bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1083: Retinoid Regulation Of Cardiac Inflammation
Authors: Gans Ian
Published: October 2023

Survivors of acute cardiac events (e.g., myocardial infarction, MI) often recover poorly due to pathological remodeling and progressive heart failure. Cell-based therapies hold potential to improve recovery but have had limited success. Our lab has found that infusion of highly proliferative cardiac cell clones from patient biopsies can improve recovery from MI in mice, but with variable efficacy stemming from unknown factors. Because Vitamin A (VA) stores are known to be mobilized to the heart in response to MI, we investigated the effect of VA on patient-derived cardiac cells. Clones were cultured and treated with 500 nanomolar retinoic acid (RA, bioactive metabolite of VA) for 48 hours. Protein was extracted and digested for proteomic analysis using liquid chromatography and mass spectrometry. To investigate interaction of RA with pro-regenerative epidermal growth factor (EGF) family receptor signaling, cells were treated with 50ng/ml recombinant neuregulin (NRG1) alone or in co-treatment with RA. Treatment with RA caused significant increase in protein expression of inflammatory mediators including IL1A/B, ICAM1, prostaglandin synthase, and NFkB2, and less studied targets including differentiation factor latexin (LXN) and orphan G-protein coupled receptor GPRC5A. Expression was validated at the transcript level. NRG1 treatment significantly modulated expression of metabolic regulators carnitine-palmitoyl transferase, phosphofructokinase, and dipeptidyl peptidase but was inhibited by co-treatment with RA. Our data suggest that RA may induce a pro-inflammatory phenotype in proliferative cardiac cells and that RA inhibits pro-regenerative NRG1/EGF receptor signaling, perhaps as part of an emergency inflammatory response to injury. While the biology underlying limited successes of cell therapies to treat heart disease is incompletely understood, managing inflammation is one strategy that has shown benefits in treatment of heart failure and reprogramming cells to therapeutic ends. Further experiments are called for to investigate an inflammatory role of VA during MI
in vivo,
and to determine how the interaction of VA and NRG1 may regulate proliferative cardiac cell fate and function after injury.
Abstract P1104: Mantarray 3D Engineered Muscle Tissue Platform Demonstrates Clinically-relevant Disease Stratification Of An
In Vitro
Human Duchenne Muscular Dystrophy Model
Authors: Luttrell Shawn , Macadangdang Jesse , Gray Kevin , Kharoufeh Samir , Worthen Christal , Luerman Greg , Geisse Nicholas
Published: October 2023

Accurately modeling disease in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac diseases, direct assessment of contractile output is the “ultimate phenotype” and a reliable metric to study overall tissue function, as other ‘proxy’ measurements or biomarkers are not good predictors of muscle strength. Human 3D engineered heart tissues (EHTs) from induced pluripotent stem cells hold great potential for modeling contractile function. However, the bioengineering strategies required to generate these predictive models present limitations for many investigators. Here, we have developed an instrument platform that utilizes 3D EHTs in conjunction with a label-free, magnetic sensing array (Mantarray). The platform enables facile and reproducible fabrication of 3D EHTs using virtually any cell source and is coupled with parallel direct measurement of contractility. This approach enables clinically relevant functional measurements of muscle, stratification of healthy vs. diseased muscle phenotypes, and facilitates therapeutic modality-agnostic compound safety and efficacy screening. We present a 3D model of Duchenne muscular dystrophy (DMD) that utilizes EHTs formed from an isogenic pair of healthy and diseased cells. DMD tissues display functional deficits across numerous metrics of contractility, including force and relaxation kinetics, though beat rate remains unchanged. EHTs remain functional over months in culture and provide a large experimental window to not only study therapeutic effect, but also disease phenotypes that may present at later stages of development and maturity. We show both acute and chronic effects of compounds in EHTs, including a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. These data demonstrate a first-and-only commercial platform that integrates individual, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Stimulation is coupled with automated assessment of muscle contraction, providing an inclusive, medium-throughput platform for disease modeling and therapeutic discovery.
Abstract P1131: Epicardially-secreted Fibronectin Promotes Hesc-cardiomyocytes' Maturation In 3d-engineered Heart Tissues.
Authors: Ong Lay Ping , Bargehr Johannes , Knight-Schrijver Vincent , Lee Jonathan , Bayraktar Semih , Colzani Maria , Marchiano Silvia , Bertero Alessandro , Murry Charles , Gambardella Laure , Sinha Sanjay
Published: October 2023

Ischaemic heart failure is due to the irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could regenerate infarcted hearts and improve cardiac function. However, these hPSC-derived cardiomyocytes remained immature. Epicardial-myocardial crosstalk underpins key events during cardiac development and maturation. Recently we showed that incorporating hPSC-derived epicardial cells improved cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). However, the key signaling pathways underpinning epicardial-myocardial crosstalk remain unclear.We found that Fibronectin (FN1) was uniquely upregulated in 3D-EHTs co-cultured with hESC-epicardium, compared to other stromal cells such as mesenchymal stem cells. Thus, we posited that epicardially-secreted fibronectin (FN1) is a key mediator of epicardial-cardiomyocyte crosstalk driving cardiomyocyte maturation.To test this hypothesis, we performed a series of loss of FN function experiments with peptide inhibition (pUR4), CRISPR/Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs. We found that the loss of epicardial-FN, at both protein and gene levels, resulted in immature hPSC-derived cardiomyocytes with decreased contractile function, inefficient Ca2+ handling, and disorganized sarcomeric apparatus. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Furthermore, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.
Abstract P1133: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene Sik1
Authors: Arrieta Adrian , Chapski Douglas , Reese Anna , Rosa-Garrido Manuel , Vondriska Thomas M
Published: October 2023

Background:
Neonatal cardiomyocyte physiological hypertrophy is temporally encoded, yet the chromatin remodeling mechanisms driving this behavior are unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAPII occupancy.

