bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 641:
Drosophila
Adipogenesis Regulates Cardiac Function
Authors: Musselman Laura P , Tuthill Bryon F
Published: October 2019

High-calorie diets are associated with obesity, type 2 diabetes, and cardiovascular disease. Recently, several investigators have proposed a model of "maximum adipose expandability," where the limited ability of animals to store fat as triglycerides results in cellular toxicity via fatty acid overflow into alternative fates, or lipotoxicity. Tissue-specific loss-of-function studies targeting metabolic pathways in the
Drosophila
fat storage organ, the fat body, were used to determine the relationship between fat body lipogenesis and cardiac physiology. Our data support a model where when caloric excess overwhelms fat body lipid storage capacity, cardiac lipotoxicity ensues. Metabolomics studies in hearts and blood from these animals may help us to identify specific lipotoxins that are “conserved” between flies and humans.
Abstract 658: RNF20/40 Regulates Cardiomyocyte Maturation
Authors: Vandusen Nathan J , Lee Julianna Y , Gu Wei-liang , Sethi Isha , Suo Shengbao , King Justin , Zheng Yanjiang , Guo Yuxuan , Pu William T
Published: October 2019

Between birth and adulthood, cardiomyocytes (CMs) undergo profound changes in size, ultrastructure, metabolism, and gene expression, a process collectively referred to as CM maturation. Although highly coordinated, the transcriptional network that governs this process is not understood. This lack of understanding is a barrier to cardiac regenerative medicine, where our current inability to mature CMs differentiated from non-myocytes limits their use for disease modeling or replacement therapy. In addition, disruption of maturation by abnormal hemodynamic loads in neonates who have undergone surgery to correct congenital heart defects likely contributes to their high incidence of heart failure in adulthood. A sound understanding of the regulatory network governing CM maturation will inspire hypothesis driven attempts to surmount these challenges.

In mice, a key hallmark of CM maturation is sarcomere isoform switching, including the well documented neonatal switch from Myosin Heavy Chain 7 (Myh7) to Myosin Heavy Chain 6 (Myh6). We have conducted and validated an in vivo high throughput CRISPR screen for transcriptional regulators of CM maturation, using the Myh7/6 isoform switch as the readout. Two top candidates from this screen, Rnf20 and Rnf40, form a complex which deposits the epigenetic mark H2bub1 (histone-2B mono-ubiquitinated on lysine 120). Defects in RNF20/40 and H2Bub1 regulation have been associated with human congenital heart disease, but their mechanistic function in the heart has not been studied. We performed ChIP and RNA-sequencing experiments in control and RNF loss-of-function models to characterize the role of H2Bub1 in transcriptional control of CM maturation. The resulting mechanistic insights into how gene expression is coordinately controlled during maturation will inform efforts to improve CM production protocols and develop targeted therapies.
Abstract 702: Fusion of Cardiac Progenitor Cell and Cardiomyocyte Induces Cardiomyocyte Division
Authors: Chen Zhongming , Bender Ingrid , van Berlo Jop
Published: October 2019

Recent evidence has shown that cardiac progenitor cells (CPCs) can fuse with existing cardiomyocytes (CMs). The fusion frequency significantly increases during heart injury or drug treatment, but the role of CPC-CM fusion in heart regeneration is largely unknown. In this study, we investigate CM-CPC fusion in vivo and its effects on CM proliferation in vitro using genetic lineage tracing and isolated cardiac cells from adult mice. To distinguish between nuclear fusion and membrane fusion, we utilized double fluorescent nuclear reporter mice. After one month of tamoxifen chow treatment, as many as 78% (n=4) of fused CMs displayed cytoplasm fusion, but not nuclear fusion. To further study the role of cytoplasm fusion in CM proliferation, we designed in vitro experiments where we either induce partial fusion or co-culture freshly isolated CMs with previously isolated c-kit+ cells. We find that 52% (387 of 741 cells, n=10) of the fused CMs maintain rod-shape morphology for over two months during culture, while all CMs ball up and dedifferentiate in co-culture controls (n=10) within 24 hours. Next, EdU was added to the culture media to assess DNA replication. EdU+ CMs were observed in both rod-shape and rounded CMs in 7 days. Finally, we monitored cell division of rod-shaped CMs fused with c-kit+ CPCs using intermittent live-cell imaging. We identified 2 cell division events from 20 randomly monitored rod-shape CMs in two months after fusion. These results demonstrate that fusion of c-kit+ cells with CMs are typically partial fusion events, that may play a role in regulating CM proliferation.
Abstract 706: Changes in Translational Efficacy During Zebrafish Heart Regeneration
Authors: Goldman Joseph A , Bazzini Ariel , Giraldez Antonio , Poss Kenneth
Published: October 2019

Zebrafish are able to regenerate heart muscle after catastrophic injury. Fervent research is underway to discover critical molecules with therapeutic potential, yet many principal biological processes that underlie regeneration likely remain undiscovered. Changes in the abundance of dozens to thousands of mRNA have been reported using both targeted approaches like in-situ hybridization and unbiased approaches like RNA-seq. However, whether the abundance of mRNA represents the entirety of gene expression control remains unknown. Regulation of translation may alter the abundance of a critical protein product without requiring an intervening transcription event. Here, we have taken advantage of ribosome profiling to document the changes in translation efficiency that accompany heart regeneration. Using high-throughput sequencing of the ~28 nucleotide fragments of mRNA protected from enzymatic degradation, we find thousands of transcripts that change in their association with ribosomes. We also find that key components of the translational machinery are transcriptionally induced during regeneration, supporting a general model in which regulation of translation is a fundamental component of zebrafish heart regeneration.
Abstract 707: Cortical-bone Stem Cell Therapy Alters the Inflammatory Response After Myocardial Infarction
Authors: Hobby Alexander , Berretta Remus , Borghetti Giulia , Feldsott Eric , Eaton Deborah , Kubo Hajime , Mohsin Sadia , Houser Steven
Published: October 2019

