bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 133: Exosomal Transfer of Muscle Specific Mir-499 to Endothelial and Endothelial Progenitor Cells Impairs Angiogenesis in Diabetes
Authors: Cheng Zhongjian , Garikipati Venkata Naga Srikanth , Cimini Maria , Trungcao May , Wang Chunlin , Mallaredy Vandana , Huang Grace , Yu Jia , Benedict Cindy , Verma Suresh K , Kishore Raj
Published: October 2019

Background:
miR-499, a muscle specific-miR, is enhanced in diabetic heart and has been suggested to be a therapeutic target for cardiovascular disease in diabetes. Recent studies provided the evidence that overexpressing miR-499 in ECs inhibits the capillary tube networks. Here, we tested the hypothesis that under diabetic conditions, myocyte-derived exosomes transfer miR-499 to EC/EPCs thereby impairing their angiogenic properties.

Methods and Results:
miR-499 expression is abundant and enhanced in heart, skeletal muscle, cardiomyocytes and skeletal muscle cells (SKMCs) of db/db mice. We observed that miR-499 level was also increased in diabetic EC/EPCs. In vitro, high D-glucose (25 mM) increased miR-499 level in SKMCs but not in ECs, suggesting that enhanced miR-499 in diabetic EC/EPCs is not regulated by hyperglycemia. To study whether SKMC-derived exosomes transfer miR-499 from SKMCs to EC/EPCs, we first examined the miR-499 levels in diabetic SKMC-derived exosomes and plasma-derived exosomes from db/db mice. We observed that miR-499 levels were greater in diabetic SKMC- and plasma-derived exosomes. Furthermore, by co-culture, we found that diabetic SKMCs increased miR-499 levels in human microvascular endothelial cells (ECs) and impaired EC tube formation and migration which was blocked by exosome inhibitor GW4869 (GW). We also observed that GW partially rescued diabetic SKMC- and plasma-derived exosome-mediated impairment in tube formation and migration of ECs. Overexpression of miR-499 in ECs decreased angiogenic factors and impaired tube formation/migration. Furthermore, in a hind-limb ischemia model of db/db mice, GW treatment improved ischemic hindlimb blood perfusion and angiogenesis. Our study suggests that diabetes-enhanced miR-499 in myocytes can be transferred to ECs via myocyte-derived exosomes thus impairs EC/EPC function. Mechanistically, Sex-determining region Y-box 6 (SOX6), one of the validated target of miR-499, was significantly decreased in diabetic EC/EPCs. Knockdown of SOX6 impairs tube formation and migration of ECs.

Conclusions:
Our results suggest that exosomal transfer of muscle specific miR-499 to EC/EPCs impairs angiogenesis and ischemic tissue injury repair in diabetes via suppression of SOX6.
Abstract 135: Hypoxia Induced Defects in 26S Proteasome Machinery Causes Loss Of Immunoprivilege of Allogeneic Mesenchymal Stem Cells
Authors: Abu-El-Rub Ejlal , Yan Weiang , Sequiera Glen Lester , Sareen Niketa , Dhingra Sanjiv
Published: October 2019

The poor survival of transplanted allogeneic mesenchymal stem cells (MSCs) in the recipient heart has dampened the overall enthusiasm of allogeneic MSCs based clinical trials. Outcome of recent studies suggests that the phenotype of transplanted cells changes from immunoprivileged to immunogenic state that leads to rejection of implanted cells by host immune system. In this study, we present a novel mechanism of immune switch in MSCs. We found that hypoxia/or ischemic environment shifts immunological landscape of MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by absence of major histocompatibility complex class II (MHC-II) molecule. Our studies demonstrate that 26S proteasome-mediated degradation of MHC-II prevents its expression on cell surface in MSCs and preserves their immunoprivilege. The exposure to hypoxia results in dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This promotes accumulation of MHC-II and increases immunogenicity of MSCs. We also found that a chaperon protein HSP90α is responsible for 26S proteasome activity in normoxic MSCs. The level of HSP90α decreases in hypoxic MSCs that leads to increase in immunogenicity. Maintaining HSP90α levels in hypoxic MSCs prevents 26S inactivation and preserves immunoprivilege of MSCs. Therefore, hypoxia-induced defects in 26S proteasome assembly causes loss of immunoprivilege of allogeneic MSCs. Preserving 26S proteasome activity in MSCs maintains immunoprivilege and prevents rejection of allogeneic MSCs in the heart. (This work was supported by CIHR)
Abstract 136: The Role of Glucose as a Promoter for Cardiac Regeneration
Authors: Fajardo Viviana , Nakano Haruko , Lien Ellen , Tian Rong , Chen Bao , Clark Peter , Nakano Austin
Published: October 2019