Hypothesis:
BMAL1 regulates temporal chromatin remodeling and expression of genes critical to neonatal cardiac hypertrophy.

Methods:
RNAPII and BMAL1 ChIP-seq and ATAC-seq data were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. Bmal1 was knocked down in neonatal rat ventricular myocytes via siRNA and hypertrophy was assessed by measuring cell size and L-azidohomoalanine incorporation into total protein, and Nppa/Nppb RT-qPCR. Bmal1 target gene promoter accessibility was assessed via MNase-qPCR and levels of chromatin-associated nucleosome core histones H2A, H2B, H3 and H4 were measured by immunoblotting of acid soluble chromatin fractions.

Results:
RNAPII occupancy at Bmal1 target genes was increased in adult as compared to E12.5 hearts. BMAL1 ChIP-seq and ATAC-seq revealed cardiac-specific BMAL1 co-localization (other tissues served as controls) with accessibility at the pro-hypertrophic gene salt-inducible kinase 1 (Sik1). BMAL1 knockdown impaired serum-stimulated hypertrophy, indicated by 30% decrease in cell size, 50% decrease in L-azidohomoalanine incorporation into total protein, and 80% decrease in Nppa/Nppb mRNA. BMAL1 knockdown decreased mRNA and promoter accessibility of BMAL1 targets Per2 and Sik1 and decreased chromatin-associated histones by 50%. Finally, Sik1 knockdown decreased myocyte size and Nppb mRNA.

Conclusion:
These findings indicate that BMAL1 controls Sik1 expression—and possibly that of other growth inducing genes—in part by modulating nucleosome assembly at specific loci, thereby regulating neonatal myocyte physiological hypertrophy.
Abstract P1144: Single-cell RNA-Sequencing Of The Murine Cardiac Conduction System Reveals Transcriptional Regulators Associated With Conduction Development And Disease
Authors: Beyersdorf Benjamin , Atzert Felix , Wu Sean M , Goodyer William R
Published: October 2023

Rhythmic beating of the heart is driven by a small subset of heart cells, altogether known as the cardiac conduction system (CCS). The CCS consists of unique components including the sinoatrial node (SAN), the atrioventricular node (AVN), the bundle of His, bundle branches and Purkinje fiber (PF) system. Due to obstacles inherent in studying the CCS, including small cell numbers, large cell-type heterogeneity and complex anatomy, our understanding of the transcriptional networks driving CCS development remains limited. Here, we harnessed single-cell RNA-sequencing (scRNAseq) and bioinformatic analyses to uncover putative DNA-binding transcription factors (TFs) as well as transcriptional co-regulators enriched within the entire developing murine CCS. Comparison of multiple independent prior datasets, spanning all major components of the CCS, revealed previously known TFs necessary for conduction development including,
Islet 1
(
Isl1
; avg log fold change 0.34, p= 1E-192) in the SAN;
T-box transcription factor 5 (Tbx5
; avg log fold change 0.54, p= 2.1E-110) in the AVN; and
ETS variant transcription factor 1
(
Etv1;
avg log fold change 0.89, p= 6.7E-105) in the PFs, among others. Notably, an additional 16 novel DNA-binding transcription factors and 40 transcription co-regulators were enriched within specific CCS components at statistical significance. To further evaluate the transcriptional landscape of individual subcomponents within the SAN (compact versus transitional cells), we also performed scRNAseq on FACS-isolated SAN cells from embryonic day 16.5
Hcn4
-GFP transgenic mice revealing differential expression of a host of TFs, both established and novel. Finally, our murine findings were then cross-referenced to over 15 human GWAS studies related to cardiac rhythm abnormalities, revealing an association of a subset of these murine CCS-enriched transcription factors and cardiac arrhythmias in humans. In conclusion, this study provides a comprehensive gene expression atlas of transcription factors and transcriptional co-regulators within individual CCS components at single-cell resolution, laying the groundwork for future studies into the transcriptional regulation of cardiac conduction development and disease.
Abstract P1151: Exploring The Role Of Bh3-like Motif Containing Cell Death Inducer In Cardiomyocyte Death: Insights From Human Failing Hearts And Ipsc-derived Cardiomyocytes
Authors: Dubey Praveen K , Singh Sarojini , Ye Lei , pogwizd Steven , Zhang Jianyi J , Krishnamurthy Prasanna
Published: October 2023

Background:
Cardiac cell death plays a critical role in the development and progression of heart failure. Identifying new molecular targets to limit cardiac cell death could improve patient lives. BLID (BH3-Like Motif-Containing Cell Death Inducer) is a member of the BCL family of proapoptotic genes containing a BH3-like motif that induces cell death. Previous studies have shown that in many human cancers, downregulation of BLID is associated with tumor progression, while overexpression promotes cell death through a caspase-dependent mechanism. However, its role in cardiomyocyte death under various stress conditions is unknown.

