bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 288: Circular RNA CircFNDC3b Modulates Cardiac Repair After Myocardial Infarction via FUS-1/VEGF-A Axis
Authors: Garikipati Venkata N , Verma Suresh K , Cheng Zhongjian , Liang Dongming , Truongcao May M , Cimini Maria , Wang Chunlin , Benedict Cindy , Ibetti Jessica , Grisanti Laurel , Schumacher Sarah M , Gao Erhe , Rajan Sudarsan , Wilusz Jeremy E , Goukassian David , Houser Steven , Koch Walter J , Kishore Raj
Published: February 2019

Circular RNA (circRNA) is a new addition to the list of growing body of non-coding RNAs.Recent studies highlighted that circRNA are dysregulated in cardiovascular disease. However,knowledge of the role of circRNAs in ischemic cardiac injury is limited. Using global circRNAexpression profiling, we identified several circRNA transcripts that were differentially regulatedpost-MI in mice, including circFNDC3b (derived from 2 and 3 exons of cognate FNDC3b gene)which is significantly down regulated. Cell fractionation experiments revealed that circFNDC3bis highly enriched in endothelial cells of post-MI mice. Notably, we found a circFNDC3b orthologin humans, which was also significantly down regulated in ischemic cardiomyopathy patients.Further, gene profile analysis of circFNDC3b overexpression in cardiac endothelial cellsdemonstrated an increase in angiogenic genes. Among them, vascular endothelial growthfactor-A (VEGF-A) was significantly elevated concomitant with reduced in vitro apoptosis ofcardiomyoblasts and endothelial cells, which also exhibited enhanced tube formation. Forcardiac overexpression of circFNDC3b, we generated AAV9 viral particles and found that in vivoover expression attenuated LV dysfunction post-MI and enhanced neovascularization.Mechanistically, circFNDC3b interacts with its potential target RNA binding protein FUS-1 (fusedin sarcoma) and regulate VEGF signaling, thereby reducing cardiomyocyte apoptosis andenhancing neovascularization and cardiac function post-MI. These results indicatethat circFNDC3b is a novel potential target to prevent cardiac remodeling and highlight theimportance of circRNAs in cardiovascular diseases.
Abstract 293: Oxidative Stress in Obese Mice and Potential Melatonin Cardioprotective Effects
Authors: Rezzani Rita , Favero Gaia , Stacchiotti Alessandra , Bonomini Francesca , Rodella Luigi F
Published: February 2019

Adult obesity is a worldwide pandemic condition that leads to the metabolic syndrome and a spectrum of irreversible cardiovascular, neurological, renal and digestive morbidities and cardiovascular diseases represent the leading cause of death in obese people. Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondria and sarcoplasmic reticulum necessary for calcium flux and contraction. Melatonin, versatile and pleiotropic indoleamine with multiple actions, has also important cardioprotective properties. The present study defined mitochondria cardiomyocyte changes and relative oxidative stress pattern in
ob/ob
mice treated orally or not with melatonin at 100 mg/kg/day for 8 weeks. We observed that
ob/ob
mice mitochondria in cardiomyocytes sub-sarcolemmal and inter-myofibrillar compartments are often devoid of cristae and have an abnormal large size, so they are defined mega-mitochondria. Moreover, in
ob/ob
mice, we observed also alteration of peroxisome proliferator-activated receptor gamma coactivator-1α, protein that controls mitochondrial biogenesis, oxidative metabolism and inflammation, together with a deregulated endogenous antioxidant system, showing in particular altered heme oxygenase-1 cardyomiocyte expression. Melatonin oral treatment in
ob/ob
mice restores heart mitochondria and minimizes oxidative stress and inflammation. These preliminary data indicate a significant protective role of melatonin supplementation against obesity-related heart alterations.
Abstract 297: Old Drug New Tricks: Repurposing Auranofin for Acute Myocardial Infarction by Targeting Protein Tyrosine Phosphatase PTP-PEST
Authors: Yang Chiu-Fen , Chen Yi-Yun , Liu Yu-Wen , Singh Jai P , Chang Chia-Wei , Cheng Ching-Feng , Meng Tzu-Ching
Published: February 2019

Rationale:
Myocardial ischemia/reperfusion (I/R) injury is almost unavoidable since reperfusion is the only established treatment for acute myocardial infarction (AMI). To date there has no effective strategies available for reducing I/R injury.

