bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract GS102: Injection Of Human IPSC-derived Cardiac Cells Promote Cardiac Repair After Infarction In Nonhuman Primates
Authors: Cheng Yu-Che , Hsieh Marvin , Lin Chen-Ju , Chang Cindy M , Huang Ching-Ying , Puntney Riley , Moy Amy , Chien-Yu Ting , Chan Darien , Nicholson Martin , Lin Jen-Hao , Lin Po-Ju , Chen Hung-Chih , Kim Gina , Coonen Jennifer , Liu Yen-Wen , Hacker Timothy A , Kamp Timothy , Hsieh Patrick C
Published: November 2022

Introduction:
Injection of induced pluripotent stem cell-derived cardiomyocytes has been reported as a promising approach to regenerate loss myocardium and restore heart function after ischemic injury. However, immaturity of the transplanted cardiomyocytes and their poor survival rates caused by limited blood supply remain as major hurdles for clinical translation.

Hypothesis:
We tested the hypothesis that co-culture of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) with hiPSC-derived endothelial cells (ECs) promotes CM maturation in vitro, and that co-transplantation of both hiPSC-CMs and hiPSC-ECs facilitates hiPSC-CM muscularization in myocardial ischemic injured mice and non-human primates.

Methods and Results:
We examined the therapeutic effect of co-transplantation of hiPSC-CMs and hiPSC-ECs in NOD-SCID mice undergoing myocardial infarction (N = 14 / group). Mice receiving co-transplantation had an improvement in ejection fraction compared to control (4.2 ± 1.2 % vs -8.4 ± 0.9 %, P < 0.0001), and even those receiving high-dose (-0.3 ± 0.9 %, P = 0.052) and low-dose (-2.4 ± 1.1 %, P = 0.001) hiPSC-CMs alone treatment. Moreover, less arrhythmic events were observed in co-transplantation using three-lead electrogram. To be more clinically relevant, we first showed in healthy non-human primates (N = 4) that hiPSC-CM engraftment, maturation, and integration was achieved when co-transplanted with hiPSC-ECs. Furthermore, we then examined the therapeutic effect of co-transplantation of hiPSC-CMs and hiPSC-ECs in rhesus macaques undergoing ischemia-reperfusion surgery (N = 3 / group). Consistent with the mouse model, co-transplantation in rhesus macaques significantly improved the ejection fraction (10 ± 1.3 % vs -1.8 ± 2.2 %, P = 0.010), accompanied by a reduced infarct size compared to control (16 ± 1.1 % vs 23 ± 3.3 %, P = 0.091).

Conclusions:
This study demonstrates the beneficial effects of co-transplantation of hiPSC-CMs with hiPSC-ECs, promoting hiPSC-CM maturation, enhancing neovascularization, and improving cardiac function in both mouse and non-human primate hearts. Delivery of this combined cell therapy holds promise for future clinical translation.
Abstract GS103: Extracellular Vesicles Function As An Alternative Disposal Pathway For Mitochondria In The Heart
Authors: Liang Wenjing , Diao Rachel , Najor Rita , Quiles Justin , Chi Liguo , Gustafsson Asa
Published: November 2022

Cardiac myocytes require large amounts of energy supplied by mitochondrial oxidative phosphorylation to sustain contractile function in heart. Because mitochondria can turn into death-promoting organelles, cells have developed defense mechanisms against aberrant mitochondria that can cause harm to the cell. It is well established that dysfunctional mitochondria are sequestered by autophagosomes and subsequently delivered to lysosomes for degradation. Recent studies have identified that additional mechanisms of mitochondrial clearance exist in cells. However, these pathways all converge at the level of the lysosomes and it is not clear whether alternative mitochondrial quality control pathways exist when lysosomal function is compromised. We have discovered that mitochondria can also be secreted from cells in extracellular vesicles (EVs) when internal degradation pathways are impaired. Pharmacological disruption of lysosomal acidification or genetic disruption of autophagy leads to increased release of large and small EVs in vitro and in vivo. Proteomics and biochemical analyses confirm that the large EVs containing mitochondria originate from the endosomal pathway. Transmission electron microscopy confirmed the presence of mitochondria in the large EVs. Knockdown of Rab27, a small GTPase involved in trafficking of multivesicular bodies (MVB) to the plasma membrane, abrogates the release of EVs from cells. Rab7 is also present on MVB and is involved in promoting fusion between MVB and lysosomes. We found that inactivation of Rab7 on the MVBs reroutes the vesicles to the plasma membrane for secretion of cargo. Cardiac-specific deletion of Rab7 in mice also leads to increased release of large EVs from myocytes. Finally, autophagic activity in the heart is reduced with age and we confirmed that secretion of EVs is increased in aged (24-month old) mouse hearts compared to young (4-month old). Taken together, these findings suggest that secretion of impaired mitochondria in EVs is an alternative quality control pathway utilized by cells when lysosomal function is compromised.
Abstract GS104: Stress-induced Uhrf1 Epigenetically Modulates Angiogenesis And Cardiac Function In Ventricular Hypertrophy
Authors: Felicetta Arianna , Carullo Pierluigi , Cassanmagnago Giada , Cremonesi Marco , Passaretti Mauro , Colombo Federico , Cibella Javier , Basso Gianluca , Peano Clelia , Condorelli Gianluigi
Published: November 2022

