bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 341: Efficient Large-scale Sarcomere Tracking (sarctrack) to Assess HCM Variants in iPSC-CMs
Authors: Toepfer Christopher , Sharma Arun , Garfinkel Amanda , Cicconet Marcelo , Agarwal Radhika , Chopra Anant , Chen Christopher , Seidman Jonathan , Seidman Christine
Published: October 2019

Variants that drive HCM and associated adverse patient outcomes are found in the cardiac sarcomere. These variants range from those that are known to be pathogenic to those that are likely pathogenic or even variants of unknown significance (VUS). CRISPR/Cas-9 engineering has accelerated our ability to generate variants in iPSC to probe changes in cellular function and assess cellular pathogenicity in VUSs. However, iPSC-CMs are not as functionally mature as adult cardiomyocytes. For this reason we have developed a platform to assess contractile function directly at the level of the sarcomere. We use a custom built MatLab algorithm to assess sarcomere length, contraction time, relaxation time, and beat rate of individual sarcomeres within iPSC-CMs. Sarcomeres are visualised using reporter lines that have been engineered with an N-terminal TTN-GFP. We assess the contractile function of thick filament variants in MYH7 and MYBPC3. We show the ability to detect changes in key contractile parameters. This platform allows the screening of pharmacological compounds against these reporter lines with engineered variants.
Abstract 362: Live Tracking Mouse Model for Endogenous Exosomes from Heart
Authors: Luo Weijia , Dai Yuan , Chang Jiang
Published: October 2019

Exosomes are emerging new category of messengers communicating among cells, tissues and organs. Understanding the kinetics of exosome communication
in vivo
are a critical foundation for studying exosome functions and developing exosome based drug-delivery models. Current studies of exosome
in vivo
trafficking largely rely on the administration of labeled exogenous exosomes. These methods may not fully represent endogenous exosome kinetics due to
ex vivo
exosome manipulations. Here, we established the first inducible endogenous exosome tracking mouse model that tracks endogenous exosome released from cardiomyocytes
in vivo
. The ultrasensitive and stable Nano-luciferase (NanoLuc) was selected as a reporter due to its 150-fold stronger signal intensity compared to traditional Firefly and Renilla luciferases, and the longest luminescent half-life amongst all known luciferases. We fused NanoLuc reporter with exosome surface marker CD63 for specific labeling of exosomes. The cardiomyocyte-specific αMHC promoter followed by a loxP-STOP-loxP cassette was engineered for a temporal control of labeled exosomes originating from cardiomyocytes. We crossed this cardiomyocyte-specific transgenic mouse with a tamoxifen-inducible Cre mouse (R26CreERT2) to achieve an inducible expression of the reporter. The exosome labeling and distribution were assessed by a luciferase assay and non-invasive bioluminescent live imaging. As expected, CD63NanoLuc expression was tightly controlled and only detected in cardiomyocytes upon induction. The endogenous exosomes released from cardiomyocytes were labeled and detected
in vitro
in a cell culture supernatant, and
in vivo
in the animal plasma. A paracrine uptaken of the Nanoluc-labeled exosomes by cardiac fibroblasts
in vivo
was demonstrated and quantified. A signature distribution profile of the endogenous exosomes released from cardiomyocytes was exhibited. For the first time, this exosome tracking model enables elucidating the endogenous exosome trafficking pattern, and allows future studies of exosome behavior under different conditions. It provides a useful tool for the exploration of biological functions, mechanisms and clinical applications of exosomes in a broad spectrum of researches.
Abstract 368: Disease and Phenotype-relevant Genetic Variants Identified From Histone Acetylomes in Human Hearts
Authors: Anene Nzelu Chukwuemeka George , Tan Wilson , Wong Eleanor , Lee Mick , Autio Matias Ilmari , Tan Susan , Bangfen Pan , Morley Michael , Margulies Kenneth , Cappola Thomas , Loh Marie , Chambers John , Prabhakar Shyam , Foo Roger
Published: October 2019

Identifying genetic markers for heterogeneous complex diseases such as heart failure has been challenging, and may require prohibitively large cohort sizes in genome-wide association studies (GWAS) in order to meet genome-wide statistical significance. On the other hand, chromatin quantitative trait loci (QTL), elucidated by direct epigenetic profiling of specific human tissues, may contribute towards prioritising sub-threshold variants for disease-association. Here, we captured non-coding genetic variants by performing enhancer H3K27ac ChIP-seq in 70 human control and end-stage failing hearts, mapping out a comprehensive catalogue of 47,321 putative human heart enhancers. 3,897 differential acetylation peaks (FDR 5%) pointed to pathways altered in heart failure (HF). To identify cardiac histone acetylation QTLs (haQTLs), we regressed out confounding factors including HF disease status, and employed the G-SCI test to call out 1,680 haQTLs (FDR 10%). RNA-seq performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression QTLs), either in
cis
(180), or through long range interactions (81), identified by Hi-C and Hi-ChIP performed on a subset of hearts. We validated 2 of the haQTLs through base editing to show that the presence of those SNPs indeed affects gene expression in human embryonic stem cells-derived cardiomyocytes. Furthermore, a concordant relationship between the gain or disruption of transcription factor (TF) binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalisation of haQTLs with heart-related GWAS datasets. Disease-association for these new loci may indeed be mediated through modification of H3K27-acetylation enrichment and their corresponding gene expression differences.
Abstract 369: Epigenetic Regulation of Cardiomyocyte Proliferation and Regeneration by Swi/snf Complex Subunit Arid1a
Authors: Boogerd Cornelis J , Perini Ilaria , La Phit , van der Swaan Britt , Berkhout Jari B , Kooijman Lieneke , Versteeg Danielle , van Rooij Eva
Published: October 2019