Methods and Results:
Our RNAseq data from human failing and non-failing hearts revealed that mRNA expression of the BLID gene was significantly upregulated in human heart failure. We further validated our RNAseq findings by measuring the expression of BLID in RNA and protein isolated from the same set of human failing and non-failing hearts. As expected, the expression of BLID was significantly upregulated at both the mRNA and protein levels in human failing hearts compared to non-failing hearts. Furthermore, in cultured human cardiomyocytes (AC16 cells) and iPSC-derived cardiomyocytes exposed to stressors (such as hypoxia or H
2
O
2
) for 2 hours, we observed a significant upregulation of BLID expression at both the mRNA and protein levels. We also overexpressed human BLID protein in human cardiomyocytes, leading to cardiomyocyte death. To determine their localization, we fractionated the overexpressed BLID proteins into cytoplasmic, nuclear, and mitochondrial fractions. We found that BLID was localized in the cytoplasm, nucleus, and mitochondria, with higher levels in the nucleus and mitochondria. These findings suggest that increased expression of BLID in failing human hearts may lead to cardiomyocyte death.

Conclusion:
Our data suggest that increased expression of BLID in human heart failure may be a key molecule that drives cardiomyocyte death. Inhibiting its expression during heart failure could mitigate cardiomyocyte death and improve cardiomyocyte function and survival.
Abstract P1170: Changes In Endothelin Signaling In A Long-term Rat Coronary Culture Resembles Chronic Coronary Syndrome
Authors: Kazantzi Spyridoula , Edvinsson Lars , Haanes Kristian A
Published: October 2023

Background:
Coronary artery disease generates changes in the structure and function of the blood vessels. 48hrs organ culture (mimicking the acute loss of blood flow) of coronary arteries induces the upregulation of contractile endothelin type B (ETB) receptors, resembling the pattern seen in ischemic heart disease. MEK inhibitors (U0126 and Trametinib), can acutely inhibit the upregulation of these receptors, illustrating the importance of this pathway. The 48hrs organ culture model has been proven successful for translation in acute ischemic disease, but arterial changes have not been studied in a long-term chronic model which could be important for chronic coronary syndrome.

Purpose:
1) Investigate the changes of ETB receptors after incubation in a long-term optimized medium for organ culture, mimicking the reduction of blood flow in chronic coronary syndrome and 2) determine whether Trametinib, would have treatment effect on the upregulation of ETB receptors in both long term and short term culture.

Methods:
Rat coronary arteries were incubated in organ culture for 48hrs in DMEM ± trametinib (“acute”) or in a long-term optimized medium for organ culture for 14 days (“chronic”). After the incubation, arteries were mounted stainless steel wires (40 μm) on a Mulvany-Halpern arterial wire myograph and cumulative concentration response curves were made to measure arterial contractility to the ETB receptor specific agonist Sarafotoxin (S6c). Trametinib was added at time 0 (for both the acute and chronic organ culture) or 7 days delayed.

Results:
For the “acute” culture, we observed a high contractile effect in response to S6c (Emax=136.5±18%). The presence of Trametinib greatly reduced this contractile effect (Emax=10±1.9%) and arteries were comparable to fresh (Emax=0.8±0.7%). The rat coronary arteries in the “chronic” culture maintained their ability to contract in response to potassium, and S6c had a strong contractile effect (Emax=74.64±17%). Interestingly, neither trametinib addition at time 0 nor at day 7 could inhibit the functional upregulation of ETB in the chronic model.

Conclusion:
Chronic coronary syndrome may require new inhibitors of ETB receptor upregulation. This could be investigated with our new long-term rat coronary artery model.
Abstract P1178: APOE-NOTCH Axis Governs Elastogenesis During Human Cardiac Valve Remodeling
Authors: Liu Ziyi , Liu Yu , Yu Zhiyun , Pek Nicole , O'Donnell Anna , Glass Ian , Guo Minzhe , Chen Ya-Wen , Wu Joseph , Yutzey Katherine E , Miao Yifei , Gu Mingxia
Published: October 2023

Background:
Valve remodeling is a complex process involving extracellular matrix organization, development of trilaminar structures, and physical elongation of valve leaflets. However, the cellular and molecular mechanisms regulating valve remodeling and their roles in congenital valve disorders remain poorly understood.

Methods:
Semilunar valves and atrioventricular valves from healthy and age-matched human fetal hearts with pulmonary stenosis (PS) were collected. Single-Cell RNA-sequencing (scRNA-seq) was performed to determine the transcriptomic landscape of multiple valvular cell subtypes in valve remodeling and disease.