Heart failure (HF) is accompanied by cardiac hypertrophy (CH) and myocardial tissue remodeling. Epigenetic mechanisms regulate cardiomyocyte (CM) gene expression during CH: the DNA methylation profile and several histone modification marks are profoundly modulated in mouse models of HF and in the human disease. UHRF1 is an epigenetic cofactor connecting DNA methylation to histone methylation. By screening for epigenetic genes upregulated during the early phase of HF,
Uhrf1
emerged as a “hit”. We found that UHRF1 is highly expressed in the endothelial cell (EC) compartment under basal conditions in comparison to other myocardial cellular components, and its expression surges in the acute phase of CH induced through transverse aortic constriction (TAC).
In vitro
studies on HUVECs and H5V cells revealed that silencing of
Uhrf1
is associated with defects in cell cycle progression, migration and tube formation. To gain further insight on the role of UHRF1, we generated an EC-specific conditional knockout (cKO) mouse line, Cdh5-CreERT2;
Uhrf
1
fl/fl
. Compared to control,
Uhrf1
-cKO mice subjected to TAC exhibit a worsening of cardiac function associated with increased left ventricular dimension, suggesting a more rapid transition toward HF. Transcriptomic analysis of ECs revealed a robust correlation between UHRF1 depletion and alteration of cell cycle and angiogenesis, in agreement with
in vitro
results. These results were further corroborated
in vivo
through EdU injection, matrigel plug and retinal angiogenesis assays, and provided robust evidence for a key role for UHRF1 in the EC response to stress, linking the latter to neo-angiogenesis. We then defined whether the crosstalk between CMs and ECs during stress was altered by the absence of UHRF1. We found that, among other angiocrines, neuregulin-1 (Nrg-1) was strongly downregulated in ECs of
Uhrf1
fl/fl
mice subjected to TAC. In line with this evidence, ErbB2 and ERK pathways were significantly downregulated in the left ventricle of
Uhrf1
fl/fl
TAC mice, leading to increased tissue apoptosis.Our study identifies UHRF1 as a critical epigenetic regulator of stress-induced angiogenesis and, more broadly, underlines the relevance of epigenetics in modulating EC gene expression during myocardial stress.
Abstract GS112: An Epigenome-wide Association Study Paired With Cell-type-specific Data Identifies Key Regulators Of Heart Failure
Authors: Lahue Caitlin , Gural Brian , Alkhawaja Abdalla , Chapski Douglas , Rosa-Garrido Manuel , Wong Eleanor , Tan Wilson , Ren Shuxun , Foo Roger , Vondriska Thomas M , Wang Yibin , Rau Christoph
Published: November 2022

Background:
Heart Failure (HF) is driven by the interactions of multiple genetic, epigenetic, and environmental factors both within and between the cell types of the heart. Our lab uses the Hybrid Mouse Diversity Panel (HMDP), a large cohort of inbred mouse strains, to perform systems genetics analyses of isoproterenol-induced cardiac hypertrophy and failure by leveraging the benefits of a curated model organism population to identify key drivers of phenotype. We are now expanding this study into the DNA methylome, which has been convincingly linked to cardiac-associated phenotypic changes in prior studies.

Methods and Results:
Using Reduced Representational Bisulfite Sequencing, we profiled left ventricular tissue samples from 88 HMDP strains subjected to isoproterenol challenge (30 mg/kg/day for 21 days) and matched control animals. We identified nearly 170,000 CpGs whose methylation status varies across the HMDP and 179 significant associations (FDR of 5%) between CpG methylation and phenotypic variation using the epigenome-wide association study algorithm MACAU, including 37 associations linking CpG methylation in unchallenged hearts to 19 post-challenge phenotypes. To identify high-confidence candidate genes, we combined our loci with data from the Wellcome Trust Mouse Genomes Resource, transcriptomic and metabolomic data from the HMDP, Hi-C data, and cell-type-specific gene expression and methylation data from healthy and failing mouse hearts. We are systematically querying the resulting 78 candidates using
in silico
and
in vitro
approaches. These genes include
Mospd3
, which is associated with right ventricular hypertrophy and whose knockdown
in vitro
results in reduced cardiomyocyte hypertrophy and changes to hypertrophy-related gene expression. Also observed were
Atp9a
, whose expression levels are associated with significant global DNA methylation changes between control and ISO-treated animals and whose knockdown likewise causes a reduction in observed cellular hypertrophy, and
Mdga1
, whose promoter methylation status is linked to changes in 5% of expressed genes of the heart during heart failure. Further analysis and
in vivo
study of these loci will further our understanding of the role of DNA methylation in heart failure.
Abstract OEC103: ATP-dependent Citrate Lyase Drives Metabolic Remodeling In The Heart During Cancer
Authors: Liu Shijie , Gammon Seth , Tan Lin , Pham Janet , Lorenzi Philip L , Taegtmeyer Heinrich , Piwnica-Worms David , Martin James F , Karlstaedt Anja
Published: November 2022