Heart failure is the leading cause of death worldwide. Our focus is on non-genetic mechanisms by which cardiac regeneration can be lengthened or enhanced. Specifically, we are interested in the cyto-protective effects of glucose in cardiomyocyte growth, differentiation and proliferation and how this knowledge can be applied to regeneration therapies. Our preliminary data showed that glucose induces cardiomyocyte proliferation and inhibits cardiomyocyte maturation in human embryonic stem cells derived cardiomyocytes (hESC-CM) via the Pentose Phosphate Pathway in a dose dependent manner. Whether this pathway can be a therapeutic target for heart regeneration is unknown.
Our hypothesis is that glucose promotes neonatal heart regeneration in a murine model.
Non-Transmural cryoinjury was performed to the apex of the left ventricle in wild-type pups and cardiac specific overexpression of Glucose Transporter 1 Transgenic pups. In the acute phase (P1-P7), the level of cardiomyocyte cell proliferation was measured via flow cytometry analysis and immunostaining with PH3 and cTnnt. Glucose uptake by cardiomyocytes was measured by 18F-FDG assay and Glut1 immunostaining. In the chronic phase (P14, P21, P40), we quantified the level of fibrosis by histology (H&E and Picrosirius Red) and neovascularization by immunostaining with PECAM. Increased cardiomyocyte proliferation was observed in the Transgenic Glut1 pups. Myocardial glucose uptake declines from the muscular layer towards the trabecular layer, corresponding with maturation of the heart. We observed that Glut1 cardiomyocyte-specific overexpression resulted in improved cardiac repair compared to wild type (WT) mice at 21 days postnatally. Compared to wild-type, Glut1 hearts showed increased angiogenesis around the site of injury secondary to an increase in cardiomyocyte proliferation. This study is the first to demonstrate the potential role of glucose as a promoter for cardiac regeneration and reveal a potential mechanism for congenital cardiomyopathy associated with diabetic pregnancy.
Abstract 140: Efficacy Evaluation of Transplantation of Three-Dimensional Adipose-derived Stem Cell Sheet With Enhanced Angiogenesis Into Cardiovascular Disease
Authors: Joo Hyung Joon , Kim Jong-Ho , Park Chi-Yeon , Hong Soon Jun , Lim Do-Sun
Published: October 2019

Integration of adipose-derived stem cells (ASCs) and cell sheet engineering technology has received much interest in the regenerative medicine. First of all, sphere formation of ASCs (sph-ASCs) were successfully produced by poly-2-hydroxyethyl methacrylate (poly-HEMA)-coated plates as we previously reported. Sph-ASCs showed increased expression of angiogenic growth factors and cytokine secretion compared to adherent ASCs. Significantly increased expression of HIF-1α and decreased expression of cleaved caspase-3 were detected in sph-ASCs compared to adherent ASCs. Moreover, ASC sheets were formed using thermo-responsive plates and sph-ASCs were seeded to produce ASC sheets covered with sph-ASCs (sph-ASC sheets). After 48 hrs of incubation, the cross-section of sph-ASC sheets revealed mixed and multi-layered of sph-ASCs and ASC sheets. Sph-ASC sheets showed the positive expression of HIF-1α and angiogenic markers such as FGF-2, CD31, PDGFb, and CD144, especially higher in sph-ASC-covered sites. In addition, cytokine arrays of secretome revealed that sph-ASCs and sph-ASC sheets showed augmented angiogenic potential compared to adherent ASCs. Furthermore, four groups of AMI induction only (sham), intramyocardial injection of sph-ASC, ASC sheet transplantation, and sph-ASC sheet transplantation were compared using acute myocardial infarction (AMI) rat models. After 6 weeks of transplantation, echocardiography revealed that significantly improved ejection fraction and fraction shortening in sph-ASC sheet group compared to sham, sph-ASC, and ASC sheet groups. In conclusion, transplantation of sph-ASC sheets showed enhanced neovascularization in AMI animal models. These results indicated that the use of ASC sheets as a novel tissue engineering approach to improve the cardiac and vascular function.
Abstract 143: Surface Modification of Stem Cell Exosomes Myocardial Infarction Specific Peptides for Non-invasive Delivery to Ischemic Myocardium
Authors: Mallaredy Vandana
Published: October 2019

Exosomes are under extensive investigation as a next generation therapeutic agent for clinical applications in various diseases. Stem cell-derived exosomes have been shown to provide post-MI cardiac repair in small and large animals, however, their multi-dose application is limited to single intra-myocardial delivery. In this study, we investigated the heart homing peptide, CRPPR (Cys-Arg-Pro-Pro-Arg) modified mouse Embryonic Stem Cells (mESCs) exosomes as efficient strategy for intravenous delivery of exosomes to ischemic myocardium. In order to target the infarcted heart, we fabricated the exosomes with CRPPR peptide. These peptides are MI-specific peptides, identified by phage display in earlier studies. For this study, we constructed a plasmid expressing fusion protein consisting of Green Fluorescent Protein (GFP), CRPPR and a truncated lactadherin that gets expressed on the surface of exosomes. The plasmid was transfected into the mESCs, followed by puromycin selection. The modified CRPPR exosomes released from the cells were collected by ultracentrifugation and displayed intense GFP- fluorescence indicating efficient CRPPR tagging of exosomes. The CRPPR exosomes were characterized by Nanosight, Electron microscopy and western blotting. MI was performed by LAD ligation and GFP-CRPPR tagged exosomes were intravenously (i.v) injected at a dose of 10
10
particles per mouse and their tissue distribution was evaluated. Interestingly, unmodified exosomes were rapidly cleared from the circulation within 1 hr after i.v injection in mice whereas exosomes modified with CRPPR were still detectable. At tissue level, there was an increased accumulation of the CRPPR exosomes in the heart compared to the unmodified exosomes. In conclusion, we report developing heart homing exosomes that may efficiently delivers the therapeutic payload of embryonic stem cells to the ischemic region of the heart.
Abstract 153: Dexamethasone Inhibits Regeneration and Causes Ventricular Aneurysm in the Neonatal Porcine Heart After Myocardial Infarction
Authors: Ye Lei , Tao Zhonghao Tao , Loo Sze Jie , Su Liping , Tan Shihua , Abdurrachim Desiree , Lalic Janise , Lee Teck Hock , Tan Rusan , Cook Stuart Alexander
Published: October 2019

Background:
The adult mammalian heart has limited ability to repair itself following injury. Zebrafish, newts and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. More recently, we demonstrated that the hearts of neonatal pigs (2-day old) have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth. The current study aims to determine whether acute inflammation is required for CM regeneration after myocardial infarction (MI).