Objective:
To elucidate the mechanisms underlying myocardial I/R injury and to develop new strategies for attenuating the damage it causes.

Methods and Results:
Using I/R-mice model created by ligation of left anterior descending (LAD) artery, we found increases in activity of protein tyrosine phosphatases (PTPs) in myocardium during I/R. Treating the I/R-mice with a pan-PTP inhibitor PVS (phenyl vinyl sulfone) attenuated I/R damage, suggesting PTPs activation to be harmful in I/R. Through analyzing myocardial RNAseq data, we showed PTPs that are abundantly expressed in the myocardium. By exposing cardiomyocytes ablated with specific endogenous PTPs to hypoxia/reoxygenation (H/R), we found a role that PTP-PEST (PTPn12) plays to promote cell death under H/R stress. Auranofin, a drug being used in clinical practice for treating rheumatoid arthritis, may target PTP-PEST thus suppressing its activity. We recapitulated the function of auranofin as a PTP-PEST inhibitor by
in vitro
studies, and then examined its effect on myocardial I/R injury. In the mice receiving auranofin before reperfusion, myocardial PTP activity was suppressed, leading to restored phosphorylation of PTP-PEST substrates, including ErbB-2 that maintains survival signaling of the heart. In line with the suppression of PTP-PEST activity, the auranofin-treated I/R-mice had smaller infarct size and better cardiac function.

Conclusions:
PTP-PEST contributes to part of the damages resulting from myocardial I/R. The PTP inhibitor auranofin, acting through the PTP-PEST-ErbB-2 signaling axis, reduces myocardial I/R injury. Based on the finding, auranofin could potentially be used in the development of treatments to manage I/R injury in patients with AMI.
Abstract 298: Irisin is an Endogenous Negative Regulator of Pathological Cardiac Hypertrophy
Authors: Zhao Yifan , Xu Yawei , Peng Wenhui
Published: February 2019

Cardiac hypertrophy is a common pathophysiological process in various cardiovascular disease which still has no effective therapies. Irisin is a novel myokine mainly secreted by skeletal muscle which is involved in the regulation of energy metabolism. However, its role in cardiac hypertrophy is still unclear. In the present study, we found that serum irisin concentration is significantly elevated in patients with hypertension. Moreover, irisin concentration has strong correlation with left ventricular posterior wall thickness(LVPWT) and intraventricular septal thickness (IVST) which are two major parameters to measure left ventricular hypertrophy. The above results suggested that irisin may be involved in the regulation of pressure overload-induced cardiac hypertrophy. Hence, we further generated transverse aortic constriction-induced cardiac hypertrophy murine model. We found that irisin expression in heart and serum were both elevated in hypertrophic murine hearts. The in vitro study also indicated the angiotensin II treated cardiomyocytes have a higher level of irisin concentration. These data indicated that cardiomyocyte can secret irisin and the irisin concentration is elevated after hypertrophic stimuli. Furthermore, we demonstrated that cardiac hypertrophy was significantly attenuated with improved cardiac function assessed by echocardiography after irisin administration. HE and PSR staining also showed decreased cardiomyocytes size and fibrosis. Mechanistically, we demonstrated that cardiomyocytes treated with ADAM family inhibitor (TAP-2) has a lower irisin level while the expression of irisin precursor (FNDC5) has no obvious alteration. These data implied that FNDC5 was cleaved into irisin, at least partially, in a ADAM family dependent manner. Further we found that irisin administration activates AMPK and subsequently inhibited activation of mTOR. In conclusion, we demonstrated that cardiomyocytes secret irisin in a ADAM family dependent manner, administration of irisin attenuated pressure overload-induced cardiac hypertrophy and fibrosis, improved cardiac function by AMPK-mTOR signaling.
Abstract 300: MicroRNA-21 Affects Platelets and Their Releasate: A Novel Mechanism for the Anti-Fibrotic Effects of MicroRNA-21 Inhibition
Authors: Barwari Temo , Eminaga Seda , Lu Ruifang , Armstrong Paul C , Schulte Christian , Lynch Marc , Chan Melissa V , Barallobre-Barreiro Javier , Yin Xiaoke , Pechlaner Raimund , Langley Sarah , Sahraei Mahnaz , Fernández-Fuertes Marta , Zampetaki Anna , Santer Peter , Warner Timothy D , Kiechl Stefan , Willeit Johann , Fernández-Hernando Carlos , Suárez Yajaira , Shah Ajay M , Mayr Manuel
Published: February 2019