Cardiovascular diseases and cancer are the leading causes of death worldwide. Recurrent mutations of metabolic enzymes in cancer cells provide benefits for tumor growth, while creating a systemic metabolic phenotype and increasing the risk for adverse cardiac events. We recently showed that tumors producing the oncometabolite D-2-hydroxyglutarate increase ATP citrate lyase activity and histone acetylation in the heart. How cancer cells drive metabolic remodeling in the heart, and whether ATP citrate lyase is critical for cardiac adaptation during cancer, remains poorly understood. We co-cultured wildtype adult mouse ventricular cardiomyocytes with colorectal cancer cells overexpressing ATP citrate lyase. Co-culture of cardiomyocytes with colorectal cancer cells was associated with reduced expression of ATP citrate lyase, pyruvate dehydrogenase, and mitochondrial complex I, suggesting that the cancer cell environment impaired mitochondrial function in vitro. In vitro modulation of ATP citrate lyase activity abrogated the metabolic alterations in cultured cardiomyocytes indicating that ATP citrate lyase drives remodeling during cancer. Accordingly, we created mice lacking ATP citrate lyase in the heart using CRISPR-Cas 9 genomic knockout. Loss of ATP citrate lyase leads to left ventricular systolic dysfunction and causes global gene and protein expression changes in the heart. Targeted mass spectrometry-based metabolomics and lipidomics showed decreased acetyl-CoA and succinyl-CoA levels in isolated cardiomyocytes, while the abundance of cholesterol esters increased.
In vivo
[
18
F]-FDG-positron emission tomography revealed that animals lacking ATP citrate lyase increased plasma glucose uptake compared to animals with cardiac ATP citrate lyase expression. These findings are consistent with maladaptive metabolic remodeling and impaired oxidative metabolism during heart failure. Manipulating the activity of ATP citrate lyase using BMS303141 (0.5 μM) in
ex vivo
perfused wildtype rat hearts caused a rapid decline in cardiac power and increased utilization of glucose confirming our
in vivo
mouse studies. Conversely, inhibition of ATP citrate lyase during oncometabolic stress via D-2-hydroxyglutarate improved cardiac contractile function via redirection of Krebs cycle flux and provision of ATP levels. Together these data demonstrate that ATP citrate lyase regulates oxidative metabolism and structural remodeling in the heart and creates a targetable metabolic liability during cancer.
Abstract P1008: Bioelectric Sutures Generated From HiPSC-derived Cardiomyocytes Enable
In Vitro
Electrical Coupling Between Engineered Cardiac Tissues
Authors: Soepriatna Arvin H , Wei Jenny , Song Elena , Choi Bum-Rak , Gaudette Glenn R , Coulombe Kareen L
Published: November 2022

Heart attack survivors live with permanent damage in their hearts and are at higher risk of developing heart failure. Current treatments do not directly repair or restore function in the damaged tissue. Implanting engineered cardiac tissues (ECTs) to restore contractility to the damaged myocardium is a promising therapy. However, this approach is challenged by poor integration between the implanted ECT and the host heart. To address this issue, we developed a bioelectric suture capable of propagating electrical signals to improve coupling between the two interfaces. We differentiated ventricular cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) following a well-adopted small molecule modulation of the Wnt signaling pathway. Metabolic-based lactate purification was performed to increase CM purity to > 80%, evaluated by flow cytometry analysis of cardiac troponin-T. We constructed fibrin threads by coextruding fibrinogen and thrombin through a blending tip connector into a HEPES bath. Polymerized, stretched, and dried threads were coated in Matrigel, clamped to suture heads, and seeded with hiPSC-CMs mixed with 5% human ventricular cardiac fibroblasts at a density of 5x10
5
cells/cm for 72 hours. We cultured the conductive and contractile sutures for 2 weeks under 1 Hz electrical stimulation. Optical mapping was performed to characterize electrical properties of bioelectric sutures, including action potential duration (APD) and conduction velocity (CV). Bioelectric sutures were electromechanically active with APD
100
= 547 +/- 29 ms and CV = 25.1 +/- 5.9 mm/s. a-actinin and connexin-43 staining revealed that hiPSC-CMs preferentially aligned with the thread and formed significant gap junctions, suggesting electrical syncytium formation. Preliminary results and ongoing work showed that bioelectric sutures sutured to two separate ECTs electrically coupled within 3 days, enabling directed electrical propagation from one tissue to the other. Collectively, these results demonstrate that bioelectric sutures establish a CM bridge for electrical propagation, which may improve the integration of implanted ECTs with the host heart and enable the development of therapies to treat diseases of cardiac electrical conduction.
Abstract P1011: Pharmacological Targeting Of Tip60 For Heart Regeneration And Repair
Authors: Wang Xinrui , Kulik Katherine , Wan Tina C , Lauth Amelia , Lough John W , Auchampach John A
Published: November 2022

In recent decades, many cardiovascular diseases have been ascribed to acetylation-dependent regulatory mechanisms, as revealed by the beneficial effects of targeting de-acetylase proteins. By contrast, we are targeting the acetyltransferase Tip60 (Tat-interactive protein 60 kD), a pleiotropic tumor suppressor encoded by the
Kat5
gene. Using a murine genetic model, we recently reported that cardiomyocyte (CM)-specific disruption of
Kat5
markedly protected against the damaging effects of myocardial infarction (MI). To establish therapeutic relevance, we have begun to evaluate the potential cardioprotective effects of TH1834, a small MW drug designed to specifically target the acetyltransferase domain of Tip60. Daily systemic administration of TH1834 on days 3-16 post-MI efficiently preserved cardiac function for up to 28 days post-MI, which was accompanied by reduced scar formation, diminished CM apoptosis, and activation of CM cell-cycle in the absence of CM hypertrophy. Subsequent gene ontology enrichment analysis of transcriptome sequencing data revealed that, among a total of 2,051 differentially expressed genes, 154 cell-cycle regulators and 96 apoptotic markers were altered by TH1834 treatment at day 10 post-MI; significant transcriptional changes in genes involved in cardiac muscle contraction, CM differentiation processes, and responses to ischemia/hypoxia were also identified. These data demonstrate that TH1834 administration promotes retention of post-MI function via enhanced remuscularization and by inhibiting cell death, supporting the translational potential of targeting Tip60 as a novel treatment for ischemic heart disease.
Abstract P1012: Tbx18 Drives Nodal-like Self-assembly Of Pacemaker Myocytes And Non-myocytes
Authors: Fan Jinqi , Kim Tae Y , Wolfson David , Cho Hee Cheol C
Published: November 2022

Background:
We have demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes (CMs) to induced pacemaker cells. Cell-cell interactions and cell-extracellular matrix (ECM) are important regulators of microtissue organization and assembly. Here, we sought to gain a finer understanding of self-organization and assembly of pacemaker microtissue with myocytes and non-myocytes driven by TBX18.