Methods:
We examined the role of acute inflammation on regenerative capacity of the neonatal pig heart (ages: 2 days postnatal) by intramuscular injection of dexamethasone (Dex) during the first week post-MI surgery. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance (CMR) imaging. Bromodeoxyuridine pulse-chase labeling, histology, and immunohistochemistry were performed to study CM cytokinesis and to quantify myocardial fibrosis.

Results:
After MI, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day old pigs. In contrast, piglets received Dex injection developed ventricular aneurysm and did not recover function. Dex limited CM cytokinesis via inhibiting aurora-B protein expression and caused mature scar and thinned walls at infarct site.

Conclusions:
Dex suppresses acute inflammation, inhibits CM regeneration, and causes ventricular aneurysm in neonatal pig hearts after MI.
Abstract 167: Increased Mitophagy and Lysophagy in High Fat Diet and Streptozotocin Induced Diabetic Cardiomyopathy
Authors: Patel Joy R , Kobayashi Satoru , Liang Qiangrong
Published: October 2019

Heart disease and diabetes are among the leading causes of morbidity and mortality. Diabetes can result in diabetic cardiomyopathy, which further increases the risk of heart failure and mortality in diabetic patients. Diabetic cardiac injury has been associated with altered mitochondrial quality control mechanisms including mitophagy, a selective autophagy in which dysfunctional mitochondria are degraded through the lysosome. Lysophagy is the elimination of injured lysosomes by healthy lysosomes which is extremely important for all forms of autophagy. In the present study, we examined the functional status of mitophagy-lysosome system in the diabetic mouse heart. Cardiac mitophagy and lysophagy were determined by using novel dual fluorescent reporter mice, namely, mt-rosella that express mitochondria-targeted RFP-GFP fusion protein and tfgal3 that express RFP-GFP-galectin3, a galactoside-binding lectin. Mice were fed a high fat diet (60%calories by fat) for 1 month and then injected i.p. with streptozotocin (30 mg/kg/d for 3 days). Mice continued on HFD for additional 3 months. Echocardiographic data showed impaired cardiac function in the diabetic mice. Merged confocal images demonstrated that diabetes increased mitophagy flux in the heart as indicated by the red puncta from mt-Rosella and the mitochondrial LC3-II levels with and without lysosomal protease inhibitors pepstatin A and E64d. In addition, diabetes triggered lysosomal injury in the heart as shown by the increased total number of puncta (yellow plus red) from tfgal3 reporter on the merged confocal images. This was associated with accelerated lysophagy flux as shown by the red puncta, suggesting that the injured lysosomes were being actively degraded in the diabetic heart. The increased lysophagy may have contributed to the increased mitophagy flux since the enhanced elimination of injured lysosomes would be expected to help maintain a pool of healthy lysosomes which is essential for the efficient execution of all forms of autophagy. In summary, our results showed an enhanced mitophagy-lysosome system in the diabetic heart which may be an adaptive response that serves to limit diabetic cardiac injury.
Abstract 168: Remote Ischemic Preconditioning Rescues Cardiac Dysfunction in Atg5 Knockdown Mice
Authors: Wang Fangfei , He Quan , Redington Andrew N
Published: October 2019

Objective:
Remote ischemic pre-conditioning (RIPC) is highly cardioprotective in models of ischemia-reperfusion (IR) injury but has not been tested in genetically modified mice. This study was design to test whether RIPC rescues cardiomyopathy induced by knockdown of autophagy-related 5 protein (Atg5).

Approach and Results:
Atg5 knockdown was induced by feeding tamoxifen diet for two weeks in Atg5
flox/flox
Cre
+/-
(KD) mice which were created by crossing Atg5
flox/flox
and heterozygous cardiac-specific Cre recombinase transgenic (Cre
+/-
) mice. Atg5
flox/flox
Cre
-/-
mice served as controls (CONT). Cardiac function was evaluated by echocardiography 28 days after tamoxifen induction. Autophagy proteins were determined by Western blot analysis. RIPC was induced by four cycles of five minutes of hind limb ischemia with tourniquet followed by five minutes reperfusion. Tamoxifen feeding knocked down Atg5 to 32% (CONT 0.91 ± 0.09 vs KD 0.29 ± 0.01, p < 0.01, n = 7), leading to 96% decrease of autophagy activity indicated by LC3-II/LC3-I ratio (CONT 0.96 ± 0.07 vs 0.06 ± 0.03, p < 0.01, n = 7). This was associated with significant cardiac dysfunction (LV ejection fraction, CONT 59.78% ± 3.26 vs KD 47.13% ± 2.50, n = 8 and 10, p < 0.01) and increased heart weight to body weight ratio (CONT 0.50 ± 0.02 vs KD 0.57 ± 0.01, n = 8 and 10, p < 0.01). RIPC increased levels of Atg5 in Atg5 KD mice by 183% (Sham 1.00 ± 0.16 vs RIPC 1.83 ± 0.14, p < 0.01, n = 11 and 13), leading to 148% increase of autophagy activity (Sham 0.99 ± 0.13 vs RIPC 1.47 ± 0.18, p < 0.05, n = 6 and 7), and resulted in rescued cardiac function in terms of heart weight (CONT 0.50 ± 0.01 vs KD 0.53 ± 0.10, n = 11 and 13 ) and LV ejection fraction (CONT 55.64% ± 2.45 vs 52.48% ±1.61, n = 11 and 13). RIPC also suppressed mTOR phosphorylation by 62% compared with sham procedure in Atg5 KD mice (Sham 1.00 ± 0.18 vs RIPC 0.45 ± 0.08 p < 0.01 n = 11 and 13) which was increased by 650% in Atg5 KD compared with CONT mice (CONT 1.23 ± 0.14 vs KD 6.35 ± 0.09, p < 0.01, n = 7).