Adult mammalian hearts do not regenerate following ischemic injury, causing permanent damage to the myocardium, often leading to heart failure. In contrast, neonatal mouse hearts can fully regenerate after injury, however this ability is lost few days after birth. Loss of regenerative capacity coincides with profound changes in the epigenetic landscape. Yet, the mechanisms controlling cardiomyocyte proliferation remain poorly understood. To identify epigenetic mechanisms that underlie cardiomyocyte regeneration in response to ischemic injury, we subjected mice to sham or ischemia-reperfusion injury (IR) and performed RNA-Seq at multiple timepoints after injury. Multiple SWI/SNF chromatin remodeling complex subunits were upregulated after IR, including the AT-rich interactive domain-containing protein 1A (
Arid1a
), which has previously been implicated in tissue regeneration. Here, we show that
Arid1a
is abundantly expressed in cardiomyocytes during development, and is reactivated in a subset of adult cardiomyocytes after IR. Moreover,
ARID1A
is highly expressed in cardiomyocytes in human failing hearts, suggesting an important function in injury response. Cardiomyocyte-specific
Arid1a
ablation around birth (
Arid1a cKO
) in mice induced cardiomyocyte hyperplasia, and severe cardiac enlargement at 2 weeks of age.
Arid1a
cKO hearts displayed increased expression of key cell cycle genes, and HIPPO target genes, suggesting
Arid1a
is required for cell cycle withdrawal in neonatal cardiomyocytes. When
Arid1a
was inducibly removed from adult cardiomyocytes (
Arid1a icKO
), hearts had normal gross morphology without visible signs of cardiac pathology. Next, we performed IR injury on
Arid1a icKO
mice and observed increased cell cycle activity in mutant border zone cardiomyocytes. To further explore the mechanisms by which
Arid1a
functions in cardiomyocytes, we are currently performing ChIP-Seq and protein interaction studies. These data suggest that
Arid1a
regulates cardiomyocyte proliferation and function. Upregulation of
Arid1a
in cardiomyocytes after injury may suppress proliferation and regeneration. Suppression of
Arid1a
after ischemic injury may prove to be a novel target for therapeutics to enhance cardiac regeneration.
Abstract 375: Acetyl-CoA Production by Specific Metabolites Promotes Cardiac Repair After Myocardial Infarction via Mediating Histone Acetylation
Authors: Lei Ienglam , Tian Shuo , Gao Wenbin , Wang Zhong
Published: October 2019

In this study, we set out to identify an interlinked metabolic and epigenetic network that produces acetyl-CoA for histone acetylation and gene regulation, and determine whether this network promotes heart repair and protection after I/R injury. Myocardial ischemic reperfusion (I/R) injury induces dramatic metabolic changes and is accompanied with extensive epigenetic changes. Recent studies reveal that energy metabolism and chromatin epigenetics are intimately linked in cellular functions. In particular, acetyl-CoA is a building block for energy metabolism and histone acetylation. However, the acetyl-CoA mediated regulatory machinery integrating metabolic pathways and chromatin modifications has been under-explored in heart repair and protection.

We conducted a screen of energy metabolites in promoting histone acetylation and heart repair both in vivo and in vitro. Our screen identified that acetate, pyruvate, and octanoic acid (8C) but not citrate and nonanoic acid (9C) improved heart function after MI in rats. In particular, 8C administration resulted in the most significant heart functional recovery after MI. More importantly, in a more clinically relevant setting, 8C injection at the time of reperfusion 45 minutes after left anterior descending coronary (LAD) ligation showed comparable repair effect to that of 8C administration before LAD ligation, suggesting that 8C could be a very effective metabolic natural product to treat MI. Mechanistically, 8C promoted histone acetylation in CM chromatin after IR injury and inhibited CM death by activating expression of anti-oxidant genes Nrf2, HO1, and NQO1. We further established that the 8C promoted histone acetylation and heart repair was transduced by metabolic enzyme medium-chain acyl-CoA dehydrogenase (MCAD) and histone acetylase GCN5/KAT2A.

Therefore, this study has elucidated an interlinked metabolic/epigenetic network comprising 8C, acetyl-CoA, MCAD, and KAT2A in stimulating histone acetylation and anti-oxidative stress gene expression to combat heart injury. This study provides a novel strategy for treating myocardial I/R disease at the interface of metabolism and epigenetics.
Abstract 377: KLF15-TCF7L2-dependent Cardiac Transcriptional Reprogramming Induces Cardiomyocyte and Vascular Cell Remodeling in the Mammalian Heart
Authors: Noack Claudia , Iyer Lavanya , Liaw Norman , Schoger Eric , Wagner Eva , Zuehlke Kerstin , Klussmann Enno , Zimmermann Wolfram-Hubertus , Zelarayan Laura C
Published: October 2019

Integrative biochemical and omic-based approaches have refined our understanding of how cells integrate the Wnt signal at the chromatin level to yield specific cellular responses. However, this aspect remains unexplored in the heart. Since Wnt/β-catenin signaling activation is a hallmark in pathological cardiac remodeling, we aimed to characterize the specific Wnt cardiac transcriptional network regulation, amenable to therapeutic intervention in the adult heart.