Introduction:
Fibrosis is a major contributor to cardiac disease. MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis, with inhibitors of miR-21 currently undergoing clinical trials. We aimed to explore how miR-21 inhibition attenuates fibrosis using a proteomics approach.

Methods and Results:
As expected, transfection with miR-21 mimics and inhibitors altered the proliferation of murine cardiac fibroblasts. However, proteomic analysis of their conditioned media revealed limited effects on ECM secretion. Similarly, proteomic analysis of hearts from miR-21
null
mice showed no major difference in extracellular matrix (ECM) composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the community-based Bruneck study (year 2000 follow-up, n=671), we compared circulating miR-21 with a panel of 229 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors correlated with plasma miR-21 levels, including the latency-associated peptide of transforming growth factor beta-1 (TGF-β1). This correlation was confirmed by ELISA measurements of active TGF-β1 in plasma (Bruneck 2015 follow-up, n=332). In agreement with this observation in men, systemic inhibition of miR-21 with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. AntagomiR-21 treatment, however, had no effect on the platelet count or their aggregation response. In contrast, miR-21
null
mice had significantly lower platelet counts compared to littermate controls.

Conclusions:
This study reports a previously unrecognized effect of miR-21 inhibition on platelets. Both genetic deletion of miR-21 and antagomiR treatment affect platelets. The effect of miR-21 inhibition on platelet TGF-β1 release, in particular, may contribute to the anti-fibrotic effects of miR-21 inhibitors.
Abstract 305: AAV9 Mediated Cardiac Bin1 Gene Therapy Attenuates Pressure Overload-induced Heart Failure in Mice
Authors: Zhou Kang , Agvanian Sosse , Liu Yan , Hitzeman Tara , Shaw Robin M , Hong Tingting
Published: February 2019

Rationale:
Pressure overload-induced myocardial hypertrophy can progress to heart failure (HF), along with a decline in myocardial expression of cardiac BIN1 (cardiac isoform of bridging integrator 1, cBin1). T-tubule cBIN1-microdomains are critical in maintaining normal cardiac contractility and electrical stability.

Objective:
To examine whether
cBin1
gene transfer can prevent or postpone HF induced by severe pressure overload in a mouse model with transverse aortic constriction (TAC).

Methods and Results:
Two sets of experiments were designed. In Set 1, TAC was performed in adult male wild type (WT-TAC) and cardiac specific heterozygous
Bin1
deleted mice (
Bin1
HT-TAC). In Set 2, TAC was performed in adult male C57BL/6J mice three weeks after retro-orbital injection of adeno-associated virus 9 transducing cBIN1-V5 or GFP-V5 (cBIN1-TAC or GFP-TAC) and compared to sham controls. The primary endpoint measured was survival at 8 weeks with left ventricular ejection fraction >=35% by echocardiogram. The
Bin1
HT-TAC group (less cBIN1) and the cBIN1-TAC group (more cBIN1) had significantly less and more endpoint achievement than their respective set controls, indicating a robust protective effect of cBIN1. Left ventricular (LV) hypertrophy occurred in all TAC groups, yet cBIN1-TAC mice had attenuated LV dilation by echocardiogram, improved contractile reserve by pressure-volume loop, reduced pulmonary edema and less HF development. Exploring myocardial remodeling, exogenous cBIN1 retained t-tubule membrane microdomains with organized L-type calcium channels (LTCC) and ryanodine receptors (RyRs) in cBIN1-TAC mice, which otherwise were significantly disrupted in GFP-TAC mice. However, the total LTCC and RyRs protein levels were not significantly altered among groups by Western blotting.