Conclusion:
We conclude that RIPC rescues Atg5 knockdown-induced cardiomyopathy. Our data adds to the emerging data suggesting benefits of RIPC beyond acute cardioprotection in IR injury.
Abstract 197: Transcription Factor Interactome in Human iPS-derived Cardiac Progenitors is Enriched for Proteins Associated With Congenital Heart Disease
Authors: Gonzalez Teran Barbara , Pittman Maureen , Richmond-Buccola Desmond , Samse Kaitlen , Cole Bonnie , Huttenhain Ruth , McGregor Michael , Krogan Nevan , Pollard Katherine , Srivastava Deepak
Published: October 2019

Congenital heart disease (CHD) affects ~1% of live births and remains the leading cause of mortality in infants. While large-scale genetic studies have uncovered genes associated with CHD, distinguishing variants that confer risk from the background noise of inconsequential variants remains a challenge. Causative mutations in transcription factors (TF) essential for cardiovascular development, such as NKX2-5, GATA4 and TBX5, have been identified in familial cases of CHD, however they are rare. To expand our understanding of their molecular function and to test whether their interacting proteins may be enriched for variants associated with CHD. We defined the protein-protein interaction (PPI) network of NKX2-5, GATA4 and TBX5 using unbiased mass spectrometry in human iPSC-derived cardiac progenitors (iPS-CPs). This approach yielded a network of 172 proteins. An interdependent gene-regulatory role has been reported at the DNA-binding level for these 3 TF during cardiac development, and we also found interdependent protein interactomes where loss of one TF affected the interactome of the others. Interactomes for each were enriched in proteins involved in similar biological processes, such as chromatin remodeling and gene regulation, or previously unrelated processes such as splicing and mRNA transport. Integration of the iPS-CP-PPI network with the CHD-associated damaging variants found in the
Pediatric Cardiac Genomics Consortium
whole-exome sequencing cohort revealed statistically significant enrichment in the GATA4 interactome for de novo missense variants. In contrast, neither the TBX5 or NKX2-5 PPIs were enriched for either de novo missense or rare damaging variants. Finally, we developed a framework to rank PPIs with reported damaging variants for functional validation studies. Overall, this work identified novel protein interactors of TFs essential for cardiac development, offering new insights regarding their regulatory roles and the mechanisms through which they may cause CHD.
Abstract 200: Targeting the Highly Abundant Circular RNA, circHeart, in Cardiomyocytes Attenuates Pressure Overload Induced Hypertrophy
Authors: Lim Tingsen B
Published: October 2019

Objectives:
Circular RNAs (circRNAs) are single-stranded non-coding RNAs that form a covalent closed looped structure and are generated during gene transcription. In our genome-wide analysis of cardiac circRNA expression in both human and mouse hearts we identified a highly abundant circRNA, named here as
circHeart
and hypothesize an important role for
circHeart
in cardiomyocyte physiology and homeostasis.

Materials and method:
We undertook FISH to visualize the localization of
circHeart
. Online tools have predicted multiple potential miRNA binding sites on
circHeart
, including the cardiac-enriched microRNA-133a (miR-133a). We assessed the interactions of
circHeart
with these miRNAs by luciferase and pull-down assays and performed AAV9-mediated RNAi knockdown and overexpression of
circHeart
in mice
in vivo
.
In vivo
phenotype was tracked by echocardiography, electrocardiogram and histological studies. Transverse aortic constriction (TAC) was also performed.

Results:
FISH imaging showed
circHeart
localized largely to the cardiomyocyte cytosol. Luciferase and reciprocal pull-down assays suggest
circHeart
can interact with miR-133a. MiR-133a is a well-known regulator of cardiac hypertrophy and antagomir-133a treatment has been shown to induce cardiac hypertrophy
in vivo
. In our studies, while AAV9 RNAi-mediated knockdown of
circHeart
has no apparent baseline effect in the heart it suppresses pathological cardiac hypertrophy post-TAC. More strikingly, AAV9-mediated overexpression of
circHeart in vivo
resulted in mouse mortality between 3-4 days post-transduction and surviving mice showed evidence of dilated cardiomyopathy. Known targets of miR-133a were concordantly modulated in the knockdown and overexpression mouse model.

Conclusion:
CircRNAs have huge potential role as therapeutic targets and/or disease biomarkers and represent a new avenue for cardiovascular research. Taken together, our data are consistent with a mechanistic model of
circHeart
sequestering miR-133a. Inhibiting
circHeart
abundance presents a potential target for treating cardiac hypertrophy.
Abstract 202: The R21C Mutation in Troponin I Has a Founder Effect in South Lebanon and Causes Malignant Hypertrophic Cardiomyopathy
Authors: Fahed Akl , Nemer Georges , Bitar Fadi , Arnaout Samir , Abche Antoine , Ware James , Batrawi Manal , Khalil Athar , DePalma Steven , McDonough Barbara , Arabi Mariam , Seidman Jonathan , Seidman Christine
Published: October 2019

Hypertrophic Cardiomyopathy (HCM) occurs in 1 of every 500 people and has a wide phenotypic variability. In the majority of cases, HCM is caused by known mutations in genes that code for sarcomere proteins. Although gene testing is widely available for HCM, knowing the phenotype caused by different gene mutations remains a challenging task. We recruited 28 families with HCM, of which 19 (67.8%) have at least one patient with pediatric onset. Index patients from 20 families received targeted sequencing for a panel of genes including
TNNI3
, and 7 families received Sanger sequencing for the
TNNI3
. We identified a missense mutation p.R21C in
TNNI3
segregating with HCM in four families from South Lebanon. Through cascade screening, we identified 30 patients from the four families; twenty of them (67%) had a clinical diagnosis of HCM with a median age of 37 years, while 9 (30%), with a median age 21 years, had no evidence of HCM on echocardiography. An additional 27 members of the families had evidence of HCM, including 22 with SCD in the setting of no past medical history, and their carrier status for p.R21C was implied from the pedigrees. Survival analysis for 57 HCM patients with the mutation revealed a markedly decreased age at first adverse event as compared to 47 HCM patients with the
MYBPC3
p.R502W mutation. Founder mutations in HCM that cause a severe phenotype are uncommon. The p.R21C mutation in
TNNI3
is the first HCM mutation described in the Lebanese population and has a founder effect in South Lebanon. Early and more frequent screening with different imaging modalities as well as tailored management might be warranted for carriers of this mutation.
Abstract 208: Reliable Biomimetic Culture System for Pig and Human Heart Slices
Authors: Abouleisa Riham , Ou Qinghui , Jacobson Zoë , Tang Xian-Liang , Hindi Sajedah M , Kumar Ashok , Ivey Kathryn N , Giridharan Guruprasad , Al-Baz Ayman , Brittian Kenneth , Rood Benjamin , Hill Bradford G , Jones Steven P , Bolli Roberto , Mohamed Tamer M
Published: October 2019

Rationale:
Heart failure is the number one killer and drug induced cardiotoxicity is a major cause of market withdrawal. The lack of availability of culture systems for human heart tissue that is functionally and structurally viable for more than 24 hours is a limiting factor in validation of novel heart failure therapies as well as reliable cardiotoxicity testing. Therefore, there is an urgent need to develop a reliable system for culturing human heart tissue for testing drug efficacy and toxicity.

Objective:
To develop a reliable method to culture pig and human heart slices under full physiological conditions for a period of time sufficient to test therapeutic efficacy and acute drug toxicity.

Methods and Results:
Here we describe a novel biomimetic culture system that maintains full viability and functionality of human and pig heart slices (300 μm thickness) for 6 days in culture through optimization of the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices.

Conclusions:
We have developed and optimized a reliable and easily reproducible culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of cardiotoxicity in a 3D heart model.
Abstract 209: iPSC Derived Cardiomyocytes Reproduce Divergent Phenotypes Caused by a LQTS Type-1 Likely Pathogenic Mutation
Authors: Campos De Carvalho Antonio C , Santos Danubia S , Brunswick Tais H , Ferreira Raphaela P , Araujo Daiana S , Farjun Bruna , Leitão Isabela C , Dias Glauber M , Coutinho Jorge , Vaz Isadora M , Brogonovo Tamara , Cruz Fernando E , Carvalho Adriana B
Published: October 2019

The purpose of this work was to investigate if iPSC derived cardiomyocytes would reproduce the divergent phenotypes observed in a proband and mother that carried the same likely-pathogenic variant in KCNQ1 gene.

A 15 years old proband had syncope and cardiac arrest while swimming being diagnosed with LQTS based on a 12 lead ECG. The mother’s ECG showed no abnormalities. Genomic DNA from blood of daughter and mother was extracted using standard procedures. All coding exons and flanking intronic sequences of KCNQ1 (NM_000218) genes were amplified by polymerase chain reaction. Direct sequencing was performed on a 3500XL Genetic Analyzer. Data was analyzed with Geneious software for identification of mutations. For iPSC generation CD71+ CD36+ erythroblasts were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit on irradiated mouse embryonic fibroblast feeder layers. Differentiation into cardiomyocytes was accomplished using the protocol described by Lian et al. (2012). Electrophysiological recordings were obtained from cardiomyocytes after 30–45 days of differentiation. Cells were transferred to acrylic plates pretreated with Matrigel and kept in culture for 3 days before microelectrode action potential (AP) were registered.

The ECG recordings showed a corrected QT interval (QTc) of 516 ms for the daughter and 436 ms for the mother. Sequencing showed that both daughter and mother had a c.1763C>T mutation resulting in p.Ile588Thr substitution. iPSC generated from daughter, mother and an unrelated control displayed normal karyotype and expressed pluripotent genes, besides spontaneously differentiating into cells of the three germ layers. Differentiation into cardiomyocytes was ascertained by immunofluorescence and flow cytometry for troponin T. In all differentiations more than 70% of cells were cardiomyocytes. Action potentials recordings showed that ventricular cardiomyocytes from the daughter exhibited significantly longer action potential durations at 90% repolarization than the mother’s or the unrelated control’s cardiomyocytes.

Cardiomyocytes derived from iPS cells reproduced the clinical phenotypes exhibited by daughter and mother.
Abstract 213: Single-Cell RNA Sequencing Reveals Pathways Dysregulation by a NFATc1 Mutation in Patient-Specific Cardiomyocytes Derived From Inducible Pluripotent Stem Cells
Authors: Torres Natalia S , Wang Karissa , Coca Enrique , Carey Andrew , Maguire Colin , Cho Scott , Tristani-Firouzi Martin
Published: October 2019

Using a family-based approach with whole exome sequencing (WES) and custom bioinformatics tools we identified a novel mutation (M527L) in the Nuclear Factor of Activated T-Cells 1 gene (NFATc1). This nonsynonymous heterozygous substitution segregates in an autosomal dominant pattern within a family with a young onset Atrial Fibrillation (AF) phenotype. Patient specific inducible pluripotent stem cell derived-cardiomyocytes (iPSC-CM) from a patient carrying this variant showed abbreviated repolarization resulting in a shorter action potential duration compared to iPSC-CM from a healthy sibling control. Unlike familial monogenic AF where the mutation occurs in an ion channel gene, NFATc1 is a transcription factor that influences expression of multiple genes. Using single-cell RNA sequencing on 30 day post-differentiation iPSC-CM, we identified four subpopulations by K-mean clustering (k=4) of single cell transcriptomes and subsequent marker expression. We then performed gene-annotation enrichment analysis of differentially expressed genes specifically in the mutant (MT) and wild-type (WT) cardiomyocyte (CM) populations, detecting several key functional and pathological cardiac pathways affected in response to the NFATc1 mutation. These included Wnt, NF-kappaB and MAPK signaling, Ca homeostasis and gene categories that have been associated with atrial fibrillation. These results suggest that NFATc1 mutation may play an important role in the pathophysiology of familial atrial fibrillation by its broad impact on gene expression and subsequent pathway regulation.
Abstract 236: Biphasic Reprogramming of Ventricular Cardiomyocytes to Pacemaker Cells by Tbx18
Authors: Fan Jinqi , Fernandez Natasha , Cho Hee Cheol
Published: October 2019

Background:
We have previously demonstrated that an embryonic transcription factor, TBX18, suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells. Here, we sought to gain finer understanding of the reprogramming process by investigating genotype/phenotype of the de novo pacemaker cells in multiple temporal domains.

Methods:
TBX18-induced pacemaker cells (TBX18-iPMs) were created in vitro by somatic gene transfer of Adeno-TBX18 into neonatal rat ventricular myocytes (NRVMs). Control ventricular myocytes were transduced with Adeno- GFP. To investigate in vivo reprogramming, we delivered AAV9-TBX18 via tail vein of Hcn4(+/eGFP) heterozygous transgenic mice, which expresses eGFP under the Hcn4 promoter, so as to lineage-trace de novo pacemaker cells in the ventricular myocardium.

Results:
The automaticity of TBX18-iPMs was higher than that of GFP-NRVMs during D1-D3, decreased after D3 and rebound by D14. Monolayers of the iPMs revealed multiple foci of spontaneous field potentials with lower spike amplitude, spike slope, and conduction velocity, demonstrating phenotypic reprogramming to the iPMs by D14. RNAseq and qPCR revealed two distinct phases of reprogramming; an early phase that showed loss of expression of ventricular myocyte-related gene program and a late phase that showed gain of expression of nodal gene program which plateaued by D14. For example, Hcn4 gene expression began increasing during week 1 and continued the pattern during week 2. By week 3, Hcn4-positive TBX18-iPMs appeared in clusters of NRVM monolayers while GFP-NRVMs were spread evenly throughout the monolayers. We lineage-traced de novo, TBX18-reprogrammed pacemaker cells in vivo by delivering AAV9-TBX18 via tail-vein of Hcn4(+/eGFP) mice. In line with our in vitro data, in vivo pacemaker cell lineage tracing experiments indicated a progressive loss of ventricular gene program followed by gain of nodal gene program.

Conclusions:
Our data indicate that TBX18-induced reprogramming of ventricular myocytes to nodal pacemaker cells undergoes progressive changes in gene expression shedding ventricular gene/phenotype first, and then gaining nodal pacemaker genotype/phenotype, establishing fully functional nodal pacemaker by D14.
Abstract 247: Phagocytosis-mediated Activation of Smad3 in Macrophages Mediates Anti-inflammatory Transition and Protects From Adverse Post-infarction Remodeling
Authors: Chen Bijun , Huang Shuaibo , Su Ya , Graff Jonathan , Frangogiannis Nikolaos G
Published: October 2019

Macrophages are essential regulators in tissue injury, repair and fibrosis. Although activation of TGF-β signaling cascades critically modulates macrophage phenotype in vitro, the in vivo role of macrophage TGF-β signaling in homeostasis and disease is unknown. Our study examined the role of macrophage-specific TGF-β/Smad3 signaling in a mouse model of myocardial infarction (MI). TGF-βs markedly activated Smad3 in isolated macrophages, without affecting Smad-independent pathways. Infarct macrophages, but not neutrophils exhibited Smad3 activation. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-β1 release. Myeloid cell-specific Smad3 knockout mice (MyS3KO) had no baseline defects, but exhibited increased late mortality and accentuated remodeling following MI. Adverse outcome in MyS3KO mice after MI was associated with perturbations in phagocytic activity, with defective transition of macrophages to an anti-inflammatory phenotype and with scar expansion. Milk-fat globule EGF factor-8 (Mfge8) partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Perturbed anti-inflammatory transition of Smad3 null macrophages was associated with marked attenuation of phagocytosis-mediated PPAR induction. We demonstrate that Smad3 is activated in phagocytic macrophages, critically regulates their function and mediates anti-inflammatory transition, and protects the infarcted heart from adverse remodeling.
Abstract 249: ERBB2 and beta-1-Adrenergic Receptor Cross-talk Underlies Cardiac Dysfunction
Authors: Gupta Manveen K , Stenson Kate , Gabrielson Kathleen , Naga Prasad Sathyamangla V
Published: October 2019

ERBB2 is a member of the EGFR growth factor receptor family and a primary target for anti-cancer treatment in patients diagnosed with breast cancer. ERBB2 targeting is known to cause cardiotoxicity, resulting in cardiac dilation while, less is understood about the underlying mechanisms. Studies have shown a direct co-relationship between beta2-adrenergic receptors (β2ARs) and ERBB2 in the cardiac hypertrophy wherein increased ERBB2 expression is associated with elevated β2ARs. Given the key role of β1ARs in cardiac function, we assessed potential cross-talk between ERBB2 and β1ARs. Towards this end, we have used a transgenic mouse model of cardiomyocyte-specific overexpression of microRNA-7 (miR-7 Tg) that abrogates ERBB2 expression in the adult cardiomyocytes and associated with cardiac dilation. Radio-ligand binding in miR-7 Tg hearts showed selective increase in β1AR density with no changes in the β2ARs. In contrast, cardiomyocyte-specific overexpression of ERBB2 results cardiac hypertrophy and is associated with significant increase in β2AR density with no changes in β1ARs. These observations show that ERBB2 expression may differentially regulate β1AR versus β2AR providing a unique role for ERBB2 in cardiac physiology. Further, ERBB2 expression is elevated in an age-dependent manner in C57BL6 mice that may account for hypertrophic response. Consistently, miR-7 Tg mice have age-dependent cardiac dilation consistent with the loss in the ERBB2 expression. To test for such a role, C57BL6 mice were administered vehicle, AG1875 (EGFR inhibitor) or AG825 (ERBB2 inhibitor) for two weeks to assess for changes in cardiac function. Administration of AG825 resulted significant cardiac dysfunction and dilation compared to AG1875 or vehicle controls, conferring the idea that inhibition of ERBB2 and not EGFR that mediates deleterious remodeling. Proteomic analysis showed dramatic differences in the associated proteins following AG825 compared to vehicle or AG1478 and our presentation will elucidate the results on how homeostatic ERBB2 signaling and expression is key for cardiac function and how chemotherapeutic inhibition of ERBB2 underlies deleterious pathways leading to cardiotoxicity.
Abstract 252: Activated Fibroblast-specific Deletion of RhoA Reduces Cardiac Fibrosis Through Regulation of the Non-canonical p38-MAPK Signaling Pathway
Authors: Kaikkonen Leena A , Sätzler Valerie , Roy Ashbeel , Contreras Mauricio , Xu Bing , Stroll Stefanie , McCarthy Jason , Molkentin Jeffery D , Das Saumya , Kontaridis Maria
Published: October 2019

Objective:
Heart failure is a progressive disease characterized by cardiomyocyte (CM) loss, interstitial fibrosis, loss of ventricular compliance and chamber remodeling. Role of cardiac fibroblasts is implicated in the process, but specific molecular mechanisms regulating their function remain unclear. Previously, we showed that in mice, in response to stress, CM-specific deletion of RhoA, a Ras-related small G protein, accelerated cardiac dilation, with significant loss of contractile function, but it also significantly reduced cardiac fibrosis. Therefore, here, we focused on understanding the role of RhoA specifically in myofibroblast transformation and the fibrotic response.

Results:
We generated mice with activated fibroblast-specific deletion of RhoA using a tamoxifen (TMX) inducible periostin-cre promoter (RhoAfl/fl::PSTN Cre/+). 10-wk-old male RhoAfl/fl::PSTN Cre/+ or control mice were treated with angiotensin-phenylephrine (AngPE) or saline for 4 wk using osmotic minipumps and fed either with TMX or regular diet. Following TMX-AngPE treatment, the RhoAfl/fl::PSTN Cre/+ mice showed significantly decreased cardiac fibrosis, as compared to controls (0.9% vs. 3.2%, respectively). Moreover, while overall cardiac function following AngPE treatment was similar at 4 wk in all groups, hearts from the TMX-RhoAfl/fl::PSTN Cre/+ mice had significantly reduced septal thickness, as compared to controls. Expression of fetal genes, ANF, BNP, and β-MHC, as well as markers of fibrosis, including α-SMA and CTGF, were similarly induced in control and TMX-RhoAfl/fl::PSTN Cre/+ mice. However, TMX-RhoAfl/fl::PSTN Cre/+ mice had reduced expression of TGF-β1, thrombospondin-5 and Col1a2. Finally, RhoA deletion in activated fibroblasts did not affect the canonical TGFβ signaling pathway, as expected; instead, we found TMX-RhoAfl/fl::PSTN Cre/+ hearts had reduced non-canonical p38-MAPK signaling, suggestive of cellular-specific effects of RhoA regulation in the onset of cardiac disease and fibrosis.

Conclusions:
These data confirm the primacy of the RhoA pathway in the fibrotic response in vivo and identify potential novel downstream targets and possible therapeutic strategies for treatment of cardiac fibrosis and heart failure.
Abstract 257: Cortical Bone Stem Cell-Derived Exosomes Alter Wound Healing Response in Cardiac Fibroblasts and Cardiac Endothelial Cells
Authors: Schena Giana , Kubo Hajime , Yang Yijun , Feldsott Eric , Borghetti Giulia , Eaton Deborah , Johnson Jaslyn , Berretta Remus , Mohsin Sadia , Houser Steven
Published: October 2019

Rationale:
Post-myocardial infarction (MI) results in remodeling that leads to the development of heart failure. We have previously seen improvements in post-MI wound healing and scar formation as a result of administration of cortical bone stem cell derived exosomes (CBSC exosomes).

Objectives:
We sought to further elucidate the mechanism through which CBSC exosomes improved scar formation and altered wound healing through
in vitro
experimentation. We continued to explore the unique characteristics of CBSC exosomes in order to understand why they have anti-fibrotic effects.

Methods and Results:
We performed migration assays on cardiac endothelial cells and cardiac fibroblasts, and saw alterations in cell migration in both cell types upon treatment with mCBSC CM and mCBSC exosomes. We discovered that migration of CECs was reduced by 20% under the influence of mCBSC CM and mCBSC exosomes, but rescued when exosomes were removed from mCBSC CM. We discovered that the removal of exosomes from mCBSC CM slowed cardiac fibroblast migration by 40%. We saw a decrease in the quantity of pro-fibrotic mRNA in cardiac endothelial cells and cardiac fibroblasts after treatment with mCBSC exosomes. Adult cardiac endothelial cell mRNA levels of VEGFA, Col4A1, and VCAM were reduced nearly two fold from control untreated cell levels following mCBSC exosome treatment. Adult cardiac fibroblast Col1A1, Col3A1, Col4A1, and MMP2 mRNA levels were reduced one-fold from control untreated cell levels following mCBSC exosome treatment.

Conclusions:
Our findings show that the wound healing induced by CBSC treatment post-MI involves a mechanism altering the migration of endothelial cells, sustaining the migratory capabilities of fibroblasts, and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.
Abstract 262: Gene Expression Profiling of Hypertrophic and Failing Cardiomyocytes Identifies New Players Involved in Heart Failure
Authors: Vigil-Garcia Marta , Demkes Charlotte J , Eding Joep E , Versteeg Danielle , de Ruiter Hesther , Gladka Monika M , Harakalova Magdalena , Bossu Alex , Vink Aryan , Asselbergs Folkert W , van Veen Toon , van Rooij Eva
Published: October 2019

Pathological remodeling during cardiac disease is a detrimental response characterized by cardiomyocyte hypertrophy and fibroblast activation, which can ultimately lead to heart failure. Genome wide expression analysis on full heart tissue have been instrumental for the identification of molecular mechanisms at play. However, these data so far were based on signals derived from all cardiac cell types and a more specific view on molecular changes driving cardiomyocyte hypertrophy and failure could aid in the development of therapies aimed at improving maladaptive remodeling. Here, we generated a cardiomyocyte-specific reporter mouse (Myh6-Cre-tdTomato) that we exposed to pressure overload by transverse aortic banding (TAB). Using Fluorescence-activated Cell Sorting (FACS) we collected both hypertrophic (one week after TAB) and failing (eight weeks after TAB) cardiomyocytes. Using these cells, we performed RNA-seq and obtained subsets of genes and pathways differentially regulated and specific for either the hypertrophic or failing stages. Among these upregulated genes we identified known marker genes for cardiomyocyte failure, such as Nppb and Myh7, but also identified genes that so far have not been linked to a failing state. RNA-seq on failing and healthy human heart samples confirmed the increased expression for several of these genes regulated during cardiomyocyte failure and allowed us to show an expressional correlation to NPPB/MYH7. Moreover, we could recapitulate the upregulation of these novel genes in stressed human induced-pluripotent stem cell (iPSC)-derived cardiomyocytes. We discovered that phosphofructokinase-platelet (PFKP) protein, a glycolytic enzyme, is strongly induced in human failing cardiomyocytes. Currently, ongoing studies are focused on defining the functional relevance of the novel failure related genes in stressed iPS-derived cardiomyocytes. Our findings suggest that cardiomyocyte-specific transcriptomic analysis allows for the identification of hypertrophic and failing gene expression profiles and helps to unveil novel genes relevant for heart disease.
Abstract 278: Reduced HtrA2 Protein Levels in Mitochondria but Elevated Levels in Cytosol of Left Ventricular Myocardium of Dogs with Chronic Heart Failure
Authors: Gupta Ramesh C , Singh-Gupta Vinita , Sabbah Hani N
Published: October 2019

Background:
The high-temperature requirement serine peptidase 2 (HtrA2) is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent programmed cell death. When HtrA2 protein is tightly associated with mitochondria (MITO), its protease activity is masked. In contrast, once the protein is released from MITO into the cytosol (CS), its protease activity is exposed. Once active HtrA2 activates caspase-3, a pro-apoptotic enzyme, by degrading the caspase-3 inhibitor XIAP (x-linked inhibitor of apoptosis protein. Recent studies have shown that translocation of HtrA2 protein from the MITO to the CS occurs in mice hearts during experimental myocardial ischemic and reperfusion.

Hypothesis:
In this study we examined if translocation of HtrA2 from MITO to CS occurs in LV myocardium of dogs with chronic stable heart failure (HF).

Methods:
LV tissue from 7 dogs with chronic HF produced by multiple sequential intracoronary micro-embolizations (LV ejection fraction <35%) and 7 normal (NL) dogs was used to prepare crude homogenate (HG) and to isolate MITO and CS fractions. Using primary antibodies, HtrA2 protein was determined in HG, MITO and CS fractions by Western blotting coupled with chemiluminescence. GAPDH was used as an internal loading control for both HG and CS fractions. Porin was used as an internal loading control for MITO fractions. HtrA2 band intensity was normalized to GAPDH for HG and CS and to porin for MITO.

Results:
GAPDH and porin protein levels were unchanged between NL and HF dogs. Compared to NL, HtrA2 protein levels in dogs with HF were unchanged in HG (0.76 ± 0.04 vs. 0.73 ± 0.08), significantly reduced in MITO (0.17 ± 0.02 vs. 0.40 ± 0.05, p<0.05) and significantly increased in CS (0.90 ± 0.07 vs. 0.36 ± 0.05, p<0.05).

Conclusions:
Compared to NL dogs, HtrA2 protein levels are significantly reduced in MITO but increased in cytosol fractions from LV myocardium of dogs with chronic stable HF; a maladaptations that promotes cardiomyocyte apoptosis and progressive LV dysfunction. Inhibiting the translocation of HtrA2 from MITO to CS may have therapeutic potential for the preventing progressive dysfunction of the failing heart.