In the adult heart, we found the transcription factor KLF15 occupying regulatory regions of tissue remodeling genes containing Wnt transcriptional activator, TCF7L2, binding sites, which are silenced in the healthy myocardium but are active during pathological remodeling. Supporting KLF15 repressive roles, its loss resulted in cardiac TCF7L2 activation, maladaptive reprograming and failure
in vivo
. We demonstrated that KLF15 possess transcriptional age-specific repressive functions controlling Wnt signaling, cardiomyocyte de-differentiation and vascular cell (VC) remodeling. Employing different transgenic mouse models we further identified a cooperative program inducing aberrant VC remodeling, caused by a reduction of KLF15 with a concomitant TCF7L2 activation in cardiomyocytes. Furthermore, we characterized a cardiac specific Wnt transcriptional inhibitory complex consisting of KLF15 directly interacting with β-catenin and TCF7L2 and identified the amino acids critical for these interactions. Next, using a CRISPR/Cas9-mediated approach we generated
KLF15
knockout (KO) hESC lines differentiated into functional cardiomyocytes and used for engineering human myocardium (EHM) generation.
KLF15
KO EHMs showed activation of TCF7L2-dependent transcription as well as impaired function in comparison to control lines, recapitulating the
Klf15
KO mouse phenotype.

Altogether, we uncover an exquisite evolutionary conserved cardiac specific regulation mediated by KLF15 on Wnt signaling in myocardium offering a basis for designing highly specific pharmacological intervention for controlling Wnt cardiac-specific gene activation to prevent irreversible heart failure. We also underscore the significance of KLF15-Wnt dynamics in VC remodeling of the adult heart.
Abstract 400: Enhanced Neovascularization by ETV2 via Angiogenesis in Myocardial Infarction Models
Authors: Bae Seong-Ho , Lee Sangho , Kim Sangsung , Yoon Young-sup
Published: October 2019

Attempts to regenerate cardiac vessels in order to treat ischemic myocardial disease have continued to be an old goal in the field of gene therapy. However, most studies that have applied proangiogenic factors have shown that the effects were limited. We examined the therapeutic effects of the ETV2 gene, a factor induced by postnatal injury, which is involved in the differentiation and specification of endothelial cells in the developmental stages in animal models of ischemic myocardial infarction via the adenoviral expression system. The ETV2 gene, using adenoviral vectors, was efficiently delivered in normal heart and myocardial infarction models and induced gene expression. In the myocardial infarction model of the control group, the function of the heart deteriorated after induction over time, but deterioration in the adenoviral ETV2-treated myocardial infarction group was delayed. To confirm whether this effect was due to angiogenesis, the expression of proangiogenic factors was examined, and the expression of vascular endothelial growth factor and angiopoietin were significantly increased in the group treated with ETV2. This result was reproduced via ETV2 gene expression at the cellular level. In order to confirm the effects of angiogenesis more deeply, we confirmed that the dividing vascular endothelial cells expressed Ki67. As a result, it was confirmed that a significant number of Ki67-positive cells were present in the functional blood vessel region in the group treated with ETV2. Taken together, the results suggest that angiogenesis plays a role in regenerating cardiac vessels when the ETV2 gene is introduced into the myocardial infarction model, which may be considered as a candidate for treatment of ischemic heart disease.
Abstract 401: Polyploidy Increases in Murine Cardiomyocytes Following Myocardial Infarction
Authors: Broughton Kathleen M , Ebeid David , Esquer Carolina , Wang Bingyan , Moshref Maryam , Sussman Mark
Published: October 2019

Introduction:
De novo
cardiomyogenesis versus polyploidy in myocardial homeostasis, aging, and response to injury is a controversial research area of intense investigation. Our lab recently created the Fluorescent Ubiquitin Cell Cycle Indicator transgenic (FUCCI-Tg) mouse model to study cardiomyocyte (CM) cell cycle progression. Therefore, the FUCCI-Tg model was used to track CM cell cycle correlated to ploidy state in response to myocardial infarction (MI).

Hypothesis:
Adult FUCCI-Tg cardiomyocytes progress into S/G2/M phase of cell cycle by 10 days after infarction resulting in binucleation rather than
de novo
cardiomyogenesis.

Methods and Results:
CMs isolated from FUCCI-Tg were analyzed 3 and 10 days following MI using confocal microscopy and flow cytometry. At 3 days post-MI, the ratio of mono- to binucleated CMs remained unchanged from non-injury CMs. At day 10 post-MI, frequency of mononuclear CMs significantly decreased compared to normal or 3-day post-MI CMs. Coincident with nucleation state, myocytes were only found to enter S/G2/M phase at day 10 post-MI. These results were verified by visualization of FUCCI in isolated CMs using Amnis ImageStream flow cytometry. Ploidy state and CM size was assessed in the infarction / border (left ventricle (LV)) and remote zone (right ventricle (RV)) at day 10 post-MI and compared to the normal and sham LV and RV. Binucleation significantly increased in the LV after MI compared to normal LV, whereas RV CM binucleation and size significantly increased in both sham and MI at 10 days after MI compared to normal RV.

Conclusion:
Adult murine CMs enter cell cycle in response to MI but primarily undergo endomitosis / endoreplication rather than complete cell cycle reflecting increases in nucleation and/or myocyte size rather than
de novo
cardiomyogenesis. Future studies will assess CM ploidy in mouse strains purported to possess enhanced cardiomyogenesis following MI injury and the biological significance of ploidy for mediating myocardial repair.
Abstract 406: Plasminogen Regulates Mesenchymal Stem Cell-mediated Tissue Repair After Ischemia Through Cry61
Authors: Duan Hao , He Zhengqiang , Mitteer Alan , Kim Hyun-Jun , Yeo Eujing , Han Hongyu , Qin Ling , Fan Yi , Gong Yanqing A
Published: October 2019

Objective:
Stem cell transplantation has emerged as a promising strategy for improving post-ischemic tissue repair. However, the poor survival and persistence of the transplanted cells including mesenchymal stem cells (MSCs) in the hostile ischemic microenvironments represents a major therapeutic barrier. Previous work shows that plasminogen (Plg) promotes post-injury tissue repair by mobilizing hematopoietic stem cell from bone marrow to circulation. Here our studies aim to define the role of Plg in microenvironment-dependent, MSC-mediated tissue repair after hindlimb ischemia.