Methods:
2D monolayer of neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno- GFP and Adeno-TBX18 in vitro model. AAV9-TBX18 was delivered via tail vein injection of Hcn4
(+/eGFP)
transgenic mice to track the induced pacemaker myocytes.

Result:
TBX18-NRVMs showed more compact nodal-like aggregates with a higher density of CMs and surrounded by nonmyocytes. The number of alpha smooth muscle actin (αSMA) + myofibroblasts producing specific ECM and facilitating cell motility was increased by 190% in TBX18-NRVMs as well. Systemic delivery of AAV9-TBX18 via tail vein of mice created aggregation of de novo Hcn4+ induced pacemaker myocytes (iPMs) surrounded by αSMA+ myofibroblasts in vivo. Then, we treated pre-plated neonatal rat ventricular cells (NRVCs, including CMs and nonmyocytes) to explore the potential role of cell-cell communication and cell-ECM interaction in the formation of nodal-like aggregates through scRNA-seq. The data indicated that TBX18+ CM (28.6% % of all CMs) and TBX18-negative CMs (71.4% of all CMs) presented highly consistent transcriptomic profiles at day 3. No significant difference in Hcn4 transcript levels and the ratio of iPMs (defined as Hcn4+, Gja1
low
, Nkx2.5
low
, Tnni3
high
, Actn2
high
CM) was observed between them, as well as other pacemaker genes, suggesting cross-communication between them. Despite of TBX18+ fibroblasts (FBs) accounting for only 5% of all FBs, a higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control. Enrichment analysis revealed that TBX18-CMs lost ventricular specific electrical coupling and sarcomere organization (down-regulated
Gja1, Cdh2, Irx3
), but reconstructed hemidesmosome assembly and cell-ECM interaction enriched in sinoatrial node myocytes (Upregulated
Lamc1, Itgb1, Plec, Des, Actn1, and Acta2
) and activated Tgfβ signaling. Furthermore, treatment with gap junction inhibitor palmitoleic acid as well as Tgfβ receptor inhibitor A83-01 significantly suppressed the activation of quiescent fibroblasts to αSMA+ myofibroblasts and the formation of nodal-like aggregation of TBX18-NRVMs.

Conclusion:
TBX18 drives self-organization and nodal-like assembly of pacemaker microtissue with myocytes and non-myocytes through cell-cell communication of gap junction and Tgfβ signal.
Abstract P1014: Cardiomyocyte Energetic Capacity Governs Responses To Cell Cycle Stimulation
Authors: Salama Abou bakr M , Ou Qinghui , Abouleisa Riham , Mohamed Tamer , Hill Bradford G , Gebreil Ahmad , Wells Collin
Published: November 2022

Background:
Induction of cardiomyocyte (CM) proliferation is a promising strategy for cardiac regeneration. We previously showed that the overexpression of four cell cycle factors (4F), i.e. cyclin B1, CDK1, cyclin D1 and CDK4, induces CM proliferation. Temporal profiling of the single cell transcriptomes of 4F- and LacZ-overexpressing CMs showed a specific population with higher levels of CD36 that was more responsive to the 4F cell cycle induction. In addition, P7 primary CMs isolated from CD36 KO mice demonstrated 50% less response to the 4F compared with the WT controls (Abouleisa et al., Circulation, 2022). These data indicate that CD36 may be required to prime CMs to proliferate. CD36 is known to internalize fatty acids to be utilized for energy production. Here, we aim to investigate the direct effect of CM energetic capacity on their response to cell cycle stimulation.

Methods and Results:
Carnitine palmitoyl transferase -1 (CPT-1) is a rate limiting enzyme for the β-oxidation of fatty acids. Knockdown of CPT-1 using siRNA significantly reduced mitochondrial ATP production by 10% and led to a significant reduction in cell cycle markers (PHH3, EDU) in response to the 4F (PHH3; control siRNA+4F:24±1% vs CPT1 siRNA+4F:15±1%, EDU; control siRNA+4F:22±2% vs CPT1siRNA+4F:16±1%, P<0.01, n=4). Small molecule inhibition of CPT1 using etomoxir significantly decreased mitochondrial ATP production and suppressed CM responses to the 4F. Furthermore, overexpression of CPT-1 using viral vectors resulted in significant increase in ATP production and increased response to cell cycle stimulation using the 4F. Supplementing the culture medium with (1μmol) L-carnitine significantly increased mitochondrial ATP production and enhanced the CM response to the 4F. Finally, to increase ATP availability in CMs directly, we overexpressed a light-activated mitochondrial proton pump (MT-ON). CMs expressing MT-ON showed a significant increase in mitochondrial ATP levels and enhanced cell cycle induction in CMs transduced with 4F (PHH3; 4F:18±2% vs 4F+MT-ON:27±3%, EDU; 4F:18±1% vs 4F+MT-ON:30±4 %, P<0.01, n=4).