Conclusions:
Normalization of cBIN1 level can postpone, if not prevent, the transition from compensated LV hypertrophy to dilated cardiomyopathy following pressure overload. The protective mechanism is linked to cBIN1-mediated preservation of dyads at t-tubule membrane microdomains, limiting pathologic remodeling of the calcium handling apparatus during HF progression.
Abstract 308:
Wls
Inhibits Fibrosis Through Wnt Secretion From the Cardiomyocytes During Heart Regeneration
Authors: Liu Shijie , Monroe Tanner , Hill Matthew , Martin James F
Published: February 2019

Because cardiomyocytes lose their capacity for self-renewal shortly after birth, the heart cannot regenerate muscle lost due to injury or heart failure. Instead, dead myocardium is replaced with a fibrotic scar. Recent studies have demonstrated that blocking the Hippo pathway
specifically in cardiomyocytes
promotes heart functional recovery after injury, not only by stimulating cardiomyocyte proliferation, but also by preventing fibrosis. The mechanism through which YAP activates the cell cycle is widely studied; however, the mechanism through which it inhibits fibrosis is unknown. Here, we identified Wntless (
Wls
) to be a downstream target of YAP in regenerating cardiomyocytes; Wls plays a role in regulating the secretion of Wnt ligands, which influences interstitial cells through paracrine effects. By using Adeno-Associated Virus 9 (AAV9) to deliver guide RNAs (gRNAs) that target the
Wls
locus exclusively in cardiomyocytes that express the Cas9 protein, we generated a cardiomyocyte-specific Wls knock out mouse (Wls-Cas9 CKO). After myocardial infarction at the neonatal stage (regenerative stage), cardiomyocyte deletion of Wls detrimental the heart regeneration as showed decreased heart function and increased fibrosis in Wls-Cas9 CKO mice. Mechanistically, by performing nuclear RNA-Seq technology, we revealed that non-canonical Wnt ligands are enriched in cardiomyocytes suggest cardiomyocytes influence fibroblasts through non-canonical signaling during heart regeneration. We will further use the single-cell sequencing technology to investigate the cell to cell communication during heart regeneration.
Abstract 311:
In vitro
Regulation of G-protein Coupled Receptor Kinase 5 (GRK5) Induced Cardiac Hypertrophy by a Novel Interaction With the Phosphatase PHLPP2
Authors: Yeh Szu-Tsen , Zambrano Cristina , Koch Walter , Purcell Nicole
Published: February 2019