Approach and Results:
Our studies show that Plg stimulates MSC proliferation and migration under normoxia and promotes MSC survival under hypoxia. Hindlimb ischemia was induced in Plg-deficient (Plg
-/-
) and wild-type (Plg
+/+
) mice by ligation of femoral artery, followed by injection with fLuc/tdTomato
+
MSCs into the ischemic limbs. Our data show that Plg deficiency abolishes MSC survival, migration, and proliferation in the ischemic limbs, and abrogates MSC-mediated blood reperfusion, neovascularization and tissue repair after ischemia, suggesting that Plg is critical for MSC-mediated tissue repair. Furthermore, multiplex cytokine array analysis identifies Cyr61, a pro-angiogenic factor, as a downstream target of Plg in MSCs. Plg cleaves and activates Cyr61, which is required for Plg-stimulated MSC survival and migration. Finally, Plg-mediated Cry61 cleavage further promotes EC migration and neovascularization in vitro and in vivo.

Conclusions:
Our study suggests that Plg promotes MSC survival and persistence in ischemic limbs and improves tissue repair. We identify an underlying mechanism, by which Plg induces Cyr61 cleavage and activation in MSCs, enhancing MSC survival and migration and promoting EC migration and neovascularization. Thus, targeting Plg/Cyr61 may offer exciting therapeutic opportunities for strengthening MSC therapy in ischemic diseases.
Abstract 412: Wnt / β-catenin Inhibitor Differentiates Human Mesenchymal Stem Cells into Myogenic Lineage
in vitro
and Improved Cardiac Function
in vivo
in Rat Model of Myocardial Infarction
Authors: Khan Irfan , Ali Syeda R , Salim Asmat
Published: October 2019

Cardiovascular disease (CVD) is one of the major causes of morbidity and mortality. At present, available therapeutic options for the treatment of cardiovascular diseases are limited and provide solution only to reduce the symptoms of CVD. The indigenous capability of cardiac tissue to meet the degeneration is limited. The feasible option is stem cells based regenerative medicine to repair or regenerate the myocardial cells and restore normal cardiac function. The aim of the present study is to assess the potential of small molecules for the differentiation of MSCs into cardiomyocytes. In this study, MSCs were cultured
in vitro
and were characterized by immunochemistry, and flow cytometry for the presence of MSCs markers, CD90, CD73, CD44, CD29, and for tri-lineage differentiation. MSCs were treated with wiki-4 for 14 days to induce cardiac differentiation, and were characterized for the presence cardiac markers by gene and protein expression for GATA-4, α-actinin, cTnT, cTnI, and myosin heavy chain, and found positive for these markers. These induced cardiac progenitor cells were transplanted into the infarcted myocardium of rats, where they exhibited increased persistence, engraftment, and homing in the infarcted region, and expressed cardiac markers within the border zone. Transplanted group improved left ventricular wall thickness at 4 weeks post injury, and reduced infarct size. Functional performance of the hearts was analyzed through M-mode echocardiography, which results in significant (p<0.05) improvement in the heart function including left ventricular internal diameter systolic and diastolic, ejection fraction, fractional shortening, end systolic volume and diastolic volume, and stroke volume compared to control. Histological examination of the heart sections 4 weeks post MI showed that MSCs home towards the site of injury. The transplanted cells expressed cardiac markers and contributed to the cardiac tissue recovery. The results of the present study demonstrate that myocytes derived of MSCs enhance the regeneration potential of the infarcted myocardium.
Abstract 418: Ready-made Microvessels Robustly Integrate Into the Infarcted Coronary Vasculature Promoting Graft Perfusion, Enhancing Cardiac Remuscularization and Function
Authors: Nunes Vasconcelos Sara , Sun Xuetao , Wu Jun , Li Ren-Ke
Published: October 2019

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an unprecedented opportunity to remuscularize infarcted hearts. However, the majority of hiPSC-CMs die post transplantation into the ischemic environment, limiting their regenerative potential. CM death occurs in the first few days post-transplantation due to ischemia. Attempts to promote intramyocardial vascularization (i.e. delivery of growth factors or cells) have not led to significant improvements due to poor cell retention and the long time required for new vessels to form and carry blood compared to the rapid death of transplanted CMs. Here, we used ready-made microvessels harvested from adipose tissue - that form a vasculature and carry blood within the first days post subcutaneous implantation - to re-vascularize ischemic rat hearts and improve hiPSC-CM survival. We performed left anterior descending artery ligation in immunocompromised rats to model MI. hiPSC-CMs (10 x10
6
) with (CM+V) or without (CM-only, control) microvessels were delivered by intra-myocardial injection 2 weeks post MI. Cardiac function was assessed by echocardiography and pressure-volume loop. Delivery of microvessels from GFP rats allowed assessment of microvessel persistence. Compared to hiPSC-CM transplantation alone, microvessels promoted a 6-fold increase in hiPSC-CM survival, with reduction in scar size and a significantly superior functional recovery. While delivery of CM-only stabilized the heart preventing further fractional shortening decline, delivery of CM+V resulted in reversal of fractional shortening loss. This was supported by PV-loop data (ejection fraction: sham, ~20%; CM-only, ~31%; CM+V, ~39%; 4 wks). Moreover, microvessels showed unprecedented persistence and integration (>60%, 4 wks), resulting in 2-fold increase in vessel area and graft perfusion (as early as day 5 post-transplantation). This was achieved despite the very low number of cells (~2 x10
5
) delivered in the form of microvessels. These findings provide a novel approach to cell-based therapies for MI whereby incorporation of ready-made microvessels can serve as a personalized delivery system to improve functional outcomes in cell replacement therapies.
Abstract 422: Placental Cdx2 Cells Regenerate Injured Myocardium
Authors: Vadakke-madathil Sangeetha , Larocca Gina , Raedschelders Koen , Yoon Jesse , Tripodi Joseph , Najfeld Vesna , Van-Eyk Jennifer E , Chaudhry Hina W
Published: October 2019