Results:
scRNA-seq analysis of healthy human fetal valves identified a novel APOE+ elastin-producing valve interstitial cell (Elastin-VIC) subtype spatially located underneath VECs in the direction of unidirectional flow. Knockdown of
APOE
in fetal VICs resulted in significant elastogenesis defects. In pulmonary valve with PS, we observed decreased expression of
APOE
and other genes regulating elastogenesis such as
EMILIN1
and
LOXL1
, as well as elastin fragmentation. Cell-cell interaction analysis revealed that JAG1 from unidirectional VECs activates NOTCH signaling in Elastin-VICs through NOTCH3.
In vitro
Jag1 treatment in VICs increased the expression of elastogenesis-related genes and enhanced contractile functions with related gene expression. This was accompanied by activation of NOTCH signaling and elastogenesis observed in VICs co-cultured with VECs in the presence of unidirectional flow. Notably, we found that the JAG1-NOTCH3 signaling pair was drastically reduced in the PS valves. Lastly, we demonstrated that APOE is indispensable for JAG1-induced NOTCH activation in VICs, reinforcing the presence of a synergistic intrinsic and external regulatory network involving APOE and NOTCH signaling that is responsible for regulating elastogenesis during human valve remodeling.

Conclusion:
scRNA-seq analysis of human fetal valves identified a novel Elastin-VIC subpopulation, and revealed mechanism of APOE and NOTCH signaling mediated elastogenesis during valve remodeling, which provided opportunity to understand pathogenesis of elastic malformation within congenital valve disease.
Abstract P2013: Cardiac Remodeling, Amyloidosis, Neurotrophic Signaling, And Innervation Impairment In The Tg2576 Model Of Alzheimer's Disease
Authors: Elia Andrea , Parodi-Rullan Rebecca , Vazquez-Torres Rafael , Carey Ashley , Fossati Silvia
Published: October 2023

Background:
In Alzheimer's disease (AD), the characteristic cerebral amyloid β (Aβ) deposition and tau pathology are accompanied by a progressive loss of the two main neuromodulators, NGF and BDNF. This AD-mediated derangement of the brain's neuro-signaling pathway may extend to the periphery and, along with potential peripheral amyloid deposition, induce peripheral nervous system impairment, thus affecting other organs, including the heart. However, whether and how AD pathology affects cardiac physiology, neurotrophins, innervation, and heart amyloidosis is still unclear.

Method:
Here, we describe for the first time that cardiac remodeling and neuro-signaling dysregulation are present in the hearts of Tg2576 mice, a widely used model of AD and cerebral amyloidosis.

Results:
Echocardiographic analysis showed significant deterioration of left ventricle function, as demonstrated by a decline of both ejection fraction and fraction shortening in 12-month-old Tg2576 mice compared to age-matched WT littermates. The transgenic mice hearts displayed an increase in interstitial fibrosis, with an accumulation of amyloid aggregates, including Aβ, associated with severe cardiac nervous system dysfunction. Tg2576 mice exhibited a depletion in cardiac nerve fiber density, both adrenergic (stained with tyrosine hydroxylase- TH) and regenerating nerve terminals (labeled with GAP-43), compared to the WT mice. This myocardial denervation was accompanied by a decline in NGF and BDNF protein expression as well as GAP-43 expression in both the brain and heart of Tg2576 mice. Likewise, human neuroblastoma cells (SH-SY5Y) and human cardiomyocytes (AC16) challenged with human Aβ-40 or, Aβ-42 oligomers showed significant downregulation of both BDNF and GAP-43 activity.

Conclusion:
Overall, this study highlights the harmful impact of cerebral amyloidosis on the cardiac nervous system and heart physiology, providing potential therapeutic targets, such as neurotrophic factors, to prevent or limit AD-mediated degenerative mechanisms in the cardiovascular system.
Abstract P2015: A Human Flii Gene Variant Shortens Sarcomeric Actin Thin Filament Length And Predisposes To Cardiomyopathy
Authors: Kuwabara Yasuhide , York Allen , Lin Suh-chin , Sargent Michelle , Grimes Kelly , Pirruccello James , Molkentin Jeff D
Published: October 2023

Objective:
We identified a human variant in the
Flightless-I homolog
(
FLII
) gene that generates a R1243H missense change associated with cardiomyopathy in human genome-wide association studies. However, the causality and its molecular mechanism remain unclear.

Methods and Results:
To examine how the human variant resulting in R1243H amino acid substitution might be pathogenic in vivo, we generated a mouse model harboring the R1245H mutation in
Flii
locus using CRISPR/Cas9 technology. The

Flii
R1245H/R1245H

homozygotes showed reduced cardiac function by 16 weeks of age compared with control (
P
=0.01). Eight-week-old

Flii
R1245H/R1245H

mice subjected to 8 weeks of TAC showed significantly worse cardiac function with greater cardiac hypertrophy and premature lethality compared with control (
P
<0.01). Here we show that Flii functions in the heart to regulate sarcomeric actin thin filament length. Indeed, hearts from

Flii
R1245H/R1245H

mice at 16 weeks of age showed reduced thin filament length compared with controls (
P
=0.009). To further investigate the mechanistic effects of the R1245H mutation in mouse
Flii
, we examined actin polymerization dynamics in vitro between wild-type (WT) Flii and the R1245H mutant proteins. The data showed that WT Flii augmented G-actin polymerization into F-actin in a concentration dependent manner, while Flii-R1245H mutant protein antagonized polymerization (
P
<0.001). Since LMOD2 is reported to elongate sarcomeric actin thin filaments, we overexpressed LMOD2 using AAV9 system in

Flii
R1245H/R1245H

mice and conducted TAC surgeries at 8 weeks of age. AAV9-LMOD2 rescued the shortening of thin filament length in

Flii
R1245H/R1245H

mice and rescued the exacerbated TAC induced pathology associated with this homozygous mutation (P<0.05).

