bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1051: Minimally Invasive Delivery Of Synthetic Cardiac Stromal Cells In A Large Animal Model Of Myocardial Infarction
Authors: Li Yuan , Huang Ke , Cheng Ke
Published: October 2023

Cell therapies for cardiac tissue regeneration are commonly used for tissue remolding post-MI, however, due to viability, retention, and cost, acellular therapeutic components are being explored. Synthetic cardiac stromal cells (synCSCs) generated by encapsulating secreted factors from isolated human cardiac stromal cells were shown to promote cardiac repair after MI. Previously, synCSCs implantation required surgy. Reducing the risks of surgical complications is essential for translational research. It is beneficial to develop a minimally invasive approach such as intrapericardial cavity (iPC) injections. This pre-clinical study aims to elucidate the therapeutic effects of synCSC embedded in hyaluronic acid (HA) hydrogel on a large animal model of MI through iPC injection. HA hydrogel and synCSCs are fabricated according to previous studies. Video-assisted thoracoscopic surgery is performed for iPC injections in Yorkshire male pigs. Magnetic resonance imaging (MRI) and immunohistochemistry (IHC) data were collected for cardiac function analyses. This pre-clinical study aims to bridge the gap between the bench and bedside by using a minimally invasive approach of iPC injection of synCSCs embedded in the hydrogel. Figure 1 shows iPC injection of synCSCs in HA improved cardiac function and cardiomyocyte proliferation in the pig MI model. Single-cell RNA sequencing data is being analyzed to identify gene markers elevated by synCSC treatment and further uncover the molecular pathways activated by synCSC. iPC injection of synCSC provides a unique dataset in the field of cardiac tissue regeneration and offers another therapeutic option for patients suffering from MI.
Abstract P1079: Regeneration Of Primate Hearts With Human IPS Cell-derived Cardiac Spheroids
Authors: Kobayashi Hideki , Tohyama Shugo , Kuwahara Koichiro , Shiba Yuji , Fukuda Keiichi
Published: October 2023

Background:
Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac repair started with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of hiPSC-CMs into the cardiac muscle layer, which was shown to induce electrical integration, was unclear because of concerns regarding poor engraftment of dispersed cardiomyocytes (CMs) and post-transplant arrhythmias. Thus, in this study, we prepared clinical-grade purified hiPSC-CM aggregates, known as cardiac spheroids (CSs), and confirmed whether the direct injection of CSs could regenerate infarcted non-human primate hearts.

Methods:
We performed two separate experiments to explore the appropriate number of CMs derived from human induced pluripotent stem cells (hiPSCs). In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks pre-transplantation and were designated as recipients of CSs containing 2х10
7
CMs or the vehicle. The animals were killed 12 weeks post-transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6х10
7
CMs).

Results:
Recipients of CSs containing 2х10
7
CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle, with a low incidence of post-transplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits. The incidence of post-transplant arrhythmia slightly increased in recipients of CSs containing 6х10
7
CMs.

Conclusions:
We demonstrated that direct injection of hiPSC-derived CSs, prepared as clinical-grade cell products, restores the contractile functions of injured primate hearts with an acceptable risk of post-transplant arrhythmia. These findings provide strong rationale for conducting clinical trials using the equivalent CM products.
Abstract P1081: Inhibition Of
Mir-200c
Abrogates Post-infarction LV Remodeling Following A Myocardial Infarct.
Authors: Leonard Riley J , Sweat Mason , Eliason Steven , Zimmerman Kathy , Batz Ariana , Kutschke William J , Weiss Robert M , Amendt Brad A
Published: October 2023

Ischemic injury and adverse post-infarction myocardial remodeling are major causes of heart failure worldwide. Strategies designed to offset cell death with cardiomyocyte regeneration have, to date, not translated to routine clinical practice. m
ir-200c
modulates the transcripts of cardiogenic transcription factors (TFs)
Tbx5
and
Gata4
to promote myocyte maturation. We hypothesized that inhibition of
mir-200c
activity would result in postnatal myocyte de-differentiation and replication sufficient to ameliorate post-infarction decompensation after ischemic injury. To test this hypothesis, we preformed left coronary ligation on Wild-Type (WT) and transgenic mice expressing an RNA inhibitor against
miR-200c
(I-200c). Echocardiographic left ventricular (LV) ejection fraction (EF) fell from 88.41 ± 1.08% (WT) and 90% ± 1.42% (I-200c) (p=NS) to 28% ± 11.55% (WT) and 36% ± 9.35% at 1 day post injury (DPI) (p =NS). At 21DPI, LVEF was 27% ± 4.31% (WT) and 64% ± 4.25% (I-200c) [p ≤ 0.0001]. Post-infarction LV chamber dilation was inhibited in I-200c mice (1.136 ± 0.19 v 0.67 ± 0.06 p ≤ 0.004 LV Vol/mass). These results indicated that inhibiting
miR-200c
following ischemic injury can recover cardiac function and prevent LV dilation. Moreover, trichrome stain showed a decrease in fibrosis 21 DPI (WT: 57% ± 17.46 v I-200c: 12% ± 3.01 p ≤ 0.001). To understand the mechanism causing this phenomenon, we analyzed expression of cardiac progenitor markers
Tbx5
and
Gata4
. Fold change in expression of both TFs were increased at 21 DPI (WT: 1.02 ± 0.09 v I-200c: 2.86 ± 0.76 p ≤ 0.05; WT: 1.00 ± 0.02 v I-200c: 3.08 ± 0.64 p ≤ 0.05). Additionally, I-200c mice have increased fold change of the cardiac progenitor factor
Nkx2.5
(1.01 ± 0.07 v 4.16 ± 1.08 p ≤ 0.05). Tbx5, Gata4, Nkx2.5, in the context of ischemic injury, are thought to perturb post-infarct remodeling and promote CM survival.

Conclusions:
I-200c treatment abrogates post-infarction LV remodeling and significantly restores LV systolic function, which are associated with increased myocyte expression of cardiogenic progenitor markers. Future studies will determine the importance of myocyte proliferation vs. humoral modulation of surviving myocyte phenotype, toward protection of post-infarction LVs.
Abstract P1086: Induced Pluripotent Stem Cell-based Cardiac Tissue Modeling Of Mitogenic Cardiomyopathy In Alström Syndrome
Authors: Duelen Robin , Ekhteraeitousi Samaneh , Costamagna Domiziana , Caluwé Ellen , Gillijns Hilde , Jacobs Johanna , Wu Ming , Malgrange Brigitte , Devriendt Koenraad , Roderick Llewelyn , Zhang Jianyi J , Sampaolesi Maurilio , Janssens Stefan
Published: October 2023

Introduction:
Cardiomyopathies are a prominent cause of heart failure and affect millions worldwide. Mutation in the
Alström syndrome 1 (ALMS1)
gene causing
Alström syndrome (ALMS)
, reported an extremely rare type of dilated cardiomyopathy - called
mitogenic cardiomyopathy
- leading to neonatal heart failure and death in early infancy due to delayed postnatal cardiomyocyte (CM) cell cycle arrest.

Methods:
Induced pluripotent stem cells (iPSCs)
from ALMS patients with or without mitogenic cardiomyopathy were used to generate ALMS patient-specific
cardiomyocytes (iPSC-CMs)
. The iPSC-CMs were divided into two main groups based on the presence or absence of the mitogenic phenotype. After transcriptome sequencing, differentially expressed genes (FDR < 0.05, logFC > 0.5 | < -0.5) were found by testing (QL F-test) a robust fitted linear model (edgeR). Harnessing recent advances in stem cell differentiation and tissue engineering, we aimed at creating a human engineered heart tissue model as an unprecedented
in vitro
disease system to study mechanisms responsible for persistent CM proliferation.

Results:
ALMS1-deficient mitogenic iPSC-CMs exhibited an impaired ability to undergo cell cycle arrest, evidenced by a higher cell percentage in the G2/M phase (19.3% in mitogenic
vs
. 7.5% in non-mitogenic iPSC-CMs). Furthermore, increased extracellular matrix levels of the fibroblast-derived protein periostin (POSTN) were observed in co-cultures of ALMS1-deficient iPSC-CMs and ALMS1 fibroblasts. Finally, we found preliminary evidence of dysregulation of the Hippo signaling pathway, observing altered cellular localization of its key downstream effector Yes-associated protein (YAP) and dysregulated ratio between phosphor and total YAP protein levels that in combination with transcript alteration of
PPIC, SH3BP4, NTN4, LRIG3
suggest perturbations in YAP signaling in ALMS patients.

Conclusion:
ALMS1-deficient mitogenic iPSC-CMs recapitulate postnatal proliferation, observed in ALMS patients with mitogenic cardiomyopathy. Preliminary molecular analyses in these
in vitro
(multicellular) models point to an important role for altered YAP signaling and provide novel cues to enhance CM proliferation, a much-sought objective in cardiac repair.
Abstract P1089: Pontin Is Necessary And Sufficient For Driving Cardiomyocyte Proliferation In Zebrafish And Mice
Authors: Yin Hui-Min , Gao Xinlei , Chen Kaifu , Burns Caroline , Burns C. Geoffrey
Published: October 2023

A major barrier to human heart regeneration is the absence of cardiomyocyte (CM) cell division in the infarcted heart. Remarkably, injured hearts of adult zebrafish and neonatal mice are capable of robust heart regeneration achieved through CM proliferation. Pontin is an evolutionarily conserved member of the AAA+ ATPase superfamily of proteins that regulates transcription, chromatin remodeling, and cell cycle progression in many contexts. Nonetheless, the involvement of Pontin in regulating CM proliferation has not been assessed. Using loss- and gain-of-function models in zebrafish and mice, we have determined that Pontin is necessary and sufficient for driving CM proliferation. Zebrafish
pontin
mutants are devoid of CM proliferation during heart development, and CM-specific overexpression of Pontin (

pontin
OE

) is sufficient to induce myocardial hyperplasia during heart development and regeneration. In mice, myocardial-specific deletion of
Pontin
dampens CM proliferation, leading to embryonic lethality at E12.5, while CM-specific overexpression of Pontin from P0 to P7 is sufficient to increase the CM mitotic index. To decipher the molecular mechanism by which

pontin
OE

stimulates CM proliferation, we performed single-cell ATAC + RNA-sequencing analysis of hyperproliferative

pontin
OE

CMs during zebrafish regeneration. We identified significant downregulation of several anti-proliferative genes, including three in the
btg/tob
family (
btg1
,
btg2
,
tob1b
). The
btg2
and
tob1b
loci showed reduced chromatin accessibility, suggesting that they could be direct targets of Pontin’s chromatin remodeling activity. We are currently testing this hypothesis and determining if overexpression of Pontin in neonatal mice extends the regenerative window beyond P7. In conclusion, our data highlight Pontin as a novel regulator of CM proliferation, potentially by controlling the chromatin accessibility of anti-proliferative genes.
Abstract P1093: Safety And Feasibility Of Tissue-engineered Biological Ventricular Assist Devices For Ischemic Cardiomyopathy
Authors: Wu Ming , Van Mil Alain , Claus Piet , Algoet Michiel , Hrynevich Andrei , Braig Johannes , Cedillo-Servin Gerardo , Gillijns Hilde , Holemans Patricia , Dokter Inge , Jüngst Tomasz , Castilho Miguel , Malda Jos , Gröll Jürgen , Mezo Manuel , Sluijter Joost P , Oosterlinck Wouter , Prosper Felipe , Janssens Stefan P
Published: October 2023

Introduction:
Advances in material science and cell technology offer new possibilities in myocardial repair. We test the safety and feasibility of implanting epicardial cellularized patches in a porcine ischemic cardiomyopathy model, representative of human disease.

Methods:
In pigs (n=4)
,
90 min occlusion of the circumflex artery induced myocardial infarction (MI) (infarct size at 4w MRI: 16±7% of LV mass with reduced EF 49±12%). To cover the scar, hexagonal multi-layered 4x4cm scaffolds were printed using high-resolution 3D melt electrowriting, and populated with human iPSC-derived cells (90% cardiomyocytes/10% fibroblasts, totaling 90 million cells), loaded in fibrin hydrogel. Four w post-MI and after 2 w immunosuppression, we sutured patches over the infarcted epicardium and monitored arrhythmias with implantable loop recorders for 1 w. Hearts were explanted after a second MRI for histopathology.

Results:
Pre-implantation cell loss from spontaneously contracting patches was 0.34±0.15% of all iPSC-derived cells. Immunosuppression was well tolerated (normal LFTs and eGFR) and effective and no significant arrhythmias were recorded after patch implantation. MRI showed correct anatomical patch position (
Fig. 1
). Visual analysis confirmed homogeneous patch attachment and histology evidenced excellent biocompatibility, and marked engraftment of the construct with host vasculature (
Fig. 1
).

Conclusions:
We provide proof-of-concept for safety and feasibility of human iPSC-derived cellular constructs of clinically relevant size in porcine ischemic cardiomyopathy. Follow-up placebo-controlled studies are ongoing to evaluate therapeutic efficacy.
Abstract P1102: Organic Semiconductor Photomodulation Enhances Maturation Of Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Galli Camilla , Salvarani Nicolo , Ronchi Carlotta , Tullii Gabriele , Marzuoli Camilla , Malferrari Marco , Rapino Stefania , Antognazza Maria Rosa , Di Pasquale Elisa
Published: October 2023

Cardiomyocytes differentiated from pluripotent stem cells (PSC-CMs) hold a great potential for the study and the cure of cardiovascular disease; indeed they have been largely used as platform for disease modeling and drugs testing, and represent a promising source of cells for regenerative therapies. However, their immature phenotype represents a major hurdle for their full application; indeed, PSC-CMs differ from adult ones for molecular, metabolic and morpho-functional properties. Recently, a new technology based on optical excitation of organic semiconductors (OS) has been shown to be able to modulate the behavior of many cell types by targeting different cellular processes, as proliferation, angiogenesis and calcium signalling. Here, we adopted a multidisciplinary approach based on morpho-functional, metabolic and transcriptional analyses to study the effect of OS-photoexcitation on PSC-CMs, by the use of a red-light sensitive OS polymer, namely Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT), in the form of a thin film. Our results revealed a significant modulation of markers of maturation in PSC-CMs exposed to PCPDTBT and photomodulation, showing increased size, a shift of their maximal diastolic potential toward more negative values, and augmented Ca
2+
transient amplitude. Moreover, by Scanning ElectroChemical Microscopy (SECM) we found a decrease in glucose uptake and lactate release upon PCPDTBT light stimulation, potentially indicating a switch toward a more adult-like metabolism in stimulated PSC-CMs. Recently obtained RNA sequencing data are in line with these results, and will provide us with hints on the underlying molecular mechanisms. In conclusion, although not definitive, our data are in support of a potential effect of polymer-mediated optical photoexcitation in boosting PSC-CMs toward a more mature phenotype. The demonstration of a relevant effect of OS-photomodulation on PSC-CM maturation and functionality will significantly promote their full applications toward personalized medicine.This research was supported by EU Horizon 2020 FETOPEN- 2018-2020 Programme ‘LION- HEARTED’-GA #828984
Abstract P1106: Antiinflammatory Hydrogel Promotes Electrical Coupling After Myocardial Ischemia
Authors: Li Zhenhua
Published: October 2023

Increased inflammation after myocardial ischemia contributes to abnormal cardiomyocyte electrical coupling, leading to inhomogeneities in ventricular conduction and retarded conduction velocity. It is highly desirable to selectively recruit the stem cells but block the inflammation. In this work, we reported a SDF-1α-encapsulated Puerarin (PUE) hydrogel (SDF-1α@PUE) that is capable of enhancing endogenous stem cell homing and simultaneously polarizing the recruited monocyte/macrophages into a repairing phenotype. These macrophages influenced the preservation of Cx43 protein content in heart which modulate intercellular coupling and improved cardiomyocyte electrical conduction. Furthermore, by taking advantage of the improved “soil”, the recruited stem cells mediated an improved cardiac function by preventing deterioration, promoting neovascular architecture, and reducing infarct size. Our findings demonstrated a promising therapeutic platform for MI which not only facilitates heart regeneration but also reduces the risk of cardiac arrhythmias.
Abstract P1107: Elmsan1 Regulates Differentiation And Maturation Of Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells
Authors: LU YU-AN , Sun Jiacheng , Wang Lu , Wang Meimei , Matthews Rachel , Zhang Jianyi J , Zhou Yang
Published: October 2023

Introduction:
ELMSAN1 is a scaffolding protein of the mitotic deacetylase complex (MiDAC) that is critical for early embryonic development. Studies on Elmsan1 knockout mice show that its absence results in heart malformation and embryo lethality. Despite this, the direct role of ELMSAN1 in heart development and formation remains unknown. To gain insights into this role, we differentiated human-induced pluripotent stem cell (hiPSC) into cardiomyocyte (hiPSC-CM) to mimic early cardiogenesis and assess the involvement of ELMSAN1 in CM maturation.

Methods and Results:
We utilized shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout techniques to generate ELMSAN1-deficient hiPSC, which were differentiated into CM for characterization. We showed that ELMSAN1 depletion caused a delay in the deactivation of pluripotency genes during early differentiation, resulting in decreased expression of cardiac-specific markers (TNNT2, MHY6), and reduced differentiation efficiency of CM. We also observed the impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels in ELMSAN1-deficient CM. Moreover, through a series of structural and functional assessments, we found these cells exhibited incomplete sarcomere Z-line structure and length (1.3 μm in KD vs. 1.5 μm in control), decreased calcium amplitude, increased time to peak value, faster beat rate with a shorter beat period, decreased field potential duration (300 ± 10 ms in KD vs. 500 ± 10 ms in control), and prolonged propagation delay. Importantly, we confirmed similar immaturity and functional changes in ELMSAN1 knockout CM, excluding the potential off-target effect. Mechanistically, we discovered that ELMSAN1 depletion resulted in decreased levels of H3K27ac in both hiPSC and CM, indicating an unexpected role of ELMSAN1 in maintaining global H3K27ac and gene expression.

Conclusion:
Our findings offer compelling evidence for the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC-CM. Notably, we were the first to identify the impact of ELMSAN1 on various aspects of hiPSC-CM generation, including cardiac differentiation, sarcomere formation, calcium handling, and electrophysiological maturation.
Abstract P1117: Personalized Care Using Patient-derived Cardiac Spheroids For Heart Failure Patients
Authors: Gentile Carmine
Published: October 2023

Preclinical studies using currently available
in vitro
and
in vivo
models fail at fully recapitulating the
in vivo
human heart microenvironment and its pathophysiology, limiting their translation from the bench to the bedside. Our laboratory has developed cardiac spheroids (CSs) as advanced
in vitro
models of the human heart using cardiac myocytes, endothelial cells and fibroblasts at ratios approximating the ones found in the human heart. Thanks to patient-derived induced pluripotent stem cells (iPSCs), we have quickly moved to personalized CSs to establish advanced pathophysiological
in vitro
models of heart failure (HF). CSs have been used to evaluate short- and long-term effects of ischaemic/reperfusion (I/R) injury typical of myocardial infarction (MI), drug-induced toxicity, SARS-CoV-2 exposure and pregnancy-induced HF. This was achieved using our established protocols to induce I/R-mimic injury, as well by exposing CSs to doxorubicin (DOX), SARS-CoV-2 virus and pre-eclampsia blood samples, respectively. First, our analyses of cell toxicity ratios using calcein-AM and ethidium homodimer (staining live and dead cells, respectively) demonstrated treatment-specific loss in cell viability and increase in cell death compared to control (untreated, healthy) CSs. Other CSs stained with ethidium homodimer and cell-specific antibodies were imaged and further used for 3D rendering analyses using IMARIS software. This enabled us to identify cell-specific responses in each pathological condition. Then, our measurements of fractional shortening and contraction frequency in CSs showed a statistically significant loss of contractile function in treated CSs compared to controls. Finally, new potential targets to prevent myocardial injury were evaluated using transcriptomics analyses, with a focus on changes in expression levels for genes regulating sarcomere structure, calcium transport, cell cycle, cardiac remodelling and signal transduction. Current studies are evaluating novel potential therapeutic targets to prevent and treat myocardial injury in HF patients using CSs. This has the potential to pave the way to prevent and treat HF in a safer and personalised way, hopefully preventing undesired effects due to non-specific care.
Abstract P1123: Phf7 As A Critical Driver Of Adult Cardiac Reprogramming
Authors: Garry Glynnis A , Chen Kenian , Zhou Huanyu , Hashimoto Hisayuki , Bezprozvannaya Svetlana , Liu Ning , Bassel-Duby Rhonda S , Olson Eric N
Published: October 2023

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Further, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 is the first epigenetic factor shown to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.
Abstract P1124: Immunosuppression Drug Mediated Cardiovascular Effects
Authors: Dexheimer Ryan , Thomas Dilip , Gaddam Sadhana , Elezaby Aly , Chen Ian Y , Wang Kevin , Sayed Nazish , Sallam Karim
Published: October 2023

Heart transplantation continues to be a life-saving therapy for patients with end-stage heart failure; yet the average long-term graft survival remains at only 12.5 years. Cardiac allograft vasculopathy (CAV) and cardiac graft dysfunction are both key contributors to long-term morbidity and mortality in the heart transplant population. Studies have suggested that immunosuppressive agents may modulate cardiovascular function, yet the mechanisms for this modulation is incompletely understood. The primary classes of immunosuppressive agents utilized post-heart transplantation are mammalian target of rapamycin inhibitors (e.g. sirolimus), calcineurin inhibitors (e.g. tacrolimus), and purine synthesis inhibitors (e.g. mycophenolate mofetil). Our prior findings indicate no discernible change to cell contraction or viability, but increased fibrosis and adverse remodeling in response to tacrolimus treatment. Using cardiac organoids; composed of human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes, endothelial cells, and cardiac fibroblasts; we investigated the effects of these agents on the cardiovascular system using RNA-sequencing and proteomic analysis. Proteomic analysis of cardiac organoids was done using Luminex bead-array assay. Treatment with MPA coincided with a significant reduction of proinflammatory cytokines including the various interleukin-families and TNF-alpha/beta. Tacrolimus yielded mixed results on proteins associated with cardiac remodeling but indicated significant upregulation of proteins that have been implicated in CAV progression. In addition, RNA-sequencing findings revealed significant upregulation of genes associated with cardiac fibrosis and remodeling with tacrolimus treatment in comparison to sirolimus treatment. These findings correlate with functional profile observed on treatment of cardiac organoids. Our results provider further insight into the pathways by which differential immunosuppression drugs influence cardiovascular remodeling.
Abstract P1130: Enhanced Electrophysiological Maturation Of Human Pluripotent Stem Cell Derived Cardiomyocytes By An Optimal Combination Of Metabolic Modulators
Authors: Muraoka Naoto , Alavattam Kris , Yang Xiulan , Reinecke Hans , Klaiman Jordan , Murry Charles
Published: October 2023

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) have emerged as ideal cell source for heart regeneration. Transplantation of hPSC-CMs to the damaged heart restores contractile function via electromechanical coupling between host and graft CMs. However, a few hurdles still remain in safety for clinical application, especially a transient period of ventricular arrhythmias after transplantation into large animals. Automaticity of graft cells, related to the electrical immaturity of hPSC-CMs, most likely triggers arrhythmic events. Here, we hypothesized that modulating metabolism toward an adult-like state would control ion channel gene expression and enhance electrophysiological maturation of hPSC-CMs in vitro. We systematically optimized combination of major metabolic components, including metabolic hormones, energy source and energy sensing, to achieve ion channel gene expression and electrophysiological phenotypes that more closely resemble adult cardiomyocytes. hPSC-CMs treated with optimized media displayed slower beat rates, longer field potentials, faster conduction velocities, and improved Ca
2+
kinetics. Moreover, optimized media increased mitochondrial content, respiratory capacity and fatty acid utilization for larger energy production, induced cell cycle exit, improved global transcriptome and sarcomere structure, and increased cell size. Subsequently, the matured cells were transplanted to mouse hearts, showing the engraftment comparable to immature hPSC-CMs. These results indicate that metabolic reprogramming can enhance many elements of maturation, including electrophysiology, myofibril organization, cell cycle, and capacity for oxidative phosphorylation.
Abstract P1132: Mettl3 Induced Fibronectin M6a Mrna Methylation Facilitates Cardiac Fibrosis Following Myocardial Ischemia
Authors: Dutta Roshan K , Ranjan Prabhat , Colin Karen , Pathak Vasundhara , zhang qinkun , Lal Hind , Verma Suresh K
Published: October 2023

Objective:
Cardiac fibrosis is one of the most warranted cardiac impairments which ultimately leads to heart failure. N6-methyladenosine (m6A) is an abundant and conserved chemical modification in eukaryotic mRNA and is associated with mRNA metabolism. Recently, we showed that m6A mRNA methylation regulates the angiogenic potential of the endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac fibrosis following ischemic injury is still elusive and needs improvement in treatment strategy.

Methods:
First we analyzed the available data set for the fibrosis-regulating targets in Sham and MI hearts. Further, we studied methylation patterns in vivo by inhibiting methylation in MI mice using STM2457. Then we assessed cardiac function using echocardiography and harvested tissue samples for further biochemical analysis. We also performed in vitro experiment to investigate the expression pattern of the screened gene (Fibronectin) in hypoxic cardiac fibroblast as an ischemic model. We also performed an Actinomycin D assay to check the role of methylation on FN mRNA stability. Fibrosis and other phenotypic statuses were studied using several molecular techniques including ICC/IHC. Finally, siMETTL3 was used to study rescue experiments.

Results:
We have shown upregulated FN expression post-MI, and also several methylation sites on FN mRNA in-silico. Histological and biochemical analysis showed increased fibrosis and m6A mRNA methylation in mice post-MI. Hypoxic stress in fibroblast induces mRNA methylation, which leads to FN mRNA stabilization and deposition of FN at the scar site by activated fibroblast. In contrast, METTL3 inhibition using STM 2457 significantly improved cardiac function. METTL3 inhibition significantly reduced FN mRNA methylation and FN expression, and ultimately reduced cardiac fibrosis.

Conclusion:
We observed that increased FN mRNA (m6A) methylation enhanced cardiac fibrosis following MI. Inhibition of mRNA methylation using STM2457 in in-vivo, and siRNA in-vitro reduced hypoxia-induced FN deposition and fibrosis, which suggests STM2457 could be a potential therapeutic molecule either alone or in combination with current regimens to curb methylation-induced cardiac fibrosis.
Abstract P1137: Novel Transcriptomic Interactions In Human Cardiomyopathic Heart Failure
Authors: Qualter Gina , Rajagopalan Viswanathan , Campbell Kenneth S
Published: October 2023

Noncoding regions span ~98% of the human genome and are emerging as key players in cardiovascular pathologies. However, the role of human noncoding RNAs and their interactions with other RNAs and protein-coding genes in ischemic (ICM) and nonischemic cardiomyopathic (NICM) heart failure is not clear. Twenty-eight patients (53±11 years; mix of gender and race) were studied using Tukey’s analysis of variance and post-hoc analyses. Significantly increased left ventricular (LV) internal diameter (diastole and systole; p<0.01) and decreased ejection fraction (EF:p<1E-10) were observed in both ICM (EF:17.5%±3.5) and NICM (EF:20.6%±3.2) compared to organ donor controls (EF:56.6%±6). End-systolic volume was increased in ICM, and QTc and QRS duration were prolonged in NICM (p<0.05). Following blinded quality control validation of both LV RNA and data, blinded whole transcriptomics (n=4/group) revealed differential expression of numerous long noncoding (lnc)RNAs, micro(mi)RNAs, and mRNAs (p-corrected/adjusted; padj<0.05). We uncovered 251 novel lncRNAs, 137 novel mature miRNAs, and 286 novel mRNAs (padj<0.05). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome and Disease Ontology enrichment analyses revealed several significant (padj<0.05) pathways in cancer, neuro/macular degeneration, neuroembryological development, endocrine, inflammation, bone remodeling, vein disease, etc. Seven lncRNA-associated protein-protein interaction networks were identified in ICM (>700 confidence score). Co-expression analysis revealed highly significant lncRNA-mRNA interactions with Pearson Correlation Coefficient of <0.95 and >0.95 with p<0.00001. GO/KEGG also revealed miRNA-mRNA interactions related to bacterial/viral, parathyroid, cancer, mTOR, etc. (padj<0.05). Importantly, several genes and interactions in all the above RNA species and multiple enrichment pathways distinguished ICM versus NICM (padj<0.05). We also observed several lncRNA alternative splicing events (padj<0.0004; false discovery rate<0.05), numerous missense and silent mutations, and minimal nonsense mutations. Together, this study offers novel transcriptomic and interactomic understanding in human ICM and NICM heart failure.
Abstract P1138: Transcriptomic Insights Into How Previously Increased Cardiac Protein O-GlcNAcylation Enhances Susceptibility To Cardiac Dysfunction In A Response To Pressure-Overload
Authors: Chang Samuel F , Ha Chae-Myeong , Brahma Manoja , Potter Luke , Bakshi Sayan , Chatham John C , Wende Adam R
Published: October 2023

Increased levels of cardiac protein posttranslational β-linked N-acetylglucosamine (O-GlcNAc) modification are implicated in adverse remodeling seen in hypertrophy and diabetes. Our previous work found elevated O-GlcNAc levels in hearts from diabetic mice, and a chronic increase in O-GlcNAc caused adverse cardiac remodeling. In diabetes, increased cardiac disease risk persists even after patients return to tight glycemic control. Thus, we hypothesize that a transient increase in O-GlcNAc is sufficient to exacerbate adverse cardiac remodeling under subsequent pressure-overload, and we propose that this is due to epigenetic modifications causing persistent changes in gene expression. We used an inducible, cardiomyocyte specific, dominant negative O-GlcNAcase (dnOGA) mouse. After a 2-wk induction (which leads to a 1.8-fold increase in O-GlcNAc levels) and subsequent 2-wk washout, Control (Con) and dnOGA mice undergo transverse-aortic constriction (TAC) or Sham surgery. After 8-wks, cardiac function is measured via echocardiography, gravimetric measures at harvest, and RNA-seq analysis is done on cardiac tissue. In both Con+TAC and dnOGA+TAC vs. Sham groups, systolic function is decreased (
-12%, p < 0.001, -11%, p < 0.01
), and ventricular weight is increased (
+43%, +69%, both p < 0.0001
), consistent with cardiac hypertrophy. Interestingly, we see further exacerbation of cardiac hypertrophy (
+29%, p < 0.0001
) and pulmonary edema (
+36%, p < 0.01
) between dnOGA+TAC vs. Con+TAC groups. Also, RNA-seq results show distinct transcriptomic profiles between the experimental groups via hierarchical clustering (
|FC| > 1.50, FDR < 0.1
). A candidate gene approach shows enhanced and exacerbated changes in genes associated with cardiac disease states like heart failure and fibrosis (e.g.,
Atp2a2, Col1a1, & Nox4
), consistent with cardiac remodeling. Our results support the hypothesis that transient increase in cardiac O-GlcNAc levels increases susceptibility to subsequent cardiac pathology. Moreover, the exacerbation of pathology is consistent with epigenetic mediated effects of transient O-GlcNAc induction; and pertaining to diabetes, provide a possible mechanism into the continued increased of cardiac disease risk.
Abstract P1147: Cardinal Is A Nuclear Lncrna That Represses SRF/TCF-mediated Gene Transcription By Co-occupying SRF/TCF-bound Gene Promoters In The Heart
Authors: Perciaccante Jessica , Priyanka Fnu , Poosala Pornthida , Parks Xiaorong , Thomas Tamlyn , Anderson Douglas M
Published: October 2023

LncRNAs are an emerging class of co-transcriptional regulators important for balancing gene expression networks in the heart during development and disease. We recently characterized a cardiac-specific lncRNA named CARDINAL, which is transcribed adjacent to the SRF-coactivator myocardin. Myocardin, and members of the ternary complex factor (TCF) family (Elk1-4), compete for interaction with SRF to balance opposing transcriptional networks in the heart. Previously we showed that CARDINAL forms a stable complex with SRF and can repress SRF/TCF-mediated gene transcription, suggesting CARDINAL functions as an RNA cofactor for SRF. Here we show using RNAscope in situ hybridization that CARDINAL is a nuclear lncRNA in both mouse and human cardiomyocytes. To identify CARDINAL-regulated genes, we performed Chromatin Isolation by RNA Purification (ChIRP) using anti-sense oligonucleotides specific to endogenous CARDINAL in HL-1 cardiomyocytes, followed by deep sequencing. CARDINAL transcripts were localized at ~100 genes, with the majority (76%) of CARDINAL peaks occupying the transcriptional start site (TSS) of gene promoters. Strikingly, the majority of CARDINAL ChIRP peaks overlapped with SRF-bound ChIP peaks in the cardiac genome. Homer motif analyses of CARDINAL peaks identified enrichment for the SRF CArG box and TCF binding motifs for Elk1 and Elk4. Consistent with our previous findings, CARDINAL peaks were localized to canonical SRF/TCF-regulated genes, including Egr1, Elk4, and components of the AP-1 complex, such as c-Fos, Junb, and Jund. These findings highlight CARDINAL, similar to its protein coding neighbor gene myocardin, as a co-regulator of SRF-dependent gene transcription. CARDINAL, however, in contrast to myocardin, can repress SRF/TCF-dependent gene transcription, which is likely mediated by its unique ability to co-occupy SRF/TCF-bound gene promoters.
Abstract P1163: Identifying Novel Intracellular Interactions Driving Membrane-bound Tumour Necrosis Factor-mediated Signalling In The Microvasculature
Authors: Mostafaie Yasaman , Hanchard Julia , Lidington Darcy , Bolz Steffen-Sebastian
Published: October 2023

Elevated total peripheral resistance (TPR) is a hallmark of many cardiovascular diseases and furthers disease progression. We previously demonstrated that myogenic tone, regulated by membrane-bound tumour necrosis factor (mTNF) reverse signalling, is a primary modulator of TPR. Thus, inhibiting mTNF reverse signalling should reduce TPR with the potential to improve disease outcome. However, since mTNF is a ubiquitous protein with critical biological functions, its indiscriminate inhibition can cause widespread side effects. Therefore, therapeutics aiming to reduce myogenic tone and TPR must target other elements of mTNF reverse signalling. To identify these unknown participants of mTNF reverse signalling, we used proximity-dependent biotinylation coupled to mass spectrometry (BioID). Our search identified 42 high confidence hits that interact with and/or are proximal to mTNF’s cytoplasmic domain. Gene ontology (GO) analysis revealed that molecularly, these proteins are structural constituents of the cytoskeleton involved in actin and cell adhesion molecule binding. This aligns with our underlying hypothesis that

mTNF functions as a
mechanosensor
that tethers to its receptors and initiates myogenic vasoconstriction in response to changes in transmural pressure.

GO also identified proteins involved in MAPK and RhoA signalling, which are critically involved in myogenic vasoconstriction. Our most abundant hit (fold change = 12.6, p-value = 0.0023) is a cytoskeletal protein that plays a role in maintaining the structural integrity of cells. This protein also interacts with proteins involved in various signalling cascades, namely, RhoA and calcium-dependent signalling pathways. Thus, we hypothesize that this protein
is essential for formation of signalling complexes with mTNF and interacts with other proteins to mediate reverse signalling
. We will employ a bioluminescence-based approach to quantify the strength of all interactions detected by BioID and identify small molecule inhibitors that can disrupt these interactions. These newly discovered inhibitors could potentially be used as novel therapeutics targeting myogenic tone and hence, elevated TPR to improve disease management and clinical outcome of cardiovascular diseases.
Abstract P1165: Gut Microbial Peptide Induced Cardiac Inflammation Via Cyclin Dependent/NFkB Signaling In Macrophages
Authors: Ranjan Prabhat , Dutta Roshan , Colin Karen , Maynard Craig , Verma Suresh K
Published: October 2023

Background:
Millions of people in the United States are affected by Inflammatory Bowel Disease (IBD). Recently, gut dysbiosis and impaired gut-barrier permeability are known to contribute to cardiovascular diseases in IBD patients. However, its mechanisms are poorly understood. Here, we hypothesized that gut derived bacterial metabolites (such a peptidoglycan in this case) in a mice colitis model, promotes cardiac inflammation and heart failure.