The limited regeneration of adult mammalian heart has prompted the need to recognize novel strategies that can restore contractile function in heart disease. However, in cell-based therapies the lack of an appropriate cell-type that can differentiate to cardiomyocytes
in vivo
persists as an ultimate unmet need. Our prior study demonstrates that experimental myocardial injury in pregnant mice triggers the flux of fetal cells via the maternal circulation into the injured heart where they undergo differentiation into diverse cardiac cell fates. Among those fetal cells, the expression of Caudal type homeobox2 (Cdx2); a trophoblast stem cell marker was unique. To understand the intriguing role of placental Cdx2 cells in cardiomyogenesis, we utilized a lineage-tracing strategy to label fetal-derived Cdx2 cells with enhanced green fluorescent protein (Cdx2-eGFP). Cdx2-eGFP cells were characterized and assayed for cardiac differentiation
in vitro
and
in vivo
using a mouse model of myocardial infarction. Cdx2-eGFP cells clonally proliferated and differentiated into spontaneously beating cardiomyocytes and vascular cells
in vitro
, signifying a multipotent nature compared to the Cdx2 negative cell population. When administered via tail vein to infarcted wild-type male mice, Cdx2-eGFP cells selectively and robustly homed to the injured heart and differentiated to cardiomyocytes and blood vessels, significantly improving the contractility noted by magnetic resonance imaging. Proteomics and immune transcriptomics studies of Cdx2-eGFP cells compared to embryonic stem (ES) cells reveal that they appear to retain ‘stem’-related functions of ES cells, but exhibit unique signatures for homing and survival in addition to being immunologically naive. Blocking CXCR4, during the migration of Cdx2-eGFP cells to SDF1α suggested a possible role for SDF1-CXCR4 signaling in the mechanistic basis of homing. Advancing towards a translational role, we demonstrate that CDX2 expressing cells can be isolated from the chorionic region of human term placenta. Our results herein may represent a paradigmatic shift in the way we approach early embryonic lineages and cell fate choices and will establish the translational potential of placental Cdx2 cells for cardiac repair.
Abstract 424: Postnatal Resetting of Cardiomyocytes Toward Ground State as a Principal Step in Heart Maturation
Authors: Wang Li , Li Zheng , Yao Fang , Yu Peng , Li Dandan , Ren Zongna , Zhou Bingying
Published: October 2019

The mammalian postnatal heart undergoes orchestrated and systematic changes to culminate in functional maturation, yet little is known about the cellular and molecular foundations underlying these processes. Here, we reported that cardiomyocytes (CMs) epigenetically and transcriptionally shut down cardiac gene expression immediately after birth, and reset into a ground state with potential enhancers for further programming CMs into distinct subtypes during heart maturation. Large-scale single-cell RNA-sequencing and genetic lineage tracing confirmed the presence of a postnatal ground state of CMs (pngCMs) that were in highest abundance around postnatal day 7 (P7), and recapitulated the developmental route of postnatal CM development. Importantly, this fate resetting mechanism used by CMs was conserved in neonatal heart regeneration upon surgical resection. Furthermore, P7-pngCMs exhibited remarkable regenerative capacity in infarcted hearts. Collectively, our data uncovered CM fate resetting as a pivotal mechanism in postnatal heart development and regeneration, and supported a new model to understand how organs adapt to abrupt environmental changes to gain maturity upon birth.
Abstract 425: Role of MLF1 in Cardiomyocyte Cell Cycle Regulation
Authors: Xiao Feng , Savla Jainy , Cardoso Alisson C , Periera Ana M , Muralidhar Shalini , Canseco Diana , Sadek Hesham A
Published: October 2019

The neonatal mammalian heart is capable of significant regeneration after cardiac injury. However, that ability is lost in the early postnatal period, coinciding with the development of cardiomyocyte cell cycle arrest. Myeloid leukemia factor 1 (MLF1) is a protein expressed in hematopoietic cells, skeletal muscle and cardiac muscle. In hematopoietic cells, MLF1 is thought to be a negative cell cycle regulator, possibly acting through a p53-dependent mechanism. However, the role of MLF1 in the cardiovascular system is not well understood. Here, we describe MLF1 as a regulator of cardiomyocyte cell cycle progression. We first examined the expression pattern of MLF1 in neonatal mouse heart. MLF1 protein specifically expresses in the nucleus, and the expression level increases from P1 to P7 as revealed by quantitative PCR and Western Blot. To elucidate a potential role of Mlf1 in cardiomyocyte proliferation, we silenced Mlf1 in neonatal rat ventricle myocytes (NRVMs) which showed increased cardiomyocyte proliferation by staining with a mitosis marker phospho-Histone 3 (pH3). Then we generated an inducible cardiac-specific Mlf1 knockout mouse model and assessed for the prolongation of the proliferative window by echocardiography, cell size analysis, and immunostaining for cell cycle markers. Staining the Mlf1 KO hearts with proliferation markers, pH3 to determine mitosis and Aurora B kinase to determine cytokinesis, indicated that deletion of Mlf1 resulted in increased cardiomyocyte proliferation. It also showed improvement in left ventricular systolic function following myocardial infarction by echocardiography. In contrast, overexpression of MLF1 in heart showed decreased cardiac function as quantified by ejection fraction. These results identify MLF1 as a negative cell cycle regulator of cardiomyocyte proliferation.
Abstract 443: Long-Term Testosterone Deficiency Modifies Frailty and Cardiac Structure and Function in Aging Male Mice
Authors: Banga Shubham , Heinze-Milne Stefan , Howlett Susan E
Published: October 2019

Objective:
Low circulating testosterone levels are correlated with heart diseases in older adults. The belief that low testosterone contributes to poor health and promotes frailty has dramatically increased testosterone prescriptions in recent years. Still links between low testosterone, frailty and the heart are unclear in part because preclinical studies typically use young, healthy animals. Here we investigated effects of chronic testosterone deficiency on frailty, body composition and cardiac structure/function in aging mice.