Ischemic heart diseases like myocardial infarction are the largest contributors to cardiovascular disease world-wide. The resulting cardiac cell death impairs function of the heart and can lead to heart failure and death. Re-perfusion of the ischemic tissue is necessary but causes damage to the surrounding tissue by re-perfusion injury and initiates a sterile inflammatory response. Cortical bone stem cells (CBSCs) have been shown to increase pump function and decrease scar size in a large animal swine model of myocardial infarction. To explore the potential cause of these changes, we hypothesized that CBSCs were altering the inflammatory response after re-perfusion thereby changing the wound healing process. To test this, we performed serial immune cell analysis of the blood and tissue from Gottingen mini-swine that underwent 90 minutes of lateral anterior descending coronary artery ischemia followed by 7 days of re-perfusion to assess changes in immune cell recruitment and phenotype. Our findings indicate that CBSCs modify the cytokines released by immune cells in the infarct, decrease macrophage infiltration of the infarct 3 days after MI, and increase the recruitment of CD4+ T-cells to the infarct zone 7 days after MI. In addition, these changes reflect a pro-healing inflammatory environment. From this data, we conclude that CBSCs are influencing immune cell recruitment dynamics and phenotype, and these changes may contribute to the decreased scar size and increased pump function seen in CBSC-treated animals.
Abstract 709: DNA Damage-free iPS-cardiomyocyte Reduces Cardiac Fibrosis Through Downmodulation of Exosomal miR-101
Authors: Kannappan Ramaswamy , Miller Jessica M , Rajasekaran Vasanthi , Rajasekaran Soorappan Namakkal , Zhang Jianyi
Published: October 2019

With the growing field of myocardial repair through transplantation of cardiomyocytes (CM) derived from human induced pluripotent stem cells (iPSC) calls for a method for reducing fibrotic tissue development at the site of cell transplantation. We tested the hypothesis of whether transplanting CMs derived from DNA damage-free (DdF) iPSCs could reduce the recruitment of myofibroblasts and reduce fibrosis at the site of transplantation. Transcription factor p53 functions are DNA damage-dependent: induce apoptosis in DNA damaged cells while promoting cell cycle in normal cells. Utilizing this discriminating nature of p53 we selected iPSCs that are DdF and differentiated them into CMs. Control CMs (Ctrl-CM) and DdF-CMs were tested for transplantation induced fibrosis in a mouse model. The inflammatory markers such as accumulation of ROS, superoxide and DNA damage were significantly reduced in DdF cells. RNA sequencing revealed that, in DdF-CMs, the inflammatory and fibrosis signaling pathways are significantly different compared to Ctrl-CMs. Transplantation of both CMs into the myocardium of ischemia-induced mice resulted in fibrosis around the transplantation site. However, the fibrotic area around transplanted sites and total fibrosis were significantly reduced in mice hearts that received DdF-CMs as evidenced by collagen accumulation and Sirus red staining of fibrotic tissue. Importantly, the number of myofibroblasts at the site of DdF-CM transplantation was significantly reduced compared to Ctrl-CM sites. In an in vitro migration study, fibroblasts preferentially migrated towards the Ctrl-CMs. Human fibroblast treatment with DdF-CM exosomes (DdF-Exo) resulted in reduced expression of fibrotic markers than Ctrl-CM exosomes (Ctrl-Exo) treatment. miRNA analysis in exosomes showed that the anti-inflammatory miR-26a is upregulated and the inflammatory miRNA101 is downregulated in DdF-Exo. Overexpression of miR-101 mimic in DdF-Exo abrogated the anti-fibrotic potential property of DdF-Exo. Our results clearly demonstrate that DdF-CMs has anti-fibrotic potential. Transplanting DdF-CMs attracted reduced myofibroblast to the engraft site and Ctrl-CMs transplantation induce fibrosis through the expression of exosomal miR-101.
Abstract 713: Engineering Complex Tissue Mechanical Environment for Cardiac Microtissues Derived From Human Induced Pluripotent Stem Cells
Authors: Ma Zhen , Wang Chenyan , Koo Sangmo , Grigoropoulos Costas
Published: October 2019

Previous studies of hiPSC-based cardiac tissue models mainly focused on uniform tissue mechanical environment, which is not sufficient to recapitulate the complex and heterogeneous tissue mechanical environment that would lead to cardiac contractile dysfunction. To create a 3D cardiac tissue model with nonuniform mechanical environment, we have developed state-of-art laser-based 3D bioprinting technology to fabricate “mechanical hybrid” scaffolds comprised of layers of parallel aligned high-aspect-ratio (100:1) fibers.We fabricated the double-hybrid (DH) scaffolds with
“1:1 thin-thick”
design by positioning the thin fibers (5 μm, low stiffness) next to the thick fiber (10 μm, high stiffness) to create a mismatch region in the center (
Fig. A
). The entire microtissue had to self-balance the force generation between high and low stiffness for synchronized contraction (
Fig. B
). This resulted in tissue hypertrophy at the high stiffness side with larger tissue cross-section comparing to low stiffness side (
Fig. C
). We also determined the local contractile velocities from each segment of the microtissues at different spatial locations, which showed a trend of exponential decay on the contractile velocities from edge to center (
Fig. D
). The difference in the decay rate between thick side of the DH scaffolds (DH-K) and thin side of the DH scaffolds (DH-N) indicated the contractile imbalance on the two sides of the entire microtissues. We also calculated the force output of the cardiac microtissues on DH-K side, which showed a HCM-like diastolic dysfunction comparing to the force output from single scaffolds with thick fibers (
Fig. E
).
Abstract 716: 2D and 3D Assessment of Angiogenesis in Cardiac Engineered Tissues Implanted on Infarcted Rat Hearts
Authors: Munarin Fabiola , Kant Rajeev J , Rupert Cassady E , Khoo Amelia , Coulombe Kareen L
Published: October 2019

A lack of efficient vessel perfusion in cardiac engineered tissues affects cell viability and limits the success of implanted cardiac tissues. To address the need for vascular infiltration, we have developed a collagen-based cardiac construct releasing pro-angiogenic growth factors. The constructs were implanted in a rodent model of myocardial infarction four days after ischemia/reperfusion injury. Angiogenesis assessment was performed 4 weeks after implantation by 2D histological analysis with RECA-1 antibody and with Microfil® (Flow Tech, MA) perfusion followed by 3D microCT analysis (voxel dimensions 10x10x10 μm). Cross-registration of microCT z-stacks with histological images allows segmentation of remote, infarct and implant regions. The heart vasculature was reconstructed in 3D with ImageJ and Imaris software, and Image J volume calculator and skeletonization algorithm have been used to measure vessels volume, length and number of branch points in each region of interest. Results from 2D histological analysis confirmed the development of capillary-like structures in the implants, visible in both cross and longitudinal sections in the histological slides. The density of cross-sectional vessels measured in the cardiac tissues was comparable to the density of vessels present in the infarct region (118 ± 77 and 93 ±74 vessels/mm
2
counted in infarct and implant regions, respectively). Furthermore, a 1.5-fold increase in vessels density was detected in the infarct zone of the treatment group with respect to the sham rats at 4 weeks. 3D reconstruction of rat heart vasculature showed evidence of the formation of new, perfused vessels in cardiac implants and presumably host-to graft connections. However, perfused vessels volume was not very different in the implants compared to the infarct regions. To the best of our knowledge, this is the first study where a 3D reconstruction of the perfused vasculature of a repaired rat heart has been conducted with a resolution of 10 μm, and that evidence of host-to-graft connections and vascular integration were shown 4 weeks after implantation. The automated quantification proposed is this study represents a non-biased method that can be further adopted by other groups to assess angiogenesis in cardiac implants.
Abstract 717: CGMP Expanded First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) Induce Significant Angiogenesis, Myocardial Regeneration and Sustained Functional Recovery Exceeding Older MSC Sources in the Rat Myocardial Infarction (MI) Model
Authors: PÃ©ter SzÃ¡raz , Johnston Alexander , Iqbal Farwah , Hoseini Banafshe , Lucato Alexandra , Mander Poonam , Wu Jun , Li Ren-Ke , Librach Clifford
Published: October 2019

Introduction:
FTM HUCPVC are a novel source of young, immunoprivileged mesenchymal stromal cells (MSC). FTM HUCPVC demonstrate significant homing, angiogenic and tissue remodelling capacities
in vitro
. We aimed to explore the therapeutic potential of cGMP compliant FTM HUCPVC for myocardial infarction (MI).

Methods:
FTM HUCPVC and term HUCPVC were expanded in cGMP compliant xeno-free (5% HPL) multilayer cultures (Corning) and Quantum Bioreactor (Terumo). One week post-MI Fox
rnu
rats (n=6) were intramyocardially injected with 3 million FTM HUCPVC, term HUCPVC, bone marrow MSC (BMSC) or basal media. Cardiac function (echocardiography, pressure/volume catheter and ECG telemetry), infarct scar (Trichrome), ECM remodelling activity (DQ collagenase substrate) vascular density (IB4) and contractile proteins (SarcA) were quantified 4 weeks and 6 months after cell implantation. Myocardial apoptosis (caspase-3) and macrophage infiltration (eNOS, CD68, CD80) were assessed at 5 days after cell treatment.

Results:
FTM HUCPVC significantly reduced apoptosis at 5 days and infarct size at 4 weeks (5.3±1.1, p<0.05) compared to other treatment groups. Histological analysis showed that the infarcted region of FTM HUCPVC treated hearts displayed significantly higher: M2 macrophage ratio, perivascular gelatinase activity, capillary density (543.4±118.6 vs. 276.5±42.69; p<0.05), abundance of contractile sarcomeric actinin (48.9±6.1 vs. 19.5±7.0; p<0.05), and Ca
2+
pump SERCA (69.9±5.9 vs. 49.8±3.7) over other experimental groups. FTM HUCPVCs induced significant and superior improvements in all measures of contractile function (ESV, EF, dPdt, tau) relative to cell-free media (p<0.001), BMSC (p<0.001) and term HUCPVC (p<0.001). Improvements in inotropy and lusitropy were maintained to 6 months (δ dPdt max 3.8±6.0; δ dPdt min 3.2±8.78) and resulted in near complete abolishment of premature ventricular contractions (<1/min).

Conclusion:
FTM HUCPVC treatment after MI induces superior structural and functional recovery compared to older MSC sources; characteristics that were maintained during xeno-free upscaling and for up to 6 months post injection. FTM HUCPVC are a promising option as a novel cell therapy for optimal cardiovascular regeneration.
Abstract 718: Reparative Macrophage Transplantation for Myocardial Repair: A Refinement of Bone Marrow Mononuclear Cell-Based Therapy
Authors: Podaru Mihai-Nicolae , Fields Laura , Kainuma Satoshi , Ichihara Yuki , Hussain Mohsin , Ito Tomoya , Kazuya Kobayashi , D'Aquisto Fulvio , Mathur Anthony , Lewis Fiona , Suzuki Ken
Published: October 2019

Background:
Reparative (alternatively activated or M2-like) macrophages play an important role in post-myocardial infarction (MI) cardiac repair. Transplantation of bone marrow mononuclear cells (BM-MNCs) is an emerging therapy for MI while its therapeutic efficacy in previous clinical trials is not satisfactory. We hypothesized that induced differentiation/polarisation of BM-MNCs to reparative macrophages before transplantation may enhance the effect of BM-MNC transplantation.

Purpose:
This study aimed to develop a robust
in vitro
protocol to produce reparative macrophages from BM-MNCs and to establish the pre-clinical proof of concept data for reparative macrophage transplantation for the treatment of MI.

Methods & Results:
Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these
in vitro
. The concomitant M-CSF+IL-4 protocol produced the highest rate and number of CD11b
+
F4/80
+
CD206
+
macrophages. Expression and secretion of tissue repair-related factors of the produced cells, including IGF-1, TGF-β1, VEGF and IL1-ra, were more extensive compared to BM-MNCs. Then, reparative macrophages, BM-MNCs or PBS only were intramyocardially injected in a mouse MI model. At 4 weeks after treatment, echocardiography demonstrated that reparative macrophage transplantation markedly improved cardiac function (left ventricular ejection fraction; 57.2±1.6%, n=11) compared to both BM-MNC transplantation (48.4±1.3%, n=9) and control group (44.4±2.0%, n=9). Histological studies showed that infarct size was the smallest after reparative macrophage transplantation in association with the greatest tissue repair in the peri-infarct myocardium, including augmented microvascular formation, reduced cardiomyocyte hypertrophy and reduced pathological interstitial fibrosis. It was also found that reparative macrophage transplantation increased host-derived reparative macrophages through TGF-β1 secretion.

Conclusion:
M-CSF+IL-4 treatment was effective in producing reparative macrophages from BM-MNCs
in vitro
. Addition of this pre-treatment improved the therapeutic effect of BM-MNC transplantation. Further pre-clinical and clinical development of this advanced cell therapy is warranted.
Abstract 721: Human Neonatal Cardiac Progenitor Cells Derived Exosomes Induce Cardiomyocytes Proliferation
Authors: Sharma Sudhish , Wang Lina , Ambastha Chetan , Mishra Rachana , Kaushal Sunjay
Published: October 2019

Background:
Human neonatal cardiac progenitor cells (ckit+/CD45-/Lin-, CPCs) improve cardiac function and attenuate adverse left ventricular remodeling after myocardial infarction (MI) through their exosomes (nEXOs). Success of cell therapy to treat injured myocardium depends mainly on reactivation of endogenous quiescent cardiomyocytes to proliferate and recover the lost myocardium. Here, we investigated the mechanism of reactivating quiescent cardiomyocyte proliferation by exosomes.

Hypothesis:
RNAseq analysis of nEXOs identified miRs which may be responsible for modulation of Hippo signaling, thereby promoting cardiomyocytes proliferation. nEXOs effectively stimulate endogenous cardiomyocyte (CM) proliferation by targeting the Hippo pathway to restore cardiac function in an injured heart.

Methods and Results:
nCPCs were conditioned for 48 hours in serum free nutrition mix (Ham’sF12) and nEXOs were purified from supernatant using size exclusion chromatography (SEC; CL2B) coupled with ultracentrifugation. nEXOs were quantified by Nanosight (NS300) and characterized by transmission electron microscopy and flow cytometry for the presence of CD63 and CD9. Our results show recovery of cardiac function (ejection fraction = 63.4% vs 40.5%, n=10, p<0.001)) and generation of myocardial mass in a rodent MI model following nEXOs’ intra-myocardial injection. We further show that nEXOs are preferentially acquired by CMs in the border zone of the infarction (m-cherry-Alix labeled EXOs). Our In vitro experiments show that miR-582-3p and miR-7641 (25nM) are the most effective miRs to induce proliferation of quiescent cardiomyocytes (40.1% and 48.8%, respectively, n=3). Increased miR-7641 expression led to a profound increase in quiescent CMs proliferation, in part through repression of the Hippo signal transduction pathway (increased YAP/pYAP ratio). By immunoblotting we show that LATs1/2 (a protein in the Hippo pathway) is directly targeted by miR-7641. nEXOs enriched with miR 7641 promote CM proliferation by 3 folds as compared to non-enriched nEXOs.

Conclusion:
Our data for the first time demonstrates the ability of nEXOs derived miRNA based therapeutic approaches to activate cardiomyocyte proliferation.
Abstract 734: NF-kB Signaling Regulates Mitochondrial Permeability Transition Pore Opening of Cardiac Myocytes via Cyclophilin D (CypD) Modulation
Authors: Kirshenbaum Lorrie A , Dhingra Rimpy , Guberman Matthew , Margulets Victoria , Aguilar Floribeth
Published: October 2019

Nuclear Factor-κB (NF-κB) is ubiquitously present transcription factor that regulates a variety of cellular functions including cell survival. Herein, we show a critical role for NF-κB signaling in regulation of mitochondrial permeability transition pore opening (mPTP) of cardiac myocytes that involves cyclophilin D (CypD). Cardiac myocytes expressing a kinase defective form of IKKβ (IKK
K-M
), the principle IKK required for NF-κB activation, displayed impaired NF-κB gene activity. Defects in NF-κB signaling coincided with an increase in mPTP opening and cell death. Interestingly, mPTP opening and cell death observed in the NF-κB defective cardiomyocytes was supressed by inhibition of mPTP modulator CypD, with cyclosporin A (CSA) or by siRNA knock down (CypDsiRNA), suggesting a link between mPTP regulation and NF-κB signaling. Earlier, we reported that doxorubicin (Dox) treatment resulted in severe ultra-structural defects including disrupted mitochondrial cristae and impaired respiration, increased mitochondrial calcium overload, mPTP opening and a widespread cell death. Interestingly, we observed a dramatic reduction in NF-κB signaling in cardiac myocytes treated with doxorubicin (18 Hrs), coupled with mitochondrial dysfunction including impaired respiration. Inhibition of CyPD suppressed doxorubicin induced cell death of cardiac myocytes. Finally restoration of NF-κB signaling in cardiac myocytes treated with doxorubicin by IKKβ, active kinase, suppressed mitochondrial calcium overload, mitochondrial perturbations, respiration and cell death. The data herein, provides the first direct evidence that impaired NF-κB signaling predispose Dox treated cardiac myocytes to cell death. Hence, interventions that preserve NF-κB survival signaling pathways in the heart may prove beneficial in reducing cardiac dysfunction and heart failure in cancer patients undergoing doxorubicin chemotherapy.
Abstract 736: Increased Cell Death in Regions of Elevated Aortic Wall Shear Stress in Bicuspid Valve Aortopathy
Authors: Maredia Ashna K , Iqbal Fatima , Guzzardi David G , Barker Alex J , McCarthy Patrick M , Fedak Paul W , Greenway Steven C
Published: October 2019

Bicuspid aortic valve (BAV) is a common congenital malformation associated with aortopathy and potential rupture. Identifying those patients at greatest risk for dissection is difficult at present. Abnormal hemodynamics in the ascending aorta creates regions of increased wall shear stress (WSS) which causes extracellular matrix dysregulation and may affect cell death. Dying cells release fragmented DNA into the blood and levels of aorta-specific cell-free DNA (cfDNA), identified by the presence of tissue-specific differentially methylated regions (DMRs), could be leveraged as a biomarker. Our objective was to determine if a relationship between elevated WSS and apoptosis in the ascending aorta existed and identify aorta-specific DMRs in the cfDNA of BAV patients. We hypothesized that areas of increased WSS would demonstrate increased cell death that would correlate with increases in aorta-specific cfDNA from patient blood.

BAV patients undergoing 4D-flow cardiac magnetic resonance imaging (CMR) and surgery for ascending aorta dilation (range 36-63 mm) were recruited. Blood was collected from 23 patients at the time of CMR and used for the isolation of plasma cfDNA. Aortic wall samples (n = 30) corresponding to regions of high and low WSS were collected at surgery from 15 patients and stained for cell death using the TUNEL assay. Publicly-available methylomes and Metilene were used to identify aorta-specific DMRs
in silico
.

Regions of elevated WSS were seen in all individuals regardless of absolute aortic dimension. These regions of elevated WSS by CMR showed significantly greater cell death when compared to the region of normal WSS in the ascending aorta (0.14 ± 0.05 vs. 0.08 ± 0.06, p=0.00006). Levels of cell death did not correlate with maximal aortic diameter. Twenty-three aorta-specific DMRs were identified. Of the four that were tested, three were found to be hypomethylated in genomic DNA from the aorta compared to 14 other tissues. Levels of aorta-specific cfDNA based on our DMRs did not correlate with maximal aortic diameter.

Increased regional cell death corresponding to elevated WSS may implicate abnormal hemodynamic flow in the progression of aortopathy and provides a biological rationale for the use of cfDNA as a biomarker for aortopathy.
Abstract 737:
N
-ethylmaleimide sensitive factor (NSF) is Essential in Necrotic Cell Death
Authors: Murray Emma K , Lombardi Alyssa A , Lambert Jonathan P , Elrod John W
Published: October 2019

Necrotic cell death is the main way in which cells die during myocardial infarction (MI) and heart failure (HF), yet the molecular mechanisms regulating necrotic cell death are poorly defined. To elucidate the key regulators of plasma membrane rupture during necrotic cell death a genome-wide shRNA loss-of-function screen was performed which identified components of SNARE-mediated membrane fusion as potential facilitators of Ca
2+
and ROS-induced necrosis. To examine if the SNARE machinery is involved in cellular necrosis we targeted N-ethylmaleimide sensitive Factor (NSF) due to its requirement in SNARE recycling, lack of gene homologs, and redox sensitivity. Deletion of NSF from 3T3 fibroblasts by CRISPR-Cas9n (Nsf
-/-
), inhibited membrane rupture and improved cell viability following Ca
2+
overload (ionomycin), ROS (H
2
O
2
), and necroptotic (TNFa, CHX, zVAD)-induced cell death but did not alter apoptotic (staurosporine) cellular demise. We next created a cardiac-specific conditional
Nsf
knockout mouse model to determine if NSF contributes to myocyte death during IR injury. Loss of NSF in cardiomyocytes did not alter baseline cardiac function or structure. Currently, studies are underway to determine if NSF contributes to necrotic cell as occurs in IR injury and heart failure. In summary, our results suggest that NSF is an important molecular component of membrane rupture and pathogenic cell death.
Abstract 752: A Novel Senolytic Drug, Seno-7284 Ameliorates Aging and Age-related Cardiometabolic Disorders
Authors: Katsuumi Goro , Shimizu Ippei , Yoshida Yohko , Suda Masayoshi , Hayashi Yuka , Ikegami Ryutaro , Wakasugi Takayuki , Nakao Masaaki , Furuuchi Ryo , Nagasawa Ayako , Mikawa Ryota , Sugimoto Masataka , Minamino Tohru
Published: October 2019

Accumulation of senescent cells is promoted in various organs as aging, and it also contributes to the progression of age-related disorders. Recent reports have demonstrated the elimination of senescent cells, so-called "senolysis" ameliorated various age-related disorders including cardiovascular diseases. However, there is currently no senolytic drug available in clinical settings. Here, we found a novel senolytic drug (termed “seno-7284”) from those already used in clinical setting and it exhibited senolytic effect in murine models of type 2 diabetes, atherosclerosis and progeroid aging. Conducting senescence-associated beta-galactosidase staining(SA-beta gal), we found that administrating seno-7284 for one week significantly reduced the accumulation of senescent cells in viscerala dipose tissue of diabetic mice induced by fed high-fat diet(Figure). This drug also ameliorated systemic glucose metabolism and adipose tissue inflammation without a reduction of body weight. Further analysis including RNA-seq analysis suggested seno-7284 stimulates the endogenous senolytic function of NK cells and CD8+ T cells via the Cxcl9-Cxcr3 axis. We also found administrating seno-7284 for two weeks also reduced the accumulation of senescent cells and atherosclerotic lesions in the aorta of western-diet-fed ApoE knock out mice. Surprisingly, this drug significantly improved the lifespan of Zmpste24 KO progeroid aging mice. Correctively, our results indicate that seno-7284 mediates its senolytic effect through the recruitment of lymphocytes. Senolytics would become a promising therapy for aging and age-related cardiometabolic disorders.
Abstract 753: Exercise Capacity, Cardiac fibrosis and Left Ventricular Remodeling in a Rat Model of Chronic Pressure Overload: Serial Echocardiography, Pressure-Volume Analysis and Gene Expression Profiling
Authors: Kim Kyung-hee , Park Yoonjee , Byun Jae Kyeong , Rha Seung-Woon
Published: October 2019

Background:
Pressure overload (PO) left ventricular (LV) hypertrophy is a known precursor of heart failure with preserved ejection fraction (EF). The aims of this study were to establish a reliable model of chronic PO in rats and verify the pathophysiological features of this model by evaluating cardiac function using serial echocardiography and a pressure-volume analysis.

Methods:
Constriction of the abdominal aorta was used to induce PO LV hypertrophy (n = 40) in rats. Twenty sham rats were compared with PO rats. Serial echo studies and exercise were performed at 2-week intervals, and invasive hemodynamic examination by a pressure-volume catheter system was performed 12 weeks after the procedure. The gene expression profiles of the left ventricle (LV) 12 weeks after the procedure were analyzed by DNA chip technology.

Results:
Serial echo revealed that the LV wall thickness began to increase after the PO and showed progressive increasing until the 8th week (LV septal wall thickness at 8 weeks 1.4mm±0.1mm for PO vs 0.6mm ±0.05mm for Sham, p <0.01). LV dimension was comparable increased until 14 th week (LV end-systolic dimension at 14 weeks, 4.91±0.50 mm 4.71±0.25 mm for PO vs Sham; LV end-diastolic dimension, 9.02±0.42 mm vs. 8.71±0.47 mm for PO vs Sham, p>0.05). The LV ejection fraction showed similar between groups until the 14th week (70.0±2.2% vs. 69.1±3.1% for PO vs. Sham). In hemodynamic analyses, the LV end-diastolic pressure and the end-diastolic pressure-volume relationship slope were greater in the PO group than sham group. When we compared LV remodeling and exercise capacity, cardiac fibrosis and exercise intolerance developed in the PO group but not in the sham group (exercise duration, 234.0 ± 80.3 vs. 597.8 ± 49.0 seconds, p < 0.05, respectively). Transcriptional profiling of cardiac apical tissues revealed that gene expression related to the cardiac fibrosis, cytoskeletal pathway and G-protein signaling genes were enriched in the PO group.

Conclusions:
We established a small animal model of chronic PO and verified its pathophysiological features. Cardiac fibrosis and cytoskeletal pathway were important pathways in the PO group and influenced exercise capacity. This model may provide a useful tool for future research on PO heart failure.
Abstract 756: Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model With Dynamically Tunable Afterload
Authors: Livingston Carissa , Ramachandran Abhinay , Corbin Elise , Vite Alexia , Bennett Alexander , Margulies Kenneth B
Published: October 2019

Introduction:
Anti-angiogenic tyrosine kinase inhibitors (TKIs) have efficacy against many solid neoplasms. Unfortunately, their on- and off-target effects frequently cause hypertension (HTN) and heart failure. Nearly all cases of cardiac dysfunction are associated with HTN, but the mechanism for this association is unclear.

Aims:
We have developed a novel in vitro 3D tissue engineering platform that uses a magnetically tunable elastomer to create dynamic alterations in afterload felt by cardiac microtissues. We are using this platform to examine the impact of increased in vitro afterload (a proxy for HTN) on cardiotoxicity of the TKI sunitinib.

Hypotheses:
1) Increased afterload augments sunitinib-induced cardiotoxicity 2) Repeat exposures to sunitinib and increased afterload will further augment this toxicity.

Methods:
Human induced pluripotent stem cell cardiomyocytes (96%) and human cardiac fibroblasts (4%) from a commercial vendor (NCardia, Inc.) were allowed to self-assemble and form cardiac microtissues. Microtissues were exposed to sunitinib (0μM-10μM) at differing levels of afterload for a minimum of 8 hours. After a washout period, tissues were either harvested for analysis or exposures to sunitinib and afterload were repeated. Outcomes measured after each drug exposure included caspase activation, contractile ability, and force generation.

Results:
The applied magnetic field produced increases in afterload from 0.509±0.032 to 0.698±0.031 μN/μm. At 36 hours after exposure to supratherapeutic concentrations of sunitinib, recovery of contractile function as measured by force generation of microtissues was greater under soft afterload conditions compared with stiff afterload conditions (2.52μN soft vs 1.16μN stiff).

Conclusion:
Our platform creates dynamic alterations in afterload in engineered cardiac microtissues. Use of this versatile platform to examine the cardiotoxic effects of sunitinib has given promising initial results. Future studies examining removal/application of afterload, repeat exposures to sunitinib, and exposure to cardioprotective drugs will allow for insight into the mechanisms, treatment, and prevention of sunitinib-induced cardiotoxicity.
Abstract 775: Interaction of Phosphate Transport and Calcification in Placenta Vasculature
Authors: Benson Ciara , Jayaraman Nirmala , Mei Ariel , Wallingford Mary C
Published: October 2019

Phosphate transport has been associated with pathological vascular calcification, but this relationship remains largely unexplored in placenta tissue. In this study we investigated the interaction of maternal-fetal phosphate transport and placental calcification using in vitro and in vivo models. Sodium-dependent phosphate transporter transcriptomics validated by quantitative RT-PCR revealed that the type III sodium-dependent phosphate transporter family members Slc20a1 and Slc20a2 are the predominant sodium-dependent symporters in both mouse and human placenta. Global loss of Slc20a2 generates multiple maternal and fetal phenotypes. We examined Slc20a2 promoter activity and protein localization of Slc20a2 throughout placentation. Slc20a2 was detected in embryonic structures that give rise to the maternal-fetal interface and in regions of the placenta that are susceptible to vascular calcification in the absence of Slc20a2. Pathologically, vascular malformations were found to precede calcium deposition in the absence of Slc20a2. Activation of osteocalcin in the placenta suggested that physiological calcification mechanisms may potentiate the mineral deposition observed with Slc20a2 loss. Histological analyses of human placenta revealed focal localization of Slc20a2 protein and distinct calcification deposition patterns. Lastly, the BeWo trophoblast model confirmed cellular competency for active, sodium-dependent phosphate symport as well as extracellular matrix calcification. Taken together, our findings support that normal growth and maintenance of the placenta requires Slc20a2. Moving forward, we now propose the working hypothesis that Slc20a2-mediated placental phosphate transport contributes to the energy requirements for rapid placental vascular development, enabling vascular patterning that maximizes surface area and minimizes tissue damage caused by aberrant flow, thereby promoting organ health and transport of nutrients, including phosphorus, across the placenta.
Abstract 777: Dual-Specificity Phosphatase 6 Deficiency Protects Against Intimal Hyperplasia After Arterial Injury
Authors: Yet Shaw-Fang
Published: October 2019

Dysregulation of MAPK activation cascades has been implicated in various diseases. Dual-specificity phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases that dephosphorylate MAPKs and modulate critical signaling pathways. Studies have shown that DUSP6 plays an important role in the heart and diet-induced obesity. However, the role of DUSP6 in vascular disease is largely unclear. Our goal is to investigate whether DUSP6 has a role in occlusive vascular disease. To this end, we used a neointima formation model by denuding endothelium of mouse femoral arteries with a guide wire. We showed that DUSP6 was expressed at very low levels in the normal uninjured mouse arteries; however, DUSP6 expression was increased in the arterial wall of injured blood vessels, suggesting a potential role of DUSP6 in vascular disease. Compared with wild-type mice, DUSP6 deficiency reduced neointima formation 4 weeks after arterial injury in mice. BrdU incorporation assays revealed that lack of DUSP6 resulted in decreased smooth muscle cell (SMC) proliferation in the neointima and media 2 weeks after injury, indicating DUSP6 may promote SMC proliferation in response to injury. Indeed, overexpressing DUSP6 in primary SMCs increased cellular proliferation, similar to that exerted by inflammatory cytokine IL-1β while knocking down DUSP6 expression with siRNA abrogated IL-1β-induced SMC proliferation. Pathway analysis showed that IL-1β-induced ERK1/2 activation preceded DUSP6 induction in SMCs. Intriguingly, ERK1/2 inhibitor U0126 abolished IL-1β-mediated ERK1/2 activation, DUSP6 induction, and SMC proliferation. Taken together, our results indicate that DUSP6 induction promotes vascular remodeling under pathological conditions. DUSP6 might be a therapeutic target for preventing/treating occlusive vascular disease.
Abstract 785: Modeling Congenital Heart Disease-Associated Variants in
GATA6
Using CRISPR/Cas9 and Human Induced Pluripotent Stem Cells
Authors: Sharma Arun , Wasson Lauren , Willcox Jon , Morton Sarah U , Gorham Joshua M , DeLaughter Daniel M , Neyazi Meraj , Schmid Manuel , Agarwal Radhika , Jang Megan , Toepfer Christopher N , Ward Tarsha , Kim Yuri , Pereira Alexandre C , DePalma Steven R , Tai Angela , Kim Seongwon , Conner David , Bruneau Benoit , Seidman Jon G , Seidman Christine E
Published: October 2019

The discovery of damaging gene mutations in congenital heart disease (CHD) patients enables identification of regulators of cardiac development. Exome sequencing identified
de novo
heterozygous loss-of-function (LoF) and missense variants in
GATA6
among CHD probands, most with outflow tract malformations. Other subjects with
GATA6
LoF mutations developed pancreatic agenesis. To elucidate the molecular basis for the predominance of this heart defect, we modeled
GATA6
mutations in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs).
GATA6
variants were introduced into isogenic hiPSCs using CRISPR/Cas9 genome editing. Genome-wide molecular profiles including chromatin accessibility (ATAC-Seq) and gene expression (single cell and bulk RNA-Seq) were evaluated during hiPSC-CM differentiation. Analyses of
GATA6
mutant hiPSC-CMs showed deficits in hiPSC-CM differentiation, chromatin accessibility and transcriptional profiles. Heterozygous
GATA6
LoF hiPSCs made hiPSC-CMs but exhibited reduced expression of second heart field genes. Homozygous
GATA6
LoF hiPSCs failed to differentiate and adopted fibroblast expression profiles. hiPSCs carrying a homozygous
GATA6
missense variant, R456G, which altered a DNA-binding domain residue, showed enhanced capacity to differentiate into neuroepithelial-like cells. Chromatin-accessibility studies confirmed that GATA6 normally binds to genes in the promoter region and other genes at distal enhancers. Human
GATA6
haploinsufficiency disrupts developmental transcriptional responses driving cardiac morphogenesis. The
HAND2
-dependent genetic program, operant during outflow tract development, is particularly sensitive to
GATA6
dosage. The mixed differentiation patterns observed in mutation-carrying hiPSCs likely contributes to vascular phenotypes observed in CHD patients.
GATA6
haploinsufficiency preferentially alters binding of distal enhancers to promoters in genes where GATA6 normally binds the enhancer rather than the promoter. We speculate that pathogenicity of GATA6 haploinsufficiency is mediated by weaker binding of GATA6 to distal enhancers than to promoter elements, altering expression of these genes in GATA6 haploinsufficient patients.
Abstract 786: A Novel Desmoplakin Mutation Contributes to Arrhythmogenic Cardiomyopathy
Authors: van Kampen Sebastiaan J , Kyriakopoulou Eirini , Han Su Ji , Goversen Birgit , Monshouwer-Kloots Jantine , Eding Joep , de Boer Teun , van Veen Toon , van Rooij Eva
Published: October 2019

Genetic alterations in desmosomal genes are associated with arrhythmogenic cardiomyopathy (ACM). This condition is characterized by sudden cardiac death, severe arrhythmias, and the presence of fibro-fatty patches within the myocardium. Despite the fact that the pathophysiology of ACM is well described, the molecular mechanisms underlying disease onset and progression are poorly understood.

In an effort to identify disease-driving mechanisms we obtained induced pluripotent stem cells (hIPS) from a patient harboring a novel heterozygous mutation in desmoplakin (
DSP
p.Tyr1188His/WT
), which we corrected to yield an isogenic control. hIPS cells carrying a known pathogenic mutation in desmoplakin (
DSP
p.Arg1113X/WT
) served as a positive control. Gene expression analyses of
DSP
p.Tyr1188His/WT
hIPS-derived cardiomyocytes revealed a dysregulation of desmosomal and ion channel-related genes. At the functional level we observed a prolonged action potential duration in
DSP
p.Tyr1188His/WT
cardiomyocytes. Importantly, we validated these findings in an independent knock-in line carrying the same heterozygous (
DSP
p.Tyr1188His/WT
) mutation. To study the
in vivo
relevance we additionally generated two corresponding mouse models harboring the mouse equivalent of the human desmoplakin mutations in the endogenous locus (
Dsp
p.Tyr1200His/WT
and
Dsp
p.Arg1125X/WT
). Both lines show altered connexin 43, phospholamban and Na
V
1.5 expression levels under baseline conditions, recapitulating the changes observed in the hIPS-derived cardiomyocytes. Moreover, Fos- and RhoA-signaling pathways are enriched in these mice compared with wildtype littermates. Since exercise can be a trigger for the development of ACM, we are currently in the process of examining the effect of intense swimming exercise in the models.

We show that the
DSP
p.Tyr1188His/WT
mutation causes cellular changes linked to ACM pathogenesis, indicating this mutation to be disease-driving. Moreover, our novel mouse models of ACM recapitulate the findings observed in our human models and show key hallmarks of ACM disease progression. Future studies will focus on the underlying pathogenic mechanisms in the presented models.
Abstract 787: Rose - A Novel Vascular Anti-inflammatory Agent
Authors: Arokiaraj Mark C , Menesson Eric
Published: October 2019

Introduction:
Inflammation is common in clinical practice and often results in significant complications. The study was performed in search of a novel anti-inflammatory agent for inflammation modulation which would be useful for cardiovascular disorders and various clinical scenarios.

Methods:
A crushed red rose extract was prepared from the petals, and it was processed for analysis. The extract was tested on HUVEC cells at various concentrations. By microscopic analysis of cells, a safe concentration was identified, and the levels below the safe limit were tested at 72 hours and seven days for selected cytokines secretion.

Results:
Majority of the tested Inflammatory cytokine secretion was reduced by the treatment of red rose extract on the cells. VEGF and angiogenic cytokine levels were reduced, but VEGF-R levels were maintained after the cell treatment. Below the safe concentration limit of the extract there were only minimal changes in the cytokines levels tested at various dilutions of the extract.The study results show a significant reduction in the secretion of anti-inflammatory cytokines by red rose extract. The effects of rose extract inhibited major inflammatory cytokines like IL-1, IL-2, RANTES, IGF-1, IL-8 etc. MCP 3 (CCL7) and PDGF BB levels were less than 10% of the baseline values. There was also a tendency in fall at 72 hours, and thereafter a rise at 7 day levels observed in the values of some cytokines.

Conclusion:
There is potential for a red rose extract treatment in the regulation of inflammatory cytokines secretion. Further studies need to be performed to identify the benefits and side effects profile.