PHLPP is a Ser/Thr phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases, which are important regulators of cell growth, proliferation, and survival. We have previously demonstrated that removal of PHLPP1 increases Akt activity in neonatal rat ventricular myocytes (NRVMs) (Miyamoto et al 2010) as well as the heart (Moc et al 2015) leading to protection from pathological hypertrophy and damage, however the effects of PHLPP2 removal on cardiomyocyte hypertrophic growth are unknown. In NRVMs, we found that removal of PHLPP2 induced hypertrophic growth basally without altering Akt activity and immunoprecipitation experiments revealed a novel interaction with an AGC kinase, G-protein coupled receptor kinase 5 (GRK5). GRK5 acts as an HDAC kinase downstream of phenylephrine (PE) to regulate hypertrophy in NRVMs. Thus, our objective was to determine whether PHLPP2 regulates PE-induced cardiomyocyte hypertrophy through its interaction with GRK5. We found that knockdown of PHLPP2, not PHLPP1, potentiated PE-induced hypertrophy (siPH2+PE (109 ± 3.5μm) vs. siCon+PE (88 ± 1.9μm)), and increased mRNA expression of fetal genes (i.e. ANF and BNP). Conversely, overexpression of PHLPP2 attenuated PE-induced hypertrophic growth (PH2+PE (66.6 ±7.7μm) vs. GFP+PE (95.5 ± 2.7μm)) as well as fetal gene re-expression. This inhibition of hypertrophic growth was dependent on phosphatase activity since a PHLPP2 mutant lacking the phosphatase domain (αPP2C) reversed the effect of PHLPP2 overexpression. To determine whether removal of PHLPP2 alters GRK5 translocation, cells were fractionated, and we found that PHLPP2 removal significantly increased basal and PE-induced nuclear accumulation of GRK5 that was blocked by overexpression of PHLPP2. Finally, knockdown studies with siGRK5 revealed that GRK5 was indeed required for the hypertrophic response elicited by PHLPP2 removal. Overall, our data revealed for the first time that PHLPP2 plays a role in regulating PE-induced cardiac hypertrophy via a GRK5 dependent pathway. Understanding the biological function and signaling pathways altered by PHLPP2 in cardiomyocytes may help delineate therapeutic targets for cardiac hypertrophy.
Abstract 315: Time-resolved Expression of Membrane-targeted Proteins During Ipsc-cardiomyocyte Differentiation
Authors: Lau Edward , Shrestha Rajani , Thomas Cody T , Williams Damon R , Wu Joseph C
Published: February 2019

Background:
The ability to create human cardiomyocytes from induced pluripotent stem cells (iPSCs) has spurred advances in disease modeling and drug testing, but there remains an ongoing need to improve the purity and functional maturity of derived cardiomyocytes. Membrane proteins are an integral component of cell signaling and specification. To better understand iPSC-cardiomyocyte differentiation and identify potential surface markers of cell maturity, we set out to examine how membrane-targeted proteins gradually remodel in differentiating cardiomyocytes.

Methods:
Human iPSCs from three donors were differentiated into cardiomyocytes in 40 independent differentiation plates. We sampled protein expression at independent time points at up to daily intervals during day 0 to day 14 post-differentiation, then sparsely up to 100 days. The extracted proteins were analyzed using a Q-Exactive HF Orbitrap high-resolution mass spectrometer to quantify how the relative quantity of membrane-targeted proteins changes over time in differentiating cells.

Results:
We identified 1095 annotated cell membrane proteins and quantified their temporal changes in abundance across 40 samples. Protein expression in the first two principal components recapitulated gradual differentiation of iPSCs into mesoderm, cardiac progenitors, and more mature cardiomyocytes. Membrane protein expression partitioned the samples into three discernible clusters, representing early (day 0-5), mid (day 6-10), and late (day 11+) differentiation time points. In total, 115 cell membrane proteins showed differential expression in late vs. early differentiation, including two popeye-domain-containing proteins (POPDC2 and BVES) known to be involved in cardiac development. In particular, we identified two proteins, dysferlin (DYSF) and alpha-1 syntrophin (SNTA1), which continued to show strong upward changes in late differentiation time points, nominating these proteins for investigation as markers for mature cells.

Conclusion:
We report the time-resolved expression profiles of over 1000 membrane-targeted proteins during iPSC-cardiomyocyte differentiation. Future work into the membrane proteome may lead to maturity markers to guide maturation efforts.
Abstract 321: Miat Long Non-coding Rna Regulates Cardiac Hypertrophy and Ribosomal Expression in Cardiomyocytes
Authors: Wang He (Iris) , Wang Zhihua , Wang Yifan , Ren Shuxun , Rau Christopher , Wang Yibin
Published: February 2019