Conclusions:
A human
FLII
variant, R1243H, shortens the sarcomeric actin thin filament, which predisposes to cardiomyopathy. Elongation of thin filament length by LMOD2 overexpression is a possible therapeutic strategy in humans harboring this R1243H mutation.
Abstract P2021: Targeting Of Cdc42-interacting Protein 4 -calcineurin Signaling Complexes Is Cardioprotective During Ischemia/Reperfusion Injury
Authors: Samuelsson Anne-Maj , Li Yang , Li Jinliang , Kapiloff Michael S
Published: October 2023

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemic/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca
2+
/calmodulin-dependent protein phosphatase calcineurin (CaN), which promotes pathological cardiac remodeling, has been problematic as a therapeutic target due to its role in cardioprotection, including during I/R injury. We have shown that a select pool of CaNAβ holoenzyme is localized within the cardiomyocyte by scaffold protein Cdc42-Interacting Protein 4 (CIP4/TRIP10), such that the CaNAβ N-terminal polyproline (PP) domain binds directly to the CIP4 C-terminal SH3 domain. Pathological cardiac remodeling induced by pressure overload was inhibited by CIP4 gene knock-out and by inhibition of CaNAβ binding to CIP4 using an adeno-associated virus (AAV) vector that expresses the CaNAβ PP peptide. We now consider whether targeting CIP4-CaNAβ signalosomes is beneficial in ischemic cardiomyopathy. To test the relevance of CIP4 in vivo to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control

CIP4
f/f

mice were subjected to 30 minutes of ischemia by transient left anterior descending coronary artery (LAD) ligation. Cardiac function and structure were evaluated 24 hours after reperfusion by echocardiography and histology. Left ventricular ejection fraction was significantly preserved, while infarct size was significantly decreased by CIP4 CKO when compared to the two control cohorts. Separate mice subjected to permanent LAD ligation showed that CIP4 CKO resulted in long-term preservation of cardiac function. To disrupt CaNAβ-CIP4 complexes in vivo, AAV has been used to express the CaNAβ PP domain peptide. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not worsen I/R injury. Together, these results suggest that PP-anchored CaNAβ does not contribute to cardioprotection. Targeting CIP4 signalosomes may constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease.
Abstract P2037: Bubr1 Is A Critical Regulator Of Cardiac Aging
Authors: Pun Renju , Thapa Aradhana , Takafuji Sylar , Suzuki Rexton , Kim Michael H , North Brian
Published: October 2023

BubR1 is a serine/threonine protein kinase with a well-established role in regulating the spindle assembly checkpoint during mitosis. Previous studies have shown that BubR1 insufficiency leads to premature aging phenotypes such as kyphosis, cataracts, loss of subcutaneous fat, and a shortened lifespan. Furthermore, BubR1 naturally declines with age a wide range of tissues due to enhanced proteosome-mediated degradation. BubR1 hypomorphic mice expressing approximately 10% of the protein die suddenly in a manner reminiscent of sudden cardiac death. Subsequent studies have further shown that these mice suffer from conduction abnormalities such as a prolonged QTc interval. In humans, the incidence of QTc interval prolongation increases with age, and is associated with sudden cardiac death. Given BubR1 protein levels decline with age, we reasoned that BubR1 is critical for the maintenance of adult cardiac structure and function and its loss with age may drive age-related pathophysiology in the heart. We generated a conditional BubR1 knockout mouse which we crossed to the cardiac-specific cre driver line,
Nkx2.5-cre
, to assess the consequence of loss of BubR1 specifically in the heart. Electrocardiography analysis of BubR1 heterozygous knockouts also have a prolonged QTc interval
in vivo
. Structural analysis of the hearts of these mice revealed that they suffer from cardiac hypertrophy as shown by an increase in both heart weight as well as ventricular cardiomyocyte size. Masson’s trichrome staining revealed that BubR1 heterozygous knockout hearts have increased fibrosis both in the ventricular space and the perivascular region. At the molecular level, these mice also show increased levels of p21 and phospho-γH2AX staining, which represent key hallmarks of cellular senescence. Transcriptomic analysis further revealed dysregulation of multiple pathways involved in hypertrophy and senescence. Therefore, reduced expression of BubR1 drives cardiac aging as observed by increases in cardiac hypertrophy, fibrosis, senescence, and DNA damage response. Our results indicate that BubR1 plays a vital role in regulating adult cardiac structure and function, and its loss with age may drive deleterious phenotypes during heart aging.
Abstract P2041: Identification Of The Nuclear Localization Signal In RBM20 And Its Function In The Pathogenesis Of Dilated Cardiomyopathy
Authors: Zhang Yanghai , Gregorich Zachery R , Wang Yujuan , Braz Camila , Zhang Jibin , Liu Yang , Liu Peiheng , Aori Nanyumuzi , Hacker Timothy A , Granzier Henk L , Guo Wei
Published: October 2023