Methods:
To induced colitis WT mice were treated with control/DSS (dextran sodium sulfate). Heart and intestine were collected to evaluate inflammatory genes expression(qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify peptidoglycan (PGN) level. The effect of PGN on NFκB inflammatory pathways was evaluated in macrophages.

Results:
Echo-data showed cardiac dysfunction in DSS mice. Deteriorated intestinal villi in Masson’s trichrome staining and decreased Occludin and increased PV-1 in western data revealed leaky gut in DSS treated mice. Remarkably, PGN level was significantly increased in blood plasma 3 weeks following DSS treatment. Real-time gene expression data of pro-inflammatory cytokines (IL-6, IL-1 and TNFα) and fibrosis associated genes (col1α1, fibronectin) were increased in DSS mice heart. Increased CD68 expression in the immunostaining data of DSS mice heart indicated the recruitment of inflammatory macrophages. Intriguingly, we observed that CDK9 expression significantly increased upon PGN treatment leading to an increase in NFκB. Next, we inhibited CDK9 before PGN treatment and surprisingly we found that CDK9 inhibition reduces the effect of PGN.

Conclusion:
Taken together, data suggest that in gut dysbiosis state, PGN leaks into the blood and causes CDK9 mediated NFκB upregulation and cardiac inflammation.
Abstract P1169: BubR1 Regulates Cardiac Development Through CamkII
In Vivo
Authors: Pun Renju , Takafuji Sylar , Suzuki Rexton , Thapa Aradhana , Kim Michael H , North Brian
Published: October 2023

Congenital heart defects (CHD), which represent the most common type of congenital defects, are found in 8 out of 1000 births each year in the US. Aneuploidy, defined as an abnormal number of chromosomes in a cell, is the largest contributor to CHD. Defects in mitotic regulators can promote aneuploidy. For instance, mutations in the mitotic checkpoint protein BubR1 can lead to mosaic variegated aneuploidy (MVA). MVA patients display varied phenotypes including growth deficiency and a shortened lifespan. In addition, some patients develop CHDs such as atrioventricular septal defects. To determine whether BubR1 regulates cardiac development we carried out timed matings to harvest heart tissue at various embryonic stages. We found that BubR1 expression dramatically increases in the mouse heart through embryonic development. We engineered a conditional BubR1 knockout mouse model using the Cre-lox system which allows for the generation of cardiac-specific
BubR1
knockout animals when crossing this

BubR1
fl/fl

mice to
Nkx2.5-cre
mice, which mediates deletion of BubR1 beginning at E7.5. Interestingly, we did not observe any homozygous cardiac-specific BubR1 knockout mice in newborn litters, suggesting that loss of BubR1 in the developing heart is embryonic lethal (n = 120,
P
< 0.01). We observed that deletion of BubR1 in the embryonic heart leads to cardiac malformations including the absence of trabeculations and reduction in the size of ventricles. Immunofluorescence studies reveal that cardiac-specific BubR1 knockout embryos have an increase in cell death as measured by the apoptosis marker cleaved-caspase 3. These embryos also show an increase in the apoptosis regulator CaMKII, whose upregulation has been previously tied to an increase in cardiomyocyte apoptosis. Therefore, we conclude that BubR1 suppresses the expression of CaMKII in the developing heart to regulate apoptosis during cardiogenesis. Taken together, our data suggest that BubR1 is a critical regulator for cardiac development
in vivo
, and its mutation or loss may contribute to the development of CHDs.
Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?
Authors: Roka-Moiia Yana , Lewis Sabrina , Italiano Joseph E , Slepian Marvin J
Published: October 2023

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “
sugar coat
,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity.

Methods:
Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates.

Results:
Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.
Abstract P2006: Cardiac Cytoskeleton Sequesters Acetylated Yap In The Cytoplasm To Inhibit Cardiomyocyte Renewal After Myocardial Infarction
Authors: Liu Shijie , Deshmukh Vaibhav , Meng Fansen , Wang Yidan , Morikawa Yuka , Steimle Jeffrey , Li Rich Gang , Wang Jun , Martin James F
Published: October 2023

The Hippo pathway effector YAP is essential in cardiomyocyte proliferation and heart regeneration. How YAP responds to physiological conditions such as myocardial infarction (MI) is unclear. Here we show YAP transiently localizes to the nucleus of border zone CMs after MI. Nuclear YAP is acetylated by P300/CBP, and SIRTs deacetylation activity is decreased due to metabolic stress resulting from the injury. These mechanisms in conjunction results in increased acetylated YAP accumulation in the cytoplasm. After MI, the cardiac cytoskeleton is stabilized and sequesters cytoplasmic acetylated YAP, which reduces YAP activity and inhibits cardiomyocyte renewal. In a genetically engineered mouse model with a mutation that abolishes YAP acetylation, the mutant heart exhibits improved cardiac regeneration ability compared to the wild-type heart due to increased nuclear YAP localization. Together, we demonstrate that the injury-induced metabolic shift results in YAP acetylation, which translocates YAP to the cytoplasm in a manner independent of the Hippo pathway, and is detrimental to cardiac regeneration after MI.