Methods:
Male C57BL/6 mice (18-21 mos) underwent a gonadectomy (GDX) or sham surgery (4-wks of age) and then were aged naturally. Sham (n=10-13) and GDX mice (n=11) were tested for frailty (frailty index (FI) tool), echocardiography, electrocardiography (ECG), blood pressure, and serum testosterone (ELISA, facial vein). Body composition was measured with dual-energy X-ray absorptiometry. Mice were anaesthetized (isoflurane) during all procedures except FI scoring.

Results:
Serum testosterone levels were lower in GDX mice than in sham controls (0.89±0.13 vs. 0.29±0.06 ng/mL; p<0.05). Systolic and diastolic blood pressures were unchanged between groups. Interestingly, GDX mice had lower lean mass than sham mice (23.5±1.2 vs. 27.4±0.7 g; p<0.05) but they were actually less frail (FI scores=0.17±0.05 vs. 0.23±0.01; p<0.05). GDX also reduced heart mass (186±11 vs. 148±15 mg; p<0.05) and prolonged the QRS complex (8.5±0.3 vs. 10.0±0.4 ms; p<0.05) without altering heart rate. While end systolic volume was reduced by GDX (p<0.05), ejection fraction, fractional shortening, and cardiac output remained unchanged.

Conclusions:
Chronic exposure to low circulating testosterone prolonged ventricular conduction time, reduced heart mass and lowered lean mass in aging male mice. This suggests that testosterone deficiency may promote electrical and contractile dysfunction in the setting of aging. However, despite adverse effects on the heart and lean muscle mass, frailty scores were actually lower in mice with chronic testosterone deficiency. This suggests that the impact of low testosterone on overall health is complex and raises questions about the benefits of testosterone supplementation in frail older adults.
Abstract 471: Exploring Haploinssuficiency Drivers in a Set of cMyBP-C Missense Mutations Causing Hypertrophic Cardiomyopathy
Authors: Suay-corredera Carmen , Pricolo Maria R , Herrero-Galán Elías , Velázquez-Carreras Diana , Sánchez-Ortiz David , García-Giustiniani Diego , Delgado Javier , Vilches Silvia , Domínguez Fernando , Barriales-Villa Roberto , Frisso Giulia , Serrano Luis , García-Pavia Pablo , Monserrat Lorenzo , Alegre-Cebollada Jorge
Published: October 2019

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations in cardiac myosin-binding protein C (cMyBP-C) are a leading cause of HCM. However, as for many other genetic diseases, it remains challenging to define whether specific gene variants found in patients are pathogenic or not. Here, we have examined pathogenicity drivers in a group of clinically annotated exonic variants of MYBPC3, the gene encoding cMyBP-C. First, we did bioinformatics predictions of RNA splicing and of protein thermodynamic stability. To validate results, we studied RNA splicing of the MYBPC3 gene using peripheral blood from variant carriers, and circular dichroism measurements on purified recombinant proteins. Our results show that around half of the pathogenic exonic mutations alter RNA splicing or protein thermodynamic stability, both of which can lead to cMyBP-C haploinsufficiency. These molecular phenotypes are not found in control, non-pathogenic variants. Remarkably, the remaining pathogenic missense mutations appear to result in stable proteins, for which the cause of pathogenicity remains unknown. We propose that examination of protein haploinsufficiency drivers can define pathogenicity of genetic variants associated with HCM, decisive for the clinical management of patients and their families.
Abstract 476: Pharmacogenetics and Drug Discovery for Anthracycline-Induced Cardiotoxicity Enabled by Sinoatrial Node-like Cells Derived From Human Pluripotent Stem Cells
Authors: Chen Shuibing
Published: October 2019

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and lack of robust
in vitro
derivation methods. We developed an efficient strategy, using a dual
SHOX2:GFP; MYH6:mCherry
knock-in reporter line, to generate and purify human pluripotent stem cell-derived SAN cells (hPSC-SAN), displaying molecular and electrophysiological characteristics of bona-fide nodal cells. We modeled cell type specific toxicity upon treatment with doxorubicin (DOXO) using hPSC-SAN generated from a library of induced pluripotent stem cells (iPSCs). We discovered 3 new genetic loci associated with increased sensitivity to DOXO-induced hPSC-SAN death. Genetic variants in these loci were associated with significantly higher early arrhythmia risk in patients receiving DOXO, confirmed by an unbiased PheWAS analysis. Finally, the
in vitro
DOXO assay enabled an unbiased drug screening platform and identification a candidate therapeutic that can partially block DOXO-mediated cardiac toxicity.
Abstract 486: Deficiency of Myocardial miR-17-92 Cluster Exacerbates Ischemic Injury in Diabetic Mice
Authors: Samidurai Arun , Roh Sean K , Kukreja Rakesh C , Salloum Fadi N , Das Anindita
Published: October 2019

Background:
Dysregulation of miRNAs is associated with the pathogenesis of cardiovascular diseases. miR-17-92 cluster has been identified as a negative regulator of inflammation in rheumatoid arthritis. In diabetes, the persistent activation of NLRP3 inflammasome contributes to cardiac dysfunction and the severity of I/R. NLRP3 inflammasome consists of cryopyrin, apoptosis-associated speck-like protein (ASC), and procaspase-1 protein. The inflammasome triggers caspase-1 induced programmed cell death known as pyroptosis. Considering the pivotal role of NLRP3 in diabetes, we investigated the anti-inflammatory role of miR-17-92 cluster against I/R injury.

Methods and Results:
Adult male wild type and cardiac-specific miR-17-92-deficient mice were fed high-fat-diet (HFD) for 16 weeks before subjecting the heart to ex vivo I (30 min)/R (1 h) in Langendorff mode. HFD-fed mice exhibited impaired glucose tolerance with increased body weight and blood glucose levels (Figs. A, B, C). Tamoxifen-treatment (20 mg/kg, i.p. for 5 days) significantly reduced cardiac miR-17 and miR-20a in HFD-fed miR-17-92-defient mice (Figs. D, E), which resulted in increased myocardial infarct size and cardiac dysfunction following myocardial I/R injury as compared to WT mice (Figs. F, G). ASC aggregation and caspase 1 activity following simulated ischemia/reoxygenation were increased in isolated adult cardiomyocytes from diabetic tamoxifen-inducible miR-17-92-deficient mice as compared to WT mice (Figs. H & I).

Conclusion:
miR-17-92 plays a critical role in attenuating exacerbated myocardial I/R injury in diabetic mice by regulating NLRP3 inflammasome.
Abstract 489: CC Chemokine Receptor 5 Protects the Heart from Inflammation in Pressure Overload-induced Cardiac Dysfunction
Authors: Ishizuka Masato , Toko Haruhiro , Harada Mutsuo , Guo Jiaxi , Bujo Satoshi , Yanagisawa-Murakami Haruka , Komuro Issei
Published: October 2019

Objective:
Sustained inflammation is a key pathological process in the development of heart failure and thus suppression of inflammation possibly leads to improvement of cardiac dysfunction. It has been suggested that CC chemokine receptor 5 (CCR5) scavenges pro-inflammatory mediators and resolves inflammation. The aim of this study is to uncover the role of CCR5 in the pathogenesis of heart failure.

Methods:
Pressure overload heart failure was induced by transverse aortic constriction (TAC). Sham or TAC operation was performed in male 10-week-old CCR5 knockout (KO) mice and wild-type (WT) controls. Cardiac function and morphology were examined by echocardiography 1 week and 4 weeks after TAC surgery. After euthanization, the heart was extracted and embedded in paraffin for the measurements of cardiac fibrosis and cardiomyocyte hypertrophy with EVG stain and immunohistochemistry using WGA, respectively. The extent of inflammation was evaluated by the immunostaining against CD45 and F4/80 antigens, and by qRT-PCR for inflammatory cytokines and chemokines.

Results:
We found that heart weight was significantly larger and fractional shortening was significantly lower in CCR5 KO mice than those in WT 4 weeks after TAC, indicating CCR5-induced protective effects on heart failure development. Whilst there was no difference observed in the extent of fibrosis, the size of cardiomyocytes from CCR5 KO heart tended to be increased compared to WT. The number of CD45-positive inflammatory cells and F4/80-positive macrophage infiltration was increased 1 week after TAC in KO mice. The RNA expression levels of monocyte chemotactic protein-1, transforming growth factor-β and tumor necrosis factor-α were significantly higher and NFκB p65 was more phosphorylated in KO mice.

Conclusions:
We revealed that the inflammation in pressure overload-induced heart failure was exacerbated by the deletion of CCR5 and, therefore, CCR5 could contribute to resolution of inflammation and be a potential therapeutic target for the heart failure treatment.
Abstract 490: Sex-Specific Inflammatory Gene Expression in the Hypertrophied and Failing Human Heart
Authors: Kararigas Georgios , Lehmann Daniel , Knosalla Christoph , Gaignebet Lea
Published: October 2019

Heart failure (HF) is a devastating syndrome with poor prognosis that is most often preceded by a left ventricular (LV) pressure overload (PO) disorder, such as aortic stenosis (AS). As pressure inside the LV increases, LV hypertrophy (LVH) develops. Persistent inflammation is known to be detrimental to the heart, which might promote HF. LVH manifests differently between men and women and there are pronounced sex differences in disease progression and outcome. However, the mechanisms that cause these differences are poorly understood. Hypothesizing significant sex differences, we aimed at studying the expression of inflammatory and remodeling genes in cardiomyocytes (CMs) isolated from the LV septum of AS patients (
n
= 34; 50% men; 67.5 ± 9.6 years old) undergoing aortic valve replacement. Echocardiography was performed 1 week before surgery and 20.06 ± 9.7 days after surgery. Ethics committee approval and patient consent were obtained. There was no significant difference between men and women in age, body mass index, systolic and diastolic blood pressure, co-morbidities and medication. Posterior wall thickness (
P
= 0.02), LV end diastolic diameter (
P
= 0.007) and mass index (
P
= 0.03) were higher in men than women. Preoperative ejection fraction (EF) was significantly lower in men than women (
P
= 0.01). Postoperative EF levels improved in men reaching those of women. Compared to female CMs, male CMs had significantly higher levels of
CTGF
(
P
= 0.03) and
NFKB1
(
P
= 0.01), as well as
ACTC1
(
P
= 0.03),
GATA4
(
P
= 0.03),
GJA1
(
P
= 0.03),
MYH6
(
P
= 0.02),
MYL4
(
P
= 0.03),
NPPA
(
P
= 0.03) and
NPPB
(
P
= 0.05). We found a negative association between gene expression and postoperative EF in men only for
GATA4
(
r
= -0.77,
P
= 0.01),
GJA1
(
r
= -0.69,
P
= 0.03),
MYH6
(
r
= -0.69,
P
= 0.04),
MYH7
(
r
= -0.82,
P
= 0.008),
MYL4
(
r
= -0.71,
P
= 0.03),
NPPA
(
r
= -0.9,
P
= 0.001) and
NPPB
(
r
= -0.79,
P
= 0.01). In end-stage HF, the levels of the chemokine receptor
CXCR4
were higher (
P
= 0.04) in LV samples of female (
n
= 5) vs. male (
n
= 4) patients. Collectively, in PO and HF, the expression of inflammatory and remodeling genes is regulated in a sex-specific manner, which may contribute to the mechanisms underlying sex differences in disease progression and outcome.
Abstract 492: Adipolin/C1q/Tnf-related Protein 12 reduces Neointimal Formation And Atherosclerotic Lesion Development By Modulation Of Macrophage Inflammatory Response And Endothelial Cell Function
Authors: Ohashi Koji , Ogawa Hayato , Enomoto Takashi , Otaka Naoya , Murohara Toyoaki , Ouchi Noriyuki
Published: October 2019

Objectives:
Obesity is causally linked to the progression of vascular disorders including atherosclerosis and post-angioplasty restenosis. Recently, we identified adipolin/C1q/Tnf-related protein 12 as an insulin sensitizing adipokine,which is downregulated in rodent models of obesity. Here, we investigated whether adipolin modulates the development of vascular diseases using loss-of-function genetic manipulations.

Methods and Results:
Under basal conditions, no significant differences were observed in body weight, organ weights, blood pressure and fasting plasma glucose levels between wild-type (WT) and adipolin-knockout (APL-KO) mice. APL-KO mice exhibited enhanced neointimal thickening after femoral artery injury compared with WT mice, which was accompanied by increased expression levels of pro-inflammatory mediators including tumor necrosis factor (TNF)-α and interleukin (IL)6 in injured vessels. Conversely, systemic administration of adipolin to WT mice using adenoviral vector expression systems ameliorated injury-induced neointimal thickening and inflammatory response. APL-KO mice also showed impaired re-reendothelialization after vascular injury compared with WT mice. Furthermore, adipolin deficiency increased atherosclerotic lesion area in an apolipoprotein E-knockout mouse model, which was accompanied by enhanced inflammatory response in the arterial vessel walls. Treatment of cultured macrophages with recombinant adipolin protein attenuated lipopolysaccharide-stimulated expression of inflammatory mediators, such as TNF-α and IL6. The anti-inflammatory actions of adipolin in macrophages were reversed by inhibition of transforming growth factor-β receptor II (TGF-βRII)/Smad2 signaling. In addition, treatment of human umbilical vein endothelial cells with adipolin protein promoted migratory activity and reduced serum starvation-induced apoptosis.

Conclusion:
These data suggest that adipolin suppresses injury-induced neointimal formation and atherosclerosis development through its abilities to modulate macrophage inflammatory responses and endothelial cell functions in vascular walls.
Abstract 497: Studying Cardiovascular Effects of Marijuana on Healthy Individuals Using Human Derived Induced Pluripotent Stem Cells
Authors: Wei Tzu-Tang " , Chandy Mark , Chen Ian Y , Wo Hung-Ta , Khanamiri Saereh , Nishiga Masataka , Seidl Fritz , Sayed Nazish , Liu Chun , Rhee June-Wha , Obal Detlef , Chour Tony , Wu Joseph C
Published: October 2019

Marijuana is the most widely used illicit drug worldwide. Epidemiological studies indicate that marijuana increases the risk of coronary artery disease (CAD). Adverse cerebrovascular and peripheral vascular effects are also associated with marijuana use. In addition, three synthetic cannabis drugs have been approved by the FDA for treating chemotherapy-induced nausea and vomiting, which also show cardiovascular side effects. Thus, both medical and recreational marijuana have adverse cardiovascular side effects. Cannabinoid CB1 receptor signaling is involved in a variety of pathophysiological processes and selective CB1 antagonists show therapeutic potential. However, the current repertoire of CB1 antagonists has psychiatric side effects and limited application. Therefore, developing new CB1 antagonists are an unmet and growing clinical need with marijuana use on the rise. Here we found compound JW-1, an isoflavone abundantly presenting in soybeans, partially docked into the CB1 receptor and inhibited CB1 activity, suggesting that compound JW-1 was a novel CB1 antagonist. Human endothelial cells were more sensitive to Δ
9
-tetrahydrocannabinol (Δ
9
-THC) than cardiomyocytes and cardiac fibroblasts. To determine the mechanism of Δ
9
-THC pathological effects on the vasculature, we generated human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) from 5 healthy individuals. CB1 receptor was expressed in all hiPSC-ECs, whilst CB2 expression was low. Δ
9
-THC induced inflammation and oxidative stress via NF-κB signaling activated in hiPSC-ECs. Knockdown of CB1 receptor with siRNA, abrogation of receptor expression with CRISPRi and compound JW-1 treatment could rescue the effect of Δ
9
-THC. Furthermore, compound JW-1 blocked Δ
9
-THC-induced endothelial dysfunction in mice models. Our investigations reveal that Δ
9
-THC causes endothelial dysfunction via the CB1 receptor. Compound JW-1 is a novel CB1 antagonist that can be used for preventing Δ
9
-THC-induced side effects.