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain (

Rbm20
ΔRS

). We show that

Rbm20
ΔRS

mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation.
In vitro
experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.
Abstract P2057: The Role Of Cardiac Neurofibromin-2 In Chronic Pressure Overload
Authors: Mareedu Satvik , Zhang Yu , Mizushima Wataru , Francisco Jamie , Guan Jin , Yang Zhi , Oka Shinichi , Zhai Peiyong , Abdellatif Maha , Del Re Dominic
Published: October 2023

Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, Newark, NJ 07103.Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 modulates heart failure due to chronic stress. We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. We found that NF2 is transiently upregulated in mouse myocardium in response to early phase of PO, and is downregulated during late phase PO. We generated cardiomyocyte-specific NF2 knockout (cKO) mice, which had normal cardiac morphology and function at baseline. Following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation and fatty acid oxidation in NF2 cKO hearts at baseline. Additionally, we observed reduced ATP content in baseline NF2 cKO hearts compared to controls. Fractionation experiments indicated that nuclear NF2 is enriched following PO stress. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments employing neonatal rat ventricular myocytes (NRMVs) confirmed that NF2 regulated expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Luciferase reporter assays demonstrated that NF2 modulates ERR function in NRVMs. Based on these findings, we propose that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.
Abstract P2087: Contributions Of A Glycolytic Switch To Hfpef In Two Mouse Models
Authors: Kamiar Ali , Williams Monique , Condor Jose Manuel , Rasmussen Monica Anne , Alitter Qusai , Webster Keith A , Kazmierczak Katarzyna , Liang Jingsheng , Szczesna-Cordary Danuta , Shehadeh Lina A
Published: October 2023

Background:
Switching from fatty acid oxidation to less efficient but more expeditious glycolysis is associated with oncometabolism and more recently cardiac hypertrophy. We hypothesized that such a switch drives HFpEF in two diverse mouse models.

Method:
WT/129J mice at age 4-weeks were subjected to biweekly p407 i.p. injections and a single i.v. injection of AAV9-cTnT-LDLR. A separate group of injected mice were fed a diet supplemented with alpha-ketoglutarate (2% aKG) at onset. 4-week-old Alport mice received either aKG diet or normal chow. Glucose uptake was measured by whole-body PET-CT imaging of 18F-FDG in four groups: (1) untreated mice (n=5), (2) LDLR/P407 mice at 1, 2, 4, 7 (n = 3) and 8 weeks treatment (n=5), (3) LDLR/P407 + aKG mice at 4-weeks treatment (n=5), and (4) 9-12-weeks Alport mice (n=5). Echocardiography was performed on untreated mice (n=14), 4-week LDLR/P407 treatment mice (n=17), and 4-week LDLR/P407 + aKG treatment mice (n=5). Mechanical and energetic measurements on skinned cardiac pupillary muscles were performed on late-stage Alport (n=8), 8-week-treated LDLR/P407 (n=9), 4-week-treated LDR/P407 + aKG mice (n=2), and untreated controls (n=8). Survival of LDLR/P407 (n=13), LDLR/P407/aKG (n=8) and control groups were recorded.

Results:
PET-CT scans showed increased cardiac glucose uptake in Alport (FC=3, p< 0.05) and LDLR/P407 mice at 1, 2, 4 (FC = 2, p<0.05), and 8 weeks (FC=3.2, p<0.001). aKG diet increased cardiac glucose uptake in LDLR/P407 at 4 weeks compared to normal chow (FC=2.15, p<0.05). Doppler and echocardiography showed worse diastolic dysfunction (E/E’) (FC = 1.8, P<.05) in LDLR/P407 αKG mice at 4-weeks versus control chow. Alport hearts favored a DRX state (FC= 1.52, p <.0001) while 8-week-LDLR/P407 hearts favored SRX (FC=1.1, p< 0.05). The SRX state was exacerbated by aKG diet in 4-week-LDLR/P407 relative to normal chow. aKG diet significantly shortened life spans of LDLR/P407 mice (p<0.001).

Conclusion:
The results support roles for oncometabolism in promoting HF and sudden death in Alport and LDLR/P407 mice, effects that were exacerbated by an aKG supplement, possibly via enhanced hypertrophic growth. Results suggest an αKGDH-dependent and phenotype-dependent redistribution of myosin energetic states.
Abstract P2095: Capillary Stalling by Neutrophils is a Novel Mechanism Underlying Myocardial Hypoperfusion in Heart Failure with Preserved Ejection Fraction
Authors: Buglione Anne E , Small David M , Rivera Daniel A , Allan-Rahill Nathaniel , Hovasapian Soseh , Schaffer Chris B , Nishimura Nozomi
Published: October 2023

Background:
Chronic inflammation and myocardial hypoperfusion are implicated in the pathogenesis of heart failure with preserved ejection fraction (HFpEF), but the cellular and molecular mechanisms are unknown. Intravital cardiac multiphoton microscopy (MPM) enables direct visualization of blood flow and inflammatory cells within the capillary bed of the actively beating heart. We hypothesize that transient arrest of neutrophils in myocardial capillaries leads to perfusion deficits, resulting in hypoxia and driving disease progression.

Methods:
Male and female 8-12-week-old C57BL/6 mice received a high fat diet and L-NAME in drinking water (HFpEF) or standard diet and water (Chow) for 15 weeks. Intravital imaging of the actively beating heart was performed in mechanically ventilated mice to visualize neutrophils and capillary perfusion. An antibody against the neutrophil surface marker Ly6G (αLy6G) was administered intraperitoneally to test the effect of neutrophil depletion.

Results:
In vivo MPM revealed increases in arrested neutrophils in myocardial capillaries in HFpEF mice compared to Chows. Myocardial hypoxia, assessed by pimonidazole staining, and diastolic function, assessed as mitral e/e’ using echocardiography, were rescued with both extended neutrophil depletion (2mg/kg every 3d for 4 wk) and acutely (24 hrs after a single dose of 4mg/kg). To further test the immediate effects of αLy6G we implemented multi-exposure laser speckle imaging for perfusion and spectral imaging for tissue and blood oxygenation with electrocardiograph-driven reconstruction.

Conclusion:
Arrested neutrophils within capillaries appear to compromise myocardial perfusion in HFpEF. Neutrophil depletion to restore blood flow improved myocardial oxygenation and diastolic function rapidly. Effects were sustained with prolonged treatment. Capillary stall perfusion deficits may be a future therapeutic target to slow disease progression and offer rapid symptomatic relief.
Abstract P2100: Highly Localized Myocardial Expression Of Brain Natriuretic Peptide In Patients With Mitral Valve Prolapse
Authors: Boeckel Jes-Niels , Erbe Stephan , Spampinato Ricardo , Meder Benjamin , Borger Michael A , Laufs Ulrich , Dieterlen Maja-Theresa , Lavall Daniel
Published: October 2023

Mitral valve prolapse (MVP) is a common cause of mitral regurgitation (MR), adverse left ventricular (LV) remodeling and arrhythmias. MVP induces mechanical stretch to the inferobasal (INF) myocardium and papillary muscles. The underlying regional and molecular pathology of MVP-induced LV remodeling remains incompletely understood. The aim was to characterize the molecular effects of mechanical stretch by comparing the regional gene expression of the INF region of LV in patients with MVP. Biopsies were obtained from the INF LV and the interventricular septum during mitral valve repair surgery for MVP-induced primary MR. Spatial transcriptome sequencing results correlated with histological staining of fibrosis in the INF and septal regions (R=0.97, P<0.0001). The extent of fibrosis was similar between the INF region and the septal control in the respective patients with MVP. The localized fibrotic areas of the INF region showed decreased expression of cardiac myocyte genes and increased expression of fibrous and EndMT markers. In the INF region, we identified a transition zone between healthy myocardium and fibrous tissue. This transition zone showed increased expression of the NPPB gene encoding brain natriuretic peptide (BNP), whereas NPPB was not increased in the septal region of the same patients, and was confirmed by qPCR (NPPB +6.4-fold, N=11, P=0.004). The INF border zone is characterized by increase expression of NPPB, IGFBP2 and MFGE8, which have been described to be associated with adverse LV remodeling in heart failure. The serum NT-proBNP levels (mean 276.8 ± 121.6 pg/ml) of the patients correlated with NPPB levels from the INF region (R=0.76, P=0.01, N=9), whereas no correlation was found with NPPB levels from the septal control regions (R=0.17, P=0.65) of the same patients.

In conclusion, spatial transcriptome analysis in MVP patients identifies a transition zone of the INF region of LV which is exposed to high mechanical stretch. This localized transition zone has a specific gene expression profile, e.g. with increased NBBP expression that correlates with increased NT-ProBNP serum concentrations. Our results indicate that mechanical stretch induces highly localized myocardial pathologies with systemic consequences.
Abstract P2101: Elucidating Heterogeneity Between Left And Right Ventricle-derived Cardiac Fibroblasts
Authors: Dewar Michael B , Shah Haisam , Langburt Dylan , Ehsan Fahad , Hacker Alison , Heximer Scott P
Published: October 2023

Cardiac fibrosis is a major risk factor for cardiovascular disease, leading to impaired electrical conduction and reduced ventricular compliance in the heart. Different pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite building evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Due to the developmental and physiologic differences between the two ventricles, we hypothesized that LV and RV-derived CFs would display transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA-seq data from the LVs and RVs of uninjured male mice revealed 442 differentially expressed genes (p<0.05, n=4) with numerous fibrosis-related genes such as
Igfbp3
,
Col8a1
,
Ctgf
,
Aspn
, and
Postn
being the most significantly different. Single-cell RNA-seq analysis of CFs from uninjured tissue identified nine subpopulations, two of which displayed LV vs RV differences. CFs marked by high expression of
Postn
,
Col8a1
, and
Ctgf
were more abundant in the LV, whereas CFs marked by high expression of
Igfbp3
,
Fgl2
, and
Sfrp2
were more abundant in the RV. Comparisons with published datasets suggest that
Postn
-high CFs are primed for differentiation into injury-induced CFs. While the
Igfbp3
-high population has not previously been described, top marker genes have mixed pro- and anti-fibrotic roles. We then used surgical models of pressure overload injury (TAC and PAB) to study changes in the transcriptome of LV and RV-derived CFs respectively at 14 days post-surgery. Single cell RNA-seq analysis showed that the LV developed a larger population of pro-fibrotic
Thbs4
+/
Cthrc1
+ injury-induced CFs while the RV uniquely showed expansion of
Igfbp3
- and
Inmt
-high CFs. Injury experiments were repeated with female mice and showed the same results. These findings demonstrate that LV and RV-derived CFs display subpopulation differences that may cause their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform whether LV and RV fibrosis require distinct treatments.
Abstract P2110: Fibroblast-specific Smad6 Restrains Adverse Remodeling And Dysfunction Following Myocardial Infarction
Authors: Venugopal Harikrishnan , Tuleta Izabela , Hanna Anis , Kubota Akihiko , Hernandez Silvia C , Humeres Claudio , Zhu Fenglan , Frangogiannis Nikolaos G
Published: October 2023

Signaling induced by the Transforming Growth Factor (TGF)-β superfamily members in fibroblasts plays a central role in repair and remodeling after myocardial infarction (MI). However, TGF-b signals need to be tightly regulated, as excessive or sustained fibroblast activation after MI can accentuate fibrosis, precipitating heart failure. The inhibitory Smads, Smad6 and Smad7, function as endogenous negative regulators that restrain TGF-β and BMP signaling. We have recently demonstrated that fibroblast-specific Smad7 restrains fibrosis after MI acting through TGF-dependent and -independent pathways. However, the role of Smad6 in tissue repair and fibrosis following injury is not known. We hypothesized that Smad6 expression in infarct fibroblasts may play an important role in repair and remodeling of the heart, regulating TGF-b and BMP-mediated responses. In a mouse model of non-reperfused MI, Smad6 expression was increased in infarct fibroblasts and myofibroblasts at 7d post-infarction. To investigate the role of Smad6 in injury and repair, we used CRISPR/Cas9 to generate Smad6
loxp
mice. These animals were bred with Col1a2CreER mice to generate fibroblast-specific conditional Smad6 KOs (FS6KO). 7d after MI, FS6KO mice had accentuated systolic and diastolic dysfunction
(p<0.01, N=10)
associated with scar expansion, accentuated fibrosis, and higher myofibroblast density in the infarct area and border zone. Fibroblast-specific Smad6 loss increased collagen denaturation in the infarct and border zone. In vitro, siRNA knockdown experiments showed that Smad6 suppresses collagen and fibronectin expression, and modulates protease and matricellular gene synthesis. Reactome analysis of RNA-Seq data suggested effects of Smad6 on pathways involved in assembly and crosslinking of collagen fibrils, collagen biosynthesis and degradation, and VEGF-dependent angiogenesis. Furthermore, Ingenuity Pathway Analysis identified signaling by Integrins, Ephrin receptors and Receptor Tyrosine Kinases as key regulators of the effects of Smad6 loss on the fibroblast transcriptome. In conclusion, fibroblast-specific Smad6 restrains adverse remodeling after MI. Ongoing studies explore the molecular mechanisms for the Smad6 effects.
Abstract P2111: Characterizing Human Cardiac Fibroblast Responsiveness To Hemodynamic Unloading In Heart Failure With Reduced Ejection Fraction
Authors: Biggs Rachel , Silverman Daniel N , Zhang Yuhua , Baicu Catalin F , Zile Michael R , Bradshaw Amy
Published: October 2023

Background:
Myocardial interstitial fibrosis is a common pathology in cardiomyopathies leading to ventricular dilation and increased hemodynamic load that result in heart failure with reduced ejection fraction (HFrEF). Previous research has shown that HFrEF patients treated with a left ventricular assist device (LVAD) undergo hemodynamic unloading resulting in at least partial cardiomyocyte recovery. However, evidence supports that these patients do not experience a regression of fibrosis and demonstrate, in some cases, a worsening of fibrosis after LVAD treatment.

Purpose:
We hypothesize that human cardiac fibroblasts (HCFs) are constitutively activated in HFrEF myocardium but remain unresponsive to hemodynamic unloading with LVAD placement.

Methods and Results:
Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). Quantification of PSR stained sections revealed a significant increase in collagen content in the HFrEF tissue (Collagen volume fraction % (CVF) = 2.8±0.2) in comparison to control tissues (CVF = 0.9±0.2) that remained elevated in LVEX hearts (CVF = 3.1±0.3). HCFs derived from biopsies received at LVAD placement were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs from healthy donors were then plated on substrates with mechanical stiffnesses reflective of either normal myocardium (2kPa) or failing myocardium (8kPa). Cells were collected at 4- and 7-day timepoints and levels of collagen I (Col I) and alpha-smooth muscle actin (α-SMA) were quantified through western blot analysis with β-actin as a loading control. Whereas control HCFs were responsive to changes in substrate stiffness producing more Col I and α-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in production on 2 vs. 8kPa.

Conclusion:
These data suggest that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and hence, might contribute to persistent increases in fibrosis in failing and unloaded hearts.