Conclusions:
These findings suggest that an energy threshold is required for CMs to progress into the cell cycle.
Abstract P1015: Construction Of A Pro-cardiogenic Extracellular Matrix: How Stem-cell Derived Epicardium Enhances Cardiac Regeneration Through Fibronectin
Authors: Lee Jonathan C , Ong Lay Ping , Bayraktar Semih , Rericha Patrick , Knight-Schrijver Vincent , Colzani Maria , Bargehr Johannes , Gambardella Laure , Sinha Sanjay
Published: November 2022

Background:
Human embryonic stem cell-derived epicardium (hESC-Epis) has been shown to improve the function of hESC-derived cardiomyocytes (hESC-CM)
in vitro
and post-transplantation into animal myocardial infarction models. However, the underlying mechanisms mediating these effects remain poorly characterised. Bulk RNA sequencing of hESC-Epis suggests a role for genes related to extracellular matrix remodeling in mediating these effects, with fibronectin (FN1) being highly implicated.

Study Objective:
To examine the role of FN1 in hESC-CM/Epi crosstalk through gain and loss of function studies.

Methods:
A tetracycline inducible FN1 knockdown system was used on hESC-CMs and Epis within the setting of 3D-engineered heart tissue. Changes in contractility and calcium handling were measured. Recombinant human plasma FN1 (rhFN) was then used to determine the effect of FN1 supplementation on hESC-CM function.

Results:
FN1 knockdown in hESC-Epis resulted in significant abrogation of the beneficial effects on hESC-CM force generation and calcium handling. Knockdown of FN1 in hESC-CMs however significantly attenuated contractility of the hESC-CMs but had no effects on calcium handling. When rhFN was added to hESC-CMs in increasing doses, hESC-CM contractility was improved in a dose dependent fashion to the same extent as seen with hESC-Epis co-cultures. However, rhFN supplementation did not result in significant improvements in calcium handling.

Conclusions:
hESC-CM and Epi crosstalk improving hESC-CM function may rely heavily on the presence of FN1. FN1 secreted by both cell types are shown to be of importance in this crosstalk with artificial supplementation of rhFN being able to recapitulate some beneficial effects of hESC-Epis on hESC-CMs. Further studies are required to determine the role of specific isoforms of secreted FN1 on hESC-CM maturity and what mechanism underlies these effects. This opens up the possibility of improving cell-based therapy for heart failure post-myocardial infarction using recombinant FN1 supplementation.
Abstract P1016: Modeling Ventricular Hypoplasia In Pulmonary Atresia With Intact Ventricular Septum Using Patient-Specific Induced Pluripotent Stem Cells
Authors: Yu Yang , Ye Shiqiao , Xu Zhaohui , Texter Karen , Garg Vidu , Zhao Mingtao
Published: November 2022

Pulmonary atresia with intact ventricular septum (PA-IVS) is a detrimental congenital heart disease where the pulmonary valve is not appropriately developed. Several hypotheses speculated to explain the pathogenesis of this disorder contains abnormal coronary arterial development, atypical blood flow through venous valve and atretic pulmonary valve formation. Conventional treatment strategy is pulmonary valve perforation, and PA-IVS patients after treatment present with varying degrees of ventricular hypoplasia: from single ventricle palliation (1v) to 1½-ventricle palliation (1.5v) and bi-ventricle repair (2v). Mechanistic studies are required to further explain the different levels of RV hypoplasia in PA-IVS patients. Here, we generated PA-IVS-specific induced pluripotent stem cells (iPSCs) from patients with a spectrum of RV hypoplasia. PA-IVS iPSC-derived cardiomyocytes (iPSC-CMs) contracted normally and displayed sarcomeric structures with intercalated cardiac troponin T and α-actinin. Early-stage PA-IVS iPSC-CMs exhibited a variety of compromised proliferation activities, which were not able to be rescued by Wnt signaling pathway activation. Transcriptomic profiling by bulk RNA seq suggested that pathways involved in the cell cycle and mitosis were downregulated in day13 PA-IVS-1v iPSC-CMs, but not in PA-IVS-2v iPSC-CMs. However, at later stage (day20), pathways involved in the regulation of cell division and mitosis was upregulated in PA-IVS-1v cardiomyocytes, indicating a possible developmental delay in the cardiomyocyte proliferation for PA-IVS-1v. Intriguingly, differentially expressed genes between PA-IVS-2v and control CMs were mostly enriched in the pathways relevant to glucose metabolism, mitochondrial biogenesis, and muscle contraction. The differentially involved pathways between PA-IVS-1v and PA-IVS-2v cardiomyocytes suggest that an intrinsic transcriptional program may lead to divergent degrees of cardiomyocyte proliferation and ventricular growth in PA-IVS. We conclude that patient iPSC-CMs can recapitulate cardiomyocyte proliferation defects which are involved in ventricular hypoplasia in PA-IVS.
Abstract P1023: Single-Cell Metabolic Reconstruction Predicts Regenerative Capacity In Cardiomyocytes
Authors: Wang Kristy , Alexander Marcus , Goodman Jack
Published: November 2022

Background:
Adult mammalian hearts have limited regenerative capacity following ischemic injury, whereas neonatal hearts can fully regenerate. Uncovering the mechanisms underlying this ability could identify potential avenues for treatment following cardiac injuries. There has been evidence that changes in the tricarboxylic acid (TCA) cycle are involved in transition of cells out of a pluripotent state. Our hypothesis is that a specific metabolic profile will be associated with regenerative capacity in cardiomyocytes (CMs), and our subsequent goal is to characterize its activity.