Cardiac hypertrophy is characterized by elevated protein synthesis and pathological cardiomyocyte enlargement. The mechanisms underlying the stress response in ribosomal remodeling at organelle level during cardiac hypertrophy is not fully established. We have identified a long non-coding RNA, Myocardial Infarction Associated Transcript (MIAT), that is significantly associated with pathological cardiac hypertrophy through a systems genetic analysis. MIAT expression is induced by hypertrophic signal, and silencing of the MIAT gene significantly blocks myocyte hypertrophy in response to hypertrophic stimuli associated with diminished global protein synthesis activities. Furthermore, MIAT inactivation significantly reduces ribosomal genes at mRNA and protein levels, and reduces protein synthesis activity at basal and post-hypertrophic stimulation
in vitro
and
in vivo
. Using a MIAT knockout mouse model, we investigated the in vivo pathophysiological impact of MIAT in pressure-overload induced cardiac hypertrophy and heart failure. Remarkably, we find that the MIAT knockout mice show significantly blunted cardiac hypertrophy and preserved cardiac function following pressure-overload. This study highlights a novel mechanism of translational regulation by a lncRNA MIAT. The outcome will expand functional role of lncRNAs in heart diseases and advance our current understanding of the complexity of regulatory circuits in cardiac hypertrophy and heart failure. It will provide novel insights to protein synthesis and ribosomal regulation and reveal potential therapeutic targets for pathological hypertrophy.
Abstract 332: Dissecting Mechanisms for Hand2-Dependent Pacemaker Cell Reprogramming
Authors: Fernandez-Perez Antonio , Bhakta MInoti , Munshi Nikhil V
Published: February 2019

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes, in both in vitro and in vivo applications. Recent evidence suggests that the supplementation of GMT with Hand2 (GHMT) can enhance this process. We previously showed that iCLM derived from GHMT are complex and heterogeneous, consisting of atrial (iALMs), ventricular (iVLMs), and pacemaker-like myocytes (iPLMs), thus underscoring the need for a systematic approach for conducting additional studies. Although chromatin and single-cell studies have explored GMT-iCLM cell fate determination in great detail, little is known about how Hand2 enhances iCLM induction, and in particular, iPLM specification. Before direct reprogramming can be used as a therapeutic strategy, the mechanistic underpinnings of specific cardiac lineage conversion must be understood. In the present study, we characterize the molecular mechanisms by which Hand2 regulates iCLM induction and iPLM diversification. We compare the transcriptional and genomic accessibility landscape of GMT and GHMT iCLMs to uncover common and distinct gene regulatory networks that drive iCLM determination. In parallel, using a series of Hand2 deletions, in combination with quantitative mass spectrometry, we identify a Hand2 interactome in reprogrammed cells enriched for iPLMs. Taken together, these complementary approaches have begun to elucidate the molecular details of GHMT-mediated cardiac reprogramming and iPLM specification.
Abstract 333: TNF Receptor Modulation of Progenitor Cells and Exosomes for Myocardial Repair
Authors: Garikipati Venkata N , Cimini Maria , Wang Chunlin , Roy Rajika , Cheng Zhongjian , Truongcao May M , Benedict Cindy , Verma Suresh K , Koch Walter J , Kishore Raj , Goukassian David A
Published: February 2019

Our published studies, using TNFR1 and TNFR2 knockout (KO) mice have demonstrated that negative effects of TNF during ischemic tissue repair including enhanced apoptosis and inflammatory cytokines expression and signaling, is largely mediated by TNFR1/p55. Our hypothesis is that inhibition of TNF-TNFR1 signaling inhibits multiple negative effects of TNF after myocardial ischemia by promoting TNF signaling through protective TNFR2 receptor and thereby augmenting EPC-mediated myocardial angiogenesis and repair and this enhanced protective effect of TNFR1 KO EPCs may involve alteration in the cargo and function of TNFR1-KO EPC derived exosomes.

Protective effect of disrupted TNF-TNFR1/p55 signaling in BM-EPCs under stress conditions in WT, p55KO and p75KO EPCs were tested in tube formation assay under hypoxia conditions and H2O2 treatment. In the absence of TNFR1 (p55KO EPCs) - EC function of BM-EPCs is enhanced under normoxia/hypoxia conditions and survival of BM-EPCs is increased under oxidative stress. To test the effect of TNFR1 and TNFR2 loss in the BM-EPCs for recovery after AMI, WT mice were subjected to AMI and WT, p75KO and p55KO BM-EPCs were injected into the myocardium immediately after AMI. Compared to WT and p75KO, injection of p55KO EPCs into WT hosts led to - increased retention of p55KO EPCs in the WT mice hearts; decreased post-MI apoptosis in WT mice; increased vascular network; significantly improved cardiac function; substantially small infarct size; the last three indicating improved cardiac remodeling by day 21 post-AMI. Further, in vitro exosome studies showed that compared to WT and p75KOs, p55KO BM-EPCs-derived exosomes showed positive activities in vitro, including - enhanced angiogenic function in HUVECs and increased survival of H9C2 cells. These effects were mediated via upregulation of miRNA-191-5p as shown by increased levels of angiogenic miR-191-5p in the exosomal cargo of p55KO EPCs and near complete inhibition of HUVEC angiogenic function in vitro by miR-191-5p-antagomiR.

Our findings suggest that decrease/loss of TNFR1 modulates both the content and function of EPC exosomes and enhance reparative and angiogenic capabilities of EPCs and EPC-mediated vascular and anatomical repair in the MI model.
Abstract 337: Long Term Safety and Efficacy of a Cardiac Graft in a Swine Model of Heart Failure
Authors: Lancaster Jordan , Koevary Jen , Daugherty Sherry , Goldman Steven
Published: February 2019

Introduction:
We have shown efficacy with cardiac grafts in the rat coronary ligation model of chronic heart failure (CHF) and feasibility of implantation and assessment in a swine model. Now we propose testing its long term safety and efficacy in a swine model of CHF. Our aims are: 1) Evaluate long term (6 months) left ventricular functional improvements after implantation of the graft in a swine coronary artery occlusion model of CHF, 2) Evaluate quality of life and functional capacity improvements of swine treated with the graft, and 3) Evaluate long term safety of the graft.

Hypothesis:
An engineered tissue comprised of differentiated cardiac myocytes and fibroblasts will effectively integrate into the host tissue, and restore cardiac function in swine with CHF.

Methods:
Male Yucatan mini swine 30-40kg were infarcted using percutaneous methods. Balloon occlusions (90 minute) are performed via catheter, distal to the first diagonal branch of the left anterior descending (LAD) coronary artery and animals. Four weeks after myocardial infarction (MI) cardiac grafts engineered with bio absorbable knitted mesh are implanted on the infarcted epicardium. Cardiac magnetic resonance imaging (MRI) is performed prior to MI, 1 month after MI, 1 month after patch placement, again at 3 month and 6 months. All swine are implanted with continuous event recorders to acquire surface electrocardiogram during the entire study. In addition, quality of life/functional capacity are assessed using FitBark collar activity monitors and treadmill exertion testing. Infarct size is determined using MRI images and 2,3,5-triphenyltetrazolium chloride staining at study end.

Results:
Medium to large transmural infarcts are generated with 90min balloon occlusion of the LAD which demonstrate compensatory remodeling of the LV as assessed via cardiac MRI. Upscale cardiac matrix grafts to 6cm diameter are easily handled by the surgeon and can be surgically implanted on the epicardium using sub-xiphoid median sternotomy. No adverse arrhythmic activity has been observed.

Conclusion:
Our cardiac graft is well suited for upscale to clinical size, can be easily handled and implanted in a swine model of CHF.
Abstract 340: Cardiomyocyte-Derived Mechanical Signals Function in a Paracrine Manner to Regulate Tissue Stiffness and Myocardial Proliferation
Authors: Zhang Caimei , Emms Sabrina , Radice Glenn
Published: February 2019

Cells sense and transduce mechanical signals through cell-cell adhesions and cell-extracellular matrix adhesions. After birth, contractile force generation and tissue stiffness increase as cardiac output increases to support the needs of the newborn organism. During this postnatal period, the N-cadherin adhesion complex assembles at the intercalated disc (ID) as integrin-fibronectin (FN) adhesions decrease, accompanying the switch from hyperplastic to hypertