form II (LC3-II), a marker of mitophagosome, were reduced by HG and/or HF in either the absence or the presence of lysosomal inhibitors, suggesting inhibited mitophagy flux. The mitophagy flux was also determined by using dual fluorescent reporter that express mitochondria-targeted RFP-GFP fusion protein. Future studies will determine if HG and HF induce diabetic cardiac injury via impaired mitophagy.
Abstract P1159: Association Of Membrane Ferritin Transporter And Transferrin Receptor Plasma Levels With Coronary Atherosclerosis
Authors: Li Jia , Yunus Rayaan , Mahmood Faraz , Bu Yifan , Song Dan , Matyal Robina , Xie Hebin , Leng Yiping , Deng Ping , Zhong Qiaoqing
Published: October 2023

Objective:
Given the potential role of iron in inflammation and atherosclerosis, we investigated the correlation between the plasma levels of membrane ferritin transporter (FPN) and soluble transferrin receptor (sTFR) with the severity of coronary artery stenosis (CAS).

Methods:
Clinical data of 85 patients admitted to our institution between August 2022 and December 2022 with suspected coronary artery disease (CAD) was utilized. Patients were stratified according to their coronary angiography results: CAD group (n=50) and control group (n=50). Patients with CAD were further divided depending on their use of statin: statin taking group (PSU, n=15) and non-statin taking group (NSU, n=35). The coronary Gensini score was used to assess the severity of CAS. Spearman’s regression analysis was used to correlate plasma FPN and sTFR concentrations with the severity of CAS.

Results:
Plasma sTFR [2,216.88 (1,922.42, 2479.01) ng/mL vs. 1,928.96 (1490.50, 2289.23) ng/mL] concentration was higher in patients with CAD, while HDL cholesterol [0.94 (0.84, 1.17) mmol/L vs. 1.14 (0.95, 1.38) mmol/L] and plasma FPN [0.71 (0.42, 2.71) ng/mL vs. 2.90 (1.32, 4.40) ng/mL] concentration was lower when compared to patients without CAD. The coronary Gensini score was positively correlated with plasma sTFR concentration (R
s
=0.24,
p
=0.045) and negatively correlated with plasma FPN concentration (R
s
=-0.28,
p
=0.021). Plasma FPN level [1.50 (0.52, 4.77) ng/mL VS 0.51 (0.28, 2.46) ng/mL, P=0.007] in PSU patients was higher than the NSU group. Levels of total cholesterol [3.71±0.28mmol/L VS 4.67±0.15mmol/L, P=0.002] and low-density lipoprotein [2.24±0.25mmol/L VS 2.96±0.10mmol/L, P=0.002] were significantly lower in the PSU group. Regression analysis indicates that low plasma FPN concentration is an independent risk factor for the development of CAD (
p
=0.038).

Conclusion:
Our results suggest that sTFR and FPN concentrations are positively and negatively correlated respectively with the severity of CAS. Furthermore, regression analysis indicates that low plasma FPN concentration is an independent risk factor for the development of CAD. While our data provides some reference for evaluating the incidence and severity of CAD, future research is needed to verify our results.
Abstract P1168: Hippo Signaling Interacts With Tgfb Pathway To Regulate Pacemaker Cell State And Fibrosis In The Sinoatrial Node
Authors: Zheng Mingjie , Li Xiao , Wang Jun
Published: October 2023

Rhythmic heartbeats are initiated by electrical impulses from pacemaker cells (PCs) within the sinoatrial node (SAN). Fibroblasts provide structure support for PCs in the SAN, yet excessive fibrosis causes cardiac diseases and arrhythmias. Here we found that Hippo signaling cell-autonomously controls the cell state of PCs and non-cell-autonomously regulates the proliferation of fibroblasts in the SAN. Conditional knockout of Hippo signaling kinases
Lats1
and
Lats2
in PCs of adult mice (
Lats1/2
CKO) leads to SAN fibrosis and cardiac arrhythmias. RNAscope and immunostaining data indicated significantly increased Tgfβ1 and pSmad3 (Tgfβ signaling effector) in the SANs of
Lats1/2
CKO compared to controls. Treatment with Tgfβ receptor inhibitor significantly reduced fibroblasts proliferation in
Lats1/2
CKO, suggesting that
Lats1/2
deletion caused fibrosis is partially mediated through Tgfβ signaling. Single-cell (sc)-multi-omics data (paired scRNA-seq and scATAC-seq) from control and
Lats1/2
CKO SANs indicated that among three subtypes of PCs (PC1-PC3, classification based on their transcriptomic profile and chromatin accessibility), PC3 had the most upregulation of Yap (Hippo signaling effector) target genes in response to
Lats1/2
deletion. Notably,
Lats1/2
CKO PC3 was featured by highly increased expression of the smooth muscle genes, suggesting a smooth-muscle-like cell state change in PC3 after
Lats1/2
deletion. Moreover, compared to controls, smooth-muscle-like PCs of
Lats1/2
CKO had increased expression of Tgfβ ligands and Smad2/3, as well as Yap/Smad co-activation of smooth-muscle genes. In response to these changes in the smooth-muscle-like PCs of
Lats1/2
CKO, fibroblasts exhibited increased expression of Tgfβ receptors. These PCs state changes and PC-fibroblasts communications likely lead to increased fibrosis and arrhythmias in
Lats1/2
CKO. Taken together, our findings suggest that the Hippo signaling crosstalk with Tgfβ pathway regulates PC state and PC-fibroblasts communication.
Abstract P1174: Nicotine Alters PHLPP Isoforms In The Heart To Increase Susceptibility To Injury
Authors: Mohammed Abdul Khaja Shameem , Guerrero Alyssa , Kulkarni Amogh , Nguyen Tan , Purcell Nicole H
Published: October 2023

Rationale:
The impact of nicotine exposure through vaping on cardiovascular (CV) protective signaling basally or following injury remains largely unknown. Recent studies have shown that PHLPP1 deletion increased activity of AKT in the heart and reduced injury whereas PHLPP2 deletion exacerbated cardiac injury. Preliminary studies
in vivo
found that systemic exposure to nicotine increased PHLPP1 and decreased PHLPP2 expression in heart. Determining the mechanism of nicotine induced PHLPP alterations will demonstrate the impact of nicotine use on CV function and physiology and whether targeting PHLPP will reduce injury.

Hypothesis:
We hypothesize that nicotine compromises metabolic adaptation of the heart to injury by altering PHLPP isoforms and dysregulating mitochondrial biogenesis and oxidative stress signaling.

Methods:
Male and female wild-type (WT, n=4) and PHLPP1 knockout (KO, n=4) 4-6 weeks of age were implanted with osmotic minipumps containing nicotine (5mg/kg/day) or saline for 7 days. For
in vitro
study, NRVMs were isolated and PHLPP isoforms were targeted with siRNA or overexpressed using adenoviruses. Cardiomyocytes were stimulated with nicotine (100nM, 48hrs).

Results:
Nicotine increased PHLPP1 expression (1.7 ± 0.25-fold vs. WT saline) in the heart and decreased PHLPP2 (0.8 ± 0.04-fold vs. WT saline) whereas the KO exhibited increased PHLPP2 (1.5 ± 0.04-fold vs KO saline) levels. Nicotine administration significantly decreased genes involved in mitochondrial biogenesis and antioxidant signaling in WT mice while significantly increasing in KO hearts. Cardiomyocytes stimulated with nicotine (48h) significantly increased PHLPP1 (1.9 ± 0.39-fold vs PBS) and ROS. Nicotine significantly decreased mitochondrial respiration as did overexpression of PHLPP1. Finally, knockdown of PHLPP1 significantly increased mitochondrial respiration.

Conclusions:
Increases in PHLPP1 in the heart and cardiomyocytes from nicotine exposure has detrimental effects on oxidative stress and mitochondrial respiration. Future studies will investigate the molecular mechanisms by which nicotine alters PHLPP protein expression in heart and whether targeting PHLPP1 could reduce cardiovascular injury induced by nicotine exposure.
Abstract P1179: Evaluation Of A Machine Learning-based Metabolic Marker For Coronary Artery Disease In The UK Biobank
Authors: Gibson Kyle M , Forrest Iain , Petrazzini Ben O , Duffy Aine , Park Joshua K , Rocheleau Ghislain , Jordan Daniel , Do Ron
Published: October 2023

A recent study has shown that an
in silico
quantitative score of CAD (ISCAD) can be built using machine learning and clinical data from electronic health records. ISCAD was shown to be associated with CAD risk, atherosclerosis, sequela and all-cause mortality. We developed a CAD-predictive learning model using only metabolic data from 93,642 individuals from the UK Biobank (median [IQR] age, 57 [14] years; 39,796 [42%] male; 5640 [6%] with diagnosed CAD), and assessed its probabilities as a quantitative metabolic risk score for CAD (M-CAD; range 0 [lowest probability] to 1 [highest probability]) in participants of the UK Biobank. The relationship of M-CAD with arterial stiffness index, ejection fraction, CAD sequelae, and key mortality measures was assessed. The model predicted CAD with an area under the receiver-operating-characteristic curve of 0.712 in the testing set. Arterial Stiffness Index increased by 0.19 and 0.27 meters/second and ejection fraction decreased by 0.2% and 1% per 0.1 increase in M-CAD in all subjects and CAD cases, respectively. Both incident and recurrent myocardial infarction increased stepwise over M-CAD quartiles when evaluated in all participants (odds ratio (OR) 15.3 [4.2%] and 12.5 [0.2%]) and participants with CAD (OR 1.78 [34%] and 2.32 [1.7%] in top quartiles as compared to the first quartile of incident and recurrent MI respectively). Likewise, the hazard ratio and prevalence of all-cause mortality, CVD-associated mortality, and CAD-associated mortality increased stepwise over M-CAD deciles when assessed in all participants (2.98 [14%], 9.34 [4.3%], 26.7 [2.7%] in the top deciles as compared to the first decile of all-cause, CVD, and CAD mortality respectively) and a subset of participants with CAD (2.32 [24%], 2.78 [14%]], 3.41 [10%] in the top deciles as compared to the first decile of all-cause, CVD, and CAD mortality respectively. Interpretation of the model with SHAP values demonstrated that features known to be CAD risk factors contributed to the model predictions in the direction expected with their documented biological effects. We demonstrate that metabolic-based machine learning can be used to build a quantitative risk score for CAD that is associated with subclinical atherosclerosis, CAD sequala and mortality.
Abstract P1185: Spatial Transcriptome Profiling In The Mouse Heart With Single-Cell Resolution
Authors: Liu Yuening , Coles Annabelle , Neininger-Castro Abigail , Rune Julia , Lee Richard T , Kosuri Pallav
Published: October 2023

Background:
The spatial organization of the diverse cells in the heart shapes biological function. For instance, during the inflammatory and fibrotic processes that are central to heart disease, many cells act mainly within their local microenvironment. Recent studies using single-cell RNA sequencing (scRNA-seq) have provided a detailed list of the cell types and states involved in development, homeostasis and in cardiovascular diseases. However, scRNA-seq requires tissue dissociation, which removes spatial information and thereby limits our ability to determine how cells interact with each other to maintain health or cause disease. As a consequence, the precise spatial organization of cell types and states in the mammalian heart remains unknown. We sought to address this knowledge gap by using multiplexed error-robust fluorescence in situ hybridization (MERFISH), a method that enables simultaneous identification and localization of single transcripts from hundreds of different genes in intact tissue sections.

Methods/Results:
We adapted MERFISH and developed a new single-cell image segmentation method to creating a single-cell atlas of the mouse heart. First, we designed and constructed a 128-gene MERFISH probe library including cell type marker genes and also genes covering a broad range of cardiac signaling pathways. We then optimized sample preparation, including cell boundary staining and tissue clearing. Once optimized, we used this approach to image 10 μm-thick sections from healthy mouse heart, resolving tens of thousands of cells and millions of transcripts. Our data thus constitute the first single-cell spatial transcriptome atlas of the mouse heart.

Conclusion:
Through our newly developed cardiac MERFISH platform, we identified tens of thousands of cells and labeled millions of individual RNA transcripts in intact sections of mouse heart. Our cellular transcriptome profile maps enable new insights into how the organization of the heart shapes its biological function.
Abstract P2023: Mitogen-activated Protein Kinase-directed Modulation Of The Non-sarcomeric Actin Cytoskeleton In Cardiomyocytes
Authors: Grimes Kelly M , Bowers Stephanie L , Swoboda Casey O , Millay Douglas P , Molkentin Jeffery D
Published: October 2023

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the widening and lengthening of cardiomyocytes, respectively. Mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinases 1/2 (ERK1/2) are known to control this directional growth of cardiomyocytes, such that activated MEK1 causes concentric hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 MEK1 inhibitor, or control GFP adenovirus. The resulting data pinpointed over 2700 sites on 324 proteins as changing their phosphorylation status with modulation of this signaling cascade. Gene ontology analysis found that cytoskeletal-associated proteins were the largest subset of proteins with phosphorylation changes. Traditional and PhosTag western blots verified that many of these cytoskeletal proteins change expression as well as their phosphorylation status in NRVMs.
In vivo
experiments showed that cytoplasmic actin expression was upregulated in MEK1 transgenic or ERK1/2 gene-deleted hearts. Furthermore, MEK1 transgenic adult cardiomyocytes had enhanced cytoplasmic actin at lateral membranes. Palladin, an actin nucleating protein, changed both phosphorylation status and isoform expression in response to modulation of MEK1-ERK1/2 signaling. Adeno-associated viruses were used to overexpress different palladin isoforms. Immunostaining for these proteins showed that localization of palladin isoforms moved from the subcortical compartment to the sarcomere, suggesting these isoforms preferentially associate with either cytoskeletal or sarcomeric actin. Future studies will further examine the signaling roles that cytoplasmic actin may play in healthy and diseased cardiomyocytes and delineate preferential binding of actin associated proteins between cytoplasmic and sarcomeric actin in cardiomyocytes.
Abstract P2026: Moderate Ethanol Consumption Causes Right Ventricular Hypertrophy And Pulmonary Hypertension Via The Pro-inflammatory Endothelin-interleukin Pathway
Authors: Donovan Mary K , Abdel-Rahman Abdel A
Published: October 2023

The substantial increase in ethanol consumption since the Covid-19 pandemic has re-emphasized the need for extensive preclinical research on the cardiac and pulmonary effects of ethanol. Current knowledge is mostly based on ethanol effects on left ventricular (LV) function and blood pressure. Therefore, this preclinical study dealt with the effects of moderate ethanol consumption on right ventricular (RV) and pulmonary artery (PA) structure and function, and the implicated mechanisms in a pulmonary hypertension susceptible rat strain. Two groups of Sprague-Dawley (CD strain) male rats (n=7-8 each) received balanced liquid diet containing 5% ethanol (w/v) or pair-fed with isocaloric liquid diet for 8 weeks. This ethanol regimen resulted in clinically relevant blood ethanol concentration (39.8
+
1.9 mg/dL). Echocardiographic analysis showed progressive decreases in PA acceleration time/ejection time and increases in mean PA pressure (hallmarks of pulmonary hypertension) in ethanol-fed rats. Further, transthoracic echocardiography in long-axis orientation showed a significant increase in the RV internal diameter/left ventricular internal diameter during diastole (RVID-d/LVID-d), indicative of RV dilation in chronic ethanol-treated rats. Echocardiographic LV Mass/body weight and post-mortem Fulton’s index were higher in ethanol-fed rats, suggesting ethanol induced simultaneous LV and RV hypertrophy. Further, ethanol increased mean arterial pressure (measured at the end of the 8-weeks study). Ex vivo biochemical analyses showed upregulation of the vasoconstrictor/pro-inflammatory protein endothelin-1 (ET-1) and its downstream target IL-6 in the RV and lungs of ethanol-fed rats. Finally, ethanol reduced lung endothelin ETB receptor expression but had no effect on the ETA receptor, leading to higher ETA/ETB receptor ratio. Collectively, this study yields new insight into ethanol-evoked RV and pulmonary artery remodeling via the upregulation of the ET-1-IL-6 pathway.
Abstract P2033: Western Diet Can Cause Adverse Cardiac Remodeling During Pregnancy By Inducing Foxo1 Expression
Authors: Yang Yijun , Kubo Hajime , Kurian Justin , Johnson Jaslyn , Li Yijia , Roy Rajika , Berretta Remus M , Koch Walter J , Khan Mohsin , Mohsin Sadia , Houser Steven R
Published: October 2023

Introduction:
Pregnancy induces physiological cardiac hypertrophy in healthy women. Our recent findings suggest that obesity can disturb this process and induce pathological cardiac hypertrophy during pregnancy both in human patients and Western Diet (WD) fed mice. Animal studies determined the pathological cardiac hypertrophy is concomitant with increased cardiomyocyte cross-sectional area (CSA), fetal gene activation and fibrosis, however, the underlying mechanisms remain unclear. The present study asks if FoxO1, as a transcriptional factor, may play a role in the adverse cardiac response to pregnancy in WD mice.

Methods:
The expression levels of FoxO1 and its downstream targeting genes were tested in Ctrl Diet (CD) and WD fed non-pregnant and postpartum animals. To further investigate the role of FoxO1 in pathological cardiac hypertrophy in WD animals during pregnancy, FoxO1 was then silenced by AAV9-shFoxO1 virus in WD mice before breeding. AAV9-scramble RNA served as control. Cardiac phenotype in AAV9 treated WD mice were characterized at post-partum day 1.

Results:
WD feeding led to increased gene expression levels of FoxO1 and its downstream targeting genes, such as CD36 and PDK4, compared with CD mice. While the gene expression levels of FoxO1 were not different between non-pregnant and pregnant WD mice, FoxO1 protein activation was significantly increased in WD mice after pregnancy determined by a decrease of pFoxO1/tFoxO1 ratio, compared with non-pregnant WD mice. Silencing FoxO1 inhibited the expression levels of FoxO1 and its downstream genes CD36 and PDK4. FoxO1 inhibition in WD fed animals prevented WD induced pathological hypertrophy during pregnancy, with decreased heart weight, cardiomyocyte CSA, attenuated fetal gene activation and fibrosis accumulation. FoxO1 silencing also led to less accumulation of lipid peroxidation product in the myocardium.

Conclusions:
FoxO1 activation in WD mice with metabolic disturbances during pregnancy can induce pathological cardiac hypertrophy observed during pregnancy.
Abstract P2034: Minimally-invasive Porcine Model For The Study Of Cardiac Remodeling In Type 4 Cardiorenal Syndrome
Authors: Mavropoulos Spyros A , Sakata Tomoki , Mazurek Renata , Ravichandran Anjali , Marx Jonas , Raisinghani Nikhil , Sahoo Susmita , Ishikawa Kiyotake
Published: October 2023

Background:
CKD is on the rise, and over half of patients die due to cardiac causes, usually HF. They present with HF not only due to volume overload, but also because of reno-cardiac interactions of unclear mechanisms termed type 4 cardiorenal syndrome (CRS4). In order to understand CRS4, we developed a pig model using minimally-invasive techniques.

Methods:
4 female Yorkshire pigs had staged embolization of the renal arteries. Clot/contrast mix was injected by catheter until no flow was seen on angiography. Echo, pressure measurements, angiography, and urine and blood collection to monitor CKD and HF progression was done monthly. At 4 months, GFR and PV loop measurements were also taken, and tissues harvested.

Results:
BUN (11.3 ± 0.3 vs 5.3 ± 0.5 mg/dL, p < 0.01) and SCr (2.1 ± 0.2 vs 1.3 ±0.1 mg/dL, p<0.01) increased at the endpoint compared to controls, and GFR declined (6 ± 4 vs 124 ± 17 mL/min, p < 0.01). Kidneys showed diffuse fibrosis on trichrome staining. LV mass (136 ± 6 vs 106 ± 6 g, p < 0.05), EDP (16 ± 2 vs 9 ± 2 mmHg, p <0.05), aortic (146 ± 9 vs 96 ± 5 mmHg, p < 0.01) and pulmonary capillary wedge pressures (12 ± 1 vs 6 ± 1 mmHg, p < 0.01) and PA (22 ± 1 vs 16 ± 1 mmHg, p < 0.05) were increased. PV loop showed increased EDPVR slope (0.36 ± 0.03 vs 0.17 ± 0.03, p < 0.01) and Tau (48.9 ± 1.9 vs 36.3 ± 1.1 ms, p < 0.01). LV was fibrotic.

Conclusions:
Repetitive clot injection successfully induced CKD, seen by increased SCr and BUN, reduced GFR, and renal fibrosis. At 4 months, the heart was hypertrophic, fibrotic, and had PV relationships indicating development of HFpEF. Therefore, we have developed a successful minimally-invasive large animal model of CRS4 for future studies.
Abstract P2046: 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 , Tripp Audrey , Phan Tina , Alissa Abdullah , Aljuaid Mossab , Madias Christopher , Homoud Munther , Galper Jonas B , Chin Michael T
Published: October 2023

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation. While mutations in sarcomere genes can account for a substantial proportion of familial cases, 40-50% 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 homozygotic 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 maximal elastance, reduced diastolic function and are more susceptible to ventricular tachycardia with programmed stimulation. Upon transverse aortic constriction, mice carrying the
Cryab

R123W

allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased 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. Studies on isolated adult cardiomyocytes reveal altered fractional shortening, calcium dyshomeostasis, prolonged action potential duration, and T-wave alternans. Crossing of CRYAB
R123W
mice with a mybpc3 knock-in model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in this model are independent of the sarcomere. Thus, our data establish the
Cryab

R123W

allele as a novel genetic model of HCM that potentially unveils additional sarcomere-independent mechanisms of cardiac pathological hypertrophy and sudden cardiac death.
Abstract P2059: Perm1 Regulates O-glcnacylation Of Pgc-1alpha In The Heart
Authors: SREEDEVI KARTHI , Yedla Shreya , Amina James , Arowa Sumaita , Shah Riyan , Do Sara , Goode Breanna , Bui Sydney , Olmos Katia , Zaitsev Alexey , Warren Junco
Published: October 2023

O-GlcNAcylation (O-GlcNAc) is a post-translational modification of proteins where N-acetylglucosamines are added to serine and threonine residues in proteins. This modification is regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. A recent study demonstrated that overexpression of OGT in the heart leads to excessive O-GlcNAcylation, resulting in mitochondrial dysfunction and the development of heart failure. However, what regulates OGT expression in the heart remains elusive. Perm1 is a striated-muscle-specific regulator of mitochondrial bioenergetics. Here, we found that Perm1 suppresses O-GlcNAcylation via gene repression of OGT. Adenovirus-mediated overexpression of Perm1 suppressed O-GlcNAcylation of proteins in H9c2 cells (47% of control, p<0.05), concurrent with downregulation of OGT (52% from control, p<0.05). Luciferase gene reporter assay revealed that Perm1 reduces the promoter activity of OGT, indicating that Perm1 acts as a gene repressor of OGT. Furthermore, phenylephrine-induced O-GlcNAcylation in cardiomyocytes was reversed by Perm1 overexpression through repressing OGT expression. Conversely, loss of Perm1 (Perm1-KO) in mice led to upregulation of OGT and the increase of O-GlcNAcylated proteins in the heart (176% and 144% from WT, respectively, both p<0.05, n=6 in WT, n=7 in KO). Concomitantly, Perm1-KO mice exhibited reduced contractility and mitochondrial function, manifested by a significant decrease in ejection fraction and succinate dehydrogenase activity, respectively (87% and 53% of WT, both p<0.05). Lastly, subcellular fractionation and co-immunoprecipitation assay of mouse hearts revealed that loss of Perm1 specifically increases O-GlcNAcylation of nuclear proteins, concurrent with excessive O-GlcNAcylation of PGC-1α, a master regulator of mitochondrial bioenergetics (2-fold increase in O-GlcNAcylated PGC-1α/total PGC-1α, p<0.05, n=3/group). Our results suggest that Perm1 is a novel regulator of O-GlcNAcylation in the heart that transcriptionally represses OGT expression, which presumably prevents excessive O-GlcNAcylation of PGC-1α and mitochondrial dysfunction under pathological stress.
Abstract P2061: The Intersection Of O-GlcNAc And Yap - A Novel Molecular Axis In Heart Failure
Authors: Lopez-Cecetaite Gabriel , Luczak Elizabeth , Umapathi Priya
Published: October 2023

Introduction:
Heart failure (HF) and pathological myocardial hypertrophy continue to grow world-wide and are strongly tied to obesity, hyperglycemia, and ischemia. These stressors lead to increased levels of O-beta-N-Acetylglucosamine modified proteins (OGN) in the heart. OGN levels are determined by the net activity of two enzymes, OGT and OGA. Our recent work showed excessive OGN was sufficient to cause HF and pathological myocardial hypertrophy. Here we test the relationship between OGN and Yes Associated Protein (YAP), a known promoter of cardiac hypertrophy.

Objectives:
Test if OGN modifies YAP in the heart and evaluate the role of OGN modified YAP in the development of pathologic cardiac hypertrophy and heart failure.

Methods:
We have shown that mouse models with myocardial OGT overexpression (OGT TG) developed pathological cardiac hypertrophy and HF. We found these animals have increased YAP activity. Myocardial OGA overexpression (OGA TG) attenuated OGN following transaortic constriction(TAC) and were protected against cardiac hypertrophy and HF. Interbreeding OGT TG with OGA TG rescued animals from pathological hypertrophy, HF and normalized YAP activity
.
We quantified levels of activated YAP protein/gene expression in hearts from WT, OGT, OGA and OGT x OGA mice. We evaluated YAP/TEAD1 target gene expression levels in OGA TG mice protected against TAC. We used an adenoviral YAP/TEAD expression reporter to quantify YAP/TEAD1 activity in cardiac myocytes, in the presence and absence of increased O-GlcNAcylation.

Results:
We found WT levels of activated (nuclear) YAP in OGA TG, increased active YAP in OGT TG and normal levels in OGT x OGA mice. YAP/TEAD1 target genes are upregulated in OGT TG hearts but interbred OGT x OGA animals have normal cardiac function and YAP/TEAD1 target gene expression. OGA TG mice who are protected against TAC induced heart failure/hypertrophy and have decreased myocardial levels of YAP/TEAD1 gene targets involved in pathological hypertrophy. Increased O-GlcNAcylation in cardiac myocytes activates YAP/TEAD activity and YAP/TEAD downstream gene expression.

Conclusion:
These preliminary data suggest a novel pathway for promoting pathological hypertrophy and HF via activation of the O-GlcNAc -YAP/TEAD1 axis.