Methods:
Previously published single-cell RNA-Seq data (GSE130699, GSE153481) from mouse hearts subjected to ischemic or sham injury at neonatal (P1) or adult (P8) stages (10987 CMs) were used to calculate metabolic reaction activity scores (RAS), which assign activity to each reaction based on gene expression (Damiani et al, 2020). We then used enrichment analysis to compare metabolic changes between experimental conditions. CMs were clustered into metabolic clusters based on RAS and compared to clusters based on total gene expression. We identified the metabolic reactions that are the best predictors of regenerative ability using LASSO regularization logistic regression.

Results:
Significant (p ≤ .05) differences in metabolism were identified between neonatal and adult CMs. Clustering based on RAS revealed four distinct metabolic profiles in neonatal CMs, but only two (regenerative and non-regenerative) in adult CMs. Compared to other CM clusters, regenerative CMs exhibited significantly upregulated TCA, nucleic acid, and folate metabolism. This subpopulation was enriched in the neonatal condition, resulting in the significant differences seen in overall CM metabolism between conditions. Further, regression using metabolic activity allowed us to predict the regenerative subpopulation of CMs with greater than 85% accuracy.

Conclusion:
In conclusion, we characterize the unique metabolic profiles of CMs that can be used to identify the regenerative subpopulation of CMs. This suggests that the different metabolic profile seen in regenerative CMs may play a significant role in their regenerative capacity and their differentiation to non-regenerative CMs.
Abstract P1024: 3d Cardiac Tissue Rings With Exogenous Extracellular Matrix As A Model For Cardiac Fibrosis
Authors: Weiss Natalie , Gershon Jacob , Hookway Tracy
Published: November 2022

Introduction:
Cardiac fibrosis, or excess deposition of extracellular matrix (ECM) within heart muscle, acts as a protective mechanism following cardiac injury, but causes long-term heart stiffening and impaired contraction.
In vitro
models aid in understanding cardiac disease mechanisms, but current fibrosis models lack (i) native-like ECM and (ii) interactions between multiple cardiac cell types.

Objective:
Our 3D model containing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) and cardiac fibroblasts (CF) with exogenous ECM will reproducibly assess contractile changes under fibrotic conditions.

Methods:
Human GCaMP-encoded iPSC-CM (day 19) and CF (3:1) were combined in a custom agarose ring mold with collagen I (Col) (0.02 or 0.5 mg/mL), laminin (Lam) (0.01 mg/mL), both Col+Lam (0.03 mg/mL), Matrigel (0.03 mg/mL), or no ECM, n=5 for each condition. Concentrations were based on
in vivo
ratios of Col and Lam in literature. After 24 hours, cells self-assembled into 3D rings (Fig. 1a).

Results:
All conditions formed robust rings; average thickness = 460 μm (Fig.1b, c). Spontaneous beating resumed 48 hours after seeding. After 3 days in culture, Ca
2+
flux videos were captured; rings without ECM displayed shorter contraction durations, as did Col+Lam rings (Fig. 1d, e). This suggests that exogenous ECM in ratios found in healthy hearts may aid in contraction.

Conclusion:
This ring platform provides a reproducible cardiac fibrosis model with native-like ECM conditions and interactions between multiple cardiac cell types. Further assessments of ring stiffness and additional ECM conditions will enable this model to be used in therapeutic development.
Abstract P1026: Creg1 Inhibits Cardiomyocyte Proliferation And Promotes Myocardial Differentiation And Compaction During Postnatal Cardiac Development
Authors: Yang NaYoung , Qi Yanmei , Chao Joshua , Lee Leonard , Li Shaohua
Published: November 2022

Introduction:
Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes the formation of compact, contracting myocardium-like structures during cardiomyogenesis from embryonic stem cells. Cell biology studies suggest that CREG1 promotes differentiation and inhibits proliferation of developing cardiomyocytes. This study aims to understand the role of CREG1 during myocardial development
in vivo
.

Methods:
Cardiac tissues were harvested from wild-type (WT) and constitutive
Creg1
knockout (KO) postnatal day 1.5 and 17 through timed mating of heterozygous
Creg1
mice.
Creg1
KO mice were compared with
Creg1
WT littermates using immunoblotting, immunofluorescence, and methylene blue staining.

Results:
Postnatal day 1.5
Creg1
KO hearts showed lower levels of expression of slow skeletal muscle troponin I (TnI) compared with WT counterparts. No significant difference in expression was seen between WT and KO
Creg1
hearts for myosin light chain 3 (MLC3), myosin heavy chain (MHC), and cardiac troponin I. Immunofluorescence staining revealed increased numbers of PCNA (Proliferating Cell Nuclear Antigen) positive nuclei in postnatal day 1.5
Creg1
KO hearts, suggesting enhanced cardiomyocyte proliferation after loss of CREG1. Methylene blue staining in postnatal day 17 hearts displayed decreased myocardial compaction in
Creg1
KO mice.

Conclusion:
CREG1 may promote cardiomyocyte differentiation and inhibit proliferation during postnatal myocardial development. Ablation of CREG1 results in cardiac compaction defects. Our findings suggest an important role for CREG1 in cardiac development.
Abstract P1028: The Epicardium As The Conductor Of Cardiovascular Cells In Cardiac Regeneration
Authors: Rericha Patrick H , Colzani Maria T , Gambardella Laure , Knight-Schrijver Vincent , Bayraktar Semih , Bargehr Johannes , Macrae Robyn , Lee Jonathan , Dillon Scott , Sinha Sanjay
Published: November 2022

Regenerative cardiovascular medicine is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects. While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and revascularization, in particular in communication with endothelial cells (EC). Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated with an emphasis on the involvement of extracellular vesicles (EVs). hESC-EPI-EVs improved responsiveness to pacing and coordination of contraction
in vitro
in hESC-CM cell culture and engineered heart tissues (EHTs). Moreover, hESC-EPI-EVs and hESC-EPI supernatant excluding EVs independently promoted tube formation in
in vitro
gel-based angiogenesis assays. hESC-EPI supernatant excluding EVs also promoted EC proliferation in MTS assays and EC migration in wound closure assays. Characterisation of the hESC-EPI-EV-cargo on a protein level revealed candidate factors involved in cardiac repair processes. Together with hESC-EPI-EV miRNA-sequencing and hESC-EPI gene expression data key paracrine signalling pathways and candidate factors to manipulate them are being identified and investigated in
in vitro
angiogenesis and CM maturation assays. Promising angiogenesis promoting candidates are being validated
in vivo
using yolk-sac membrane and chorioallantoic membrane assays. Identification of the paracrine signalling pathways involved would make it possible to effectively address selected cardiac repair processes, resulting in new approaches to therapy.
Abstract P1029: Improving Cardiomyocyte Cell Cycle Entry In Response To Direct Cell Cycle Stimulation Using A Novel Small Molecule Inhibitor Of P38
Authors: Abouleisa Riham R , Gebreil Ahmad , Salama Aboubakr B , Ou Qinghui , Abo-Dya Nader E , Hill Bradford G , Mohamed Tamer
Published: November 2022

Background:
Myocardial infarction is the leading cause of mortality globally, due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we showed that overexpression of a combination of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), induced cell division in ~15% of post-mitotic mouse, rat, and human CMs. The aim of the current study is to identify novel small molecules that further augment CM cycle induction caused by the 4F.

Methods and Results:
We performed a drug screening of 30 chemical compounds with possible cell cycle regulatory activities using 60-day-old mature hiPS-CMs overexpressing the 4F and assessed the increase in EDU (DNA synthesis marker) and PHH3 (G2-M phase marker). The top hit was N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC). NDPPC showed a dose-dependent increase in the percentage of EDU and PHH3 positive nuclei in hiPS-CMs transduced with 4F. CMs transduced with 4F and treated with 1 μM NDPPC showed a significant increase in the percentage of EDU and PHH3 compared with vehicle (V)-treated CMs (PHH3; V: 20.41±2.02%, NDPPC: 33.75±3.9%, EDU; V: 18.24±1.7%, NDPPC: 30.81±2.1% n=6, p<0.001). In Silico Drug-Target prediction showed that NDPPC could interact with p38 mitogen-activated protein kinase (P38), a critical negative regulator of mammalian cell cycle. Therefore, we overexpressed P38, in the presence and absence of NDPPC and 4F. As expected, overexpression of P38 in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect (in presence of 4F PHH3%; V: 20.38±0.97%, P38 overexpression+V: 11.02±1.3%, P38 overexpression+NDPPC: 23.62±2.79, n=4, P<0.001). Knockdown of P38 showed a significant increase in the percentage of EDU and PHH3 positive nuclei in 4F treated CMs, and treatment with NDPPC did not show any further increase in cell cycle induction. These data indicate that NDPPC enhances the 4F cell cycle response in CMs through inhibition of P38.

Conclusions:
NDPPC is a novel inhibitor for P38. NDPPC is a promising drug to promote CM cell cycle response to the 4F. Further work in vivo is needed to test whether NDPPC could improve the 4F effect on cardiac function after ischemic injury.
Abstract P1031: A Novel Transcription Factor Combination For Direct Reprogramming To A Spontaneously Contracting Human Cardiomyocyte-Like State
Authors: Romero Tejeda Marisol , Fonoudi Hananeh , Weddle Carly J , DeKeyser Jean-Marc , Epting Conrad L , Burridge Paul W
Published: November 2022

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts, but has been less successful with human cells, limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations. Despite a decade of research, direct myocardial reprogramming still relies on overexpression of reprogramming factors first identified in mice: GATA4, MEF2C, TBX5. Using the Mogrify algorithm, we identified new candidate transcription factor combinations to induce cell conversion between human fibroblasts and cardiomyocytes. We developed an automated, high-throughput method for screening transcription factors, small molecules, and growth factors utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform we identified the combination of MYOCD, SMAD6, and TBX20 (MST) that can produce 40% TNNT2+MYH6+ cells in just 6 days and consistently produce spontaneous cardiomyocyte-like calcium transients and spontaneous contraction at just 25 days. These findings indicate that human cardiac direct reprogramming is feasible at similar levels to that achieved in the mouse and represents a step forward towards the possible clinical application of this direct cardiacreprogramming approach.
Abstract P1032: Patterned Vessels In HIPSC-derived Engineered Myocardium Enhance Perfusion And Heart Regeneration In A Rat Myocardial Infarction Model
Authors: Kant Rajeev , Dwyer Kiera , Polucha Collin , Coulombe Kareen L
Published: November 2022

Globally, >6 million patients with heart failure (HF) have reduced ejection fraction due to prior myocardial infarction (MI). In the U.S., 50,000 become refractory to treatment each year and novel therapies are urgently needed to treat systolic dysfunction, as treatments to restore cardiac muscle are not available. Human embryonic or induced pluripotent stem cell-derived cardiomyocytes (hESC/hiPSC-CMs) have emerged as a leading candidate therapy for replacing CMs lost to MI and are being evaluated in preclinical models and clinical trials. Current delivery of CMs by intramuscular injection or engineered cardiac tissue is the first step in developing a new therapy but fails to address concerns of persistent ischemia in the host and implanted CMs. We hypothesize a dual remuscularization-revascularization therapy will promote vessel development to advance regeneration. We produce hiPSC-CMs and form human engineered myocardium (hEM) in custom molds by mixing CMs with 5% human primary cardiac fibroblasts in collagen. Sacrificial gelatin-alginate fibers coated with endothelial cells are embedded in the hEM to pattern patent channels 400 μm in diameter upon chelation and wash out of alginate. HEM is cultured with perfusion prior to implant in a rat model of ischemia/reperfusion MI to evaluate host-graft inosculation, perfused vascular bed morphology, graft size, and heart function at 2 weeks.
Ex vivo
optical coherence tomography reveals capillary-scale vessels in implants. Host vasculature and origin-tracking of inosculated vessels are quantitatively evaluated using micro-CT based 3D vascular reconstruction. Immunohistochemistry shows robust engraftment of hiPSC-CMs and chimeric vascular ingrowth in implants. Gene expression analysis from explanted hEM by bulk RNAseq is revealing a great benefit of vascularization on CM development of excitation-contraction coupling machinery and sarcomeres. In conclusion, remuscularization with revascularization therapy, featuring innovative approaches to pattern vessels, is enhancing multiple metrics of regeneration after hEM implantation. This study is the first of its kind and provides a new direction for development of regenerative therapies needed for the failing hearts of post-MI patients.
Abstract P1033: A Simple 2-D Assay For Measuring Contraction And Calcium Kinetics In Engineered Human Cardiomyocytes To Model Duchenne Muscular Dystrophy
Authors: Lin Brian L , Jeffreys William , Wu Alex , Kass David A
Published: November 2022

Duchenne muscular dystrophy (DMD) is caused by the loss of the dystrophin protein, resulting in progressive muscle wasting, loss of ambulation, and early mortality in the second and third decades of life. While the skeletal muscle phenotype is most prominently associated with DMD, heart failure is the leading cause of death in DMD patients, and therapies remain limited. Advancements in stem cell-derived models and assays for heart disease have exciting translational potential for DMD, such as patient-specific modeling and drug screening. Contracting 2-D monolayers and organoids are standards of cardiac engineering, but measuring contraction is challenging due in part due to multi-axial alignment of immature sarcomeres. Complicated solutions to measure contraction in stem cell models use fluorescent beads to infer contraction, transgenic systems that express fluorescent proteins, 3-D scaffolding, and/or custom hardware and software. Here, we utilize a common microscope system used for measuring mature cardiomyocyte (IonOptix) and a commercially-available module (CytoMotion) that measures changes in pixel intensity to index contraction. This platform allows simultaneous measurement of contraction motion and calcium transients just like with adult mammalian cardiomyocytes. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from a Duchenne muscular dystrophy (DMD) patient and a healthy relative, we demonstrate DMD hiPSC-CM exhibit impaired contraction and calcium handling. We have since further increased the output of our assay using a custom 24-well stimulation plate, and current studies are using this workflow to validate experimental therapies that show promise from mouse models of DMD.
Abstract P1034: Neurohormonal Pathways Drive Postnatal Thermogenesis And Loss Of Cardiac Regenerative Potential
Authors: Huang Guo
Published: November 2022

Why do mammalian organs including the heart lose regenerative potential during the perinatal window remains enigmatic. Through phylogenetic analysis of vertebrate cardiomyocyte ploidy as a proxy for cardiac regenerative potential, we uncover that certain monotreme, edentate, cetacean, chiropteran species have unusually high percentages of diploid cardiomyocytes in the adult heart. Cardiomyocyte abundance across 41 vertebrate species conforms to Kleiber's law, the 3/4-power law scaling of metabolism with bodyweight, and decreases when the standard metabolic rate and body temperature increase during the ectotherm-to-endotherm transition. Recently, following the fractal feature of the cardiovascular system, we further strengthen the link between endothermy and heart physiology. Thermogenesis increases by more than 10-fold during the ectotherm-to-endotherm transition requiring similar increases in blood flow and cardiac function that may impede heart regenerative potential. Moreover, we report sympathetic nerve-adrenergic receptor and thyroid hormone signaling as two major pathways promoting both thermogenesis and cardiomyocyte cell cycle arrest. Combined inhibition of postnatal adrenergic and thyroid hormone signaling results in juvenile mice with strikingly low body temperatures (~25
°
C), significantly elevated abundances of diploid cardiomyocytes (~60%), dramatically increased cardiomyocyte proliferation, and enhanced cardiac regenerative abilities when analyzed at postnatal day 14. Intriguingly, both neurohormonal pathways seem to be inactive in adult zebrafish hearts. Altogether, our findings support critical roles of two major thermogenic pathways in suppressing heart regenerative capacity, and implicate that the limited regenerative capacity in various adult mammalian tissues may be a tradeoff for the acquisition of endothermy in ontogeny and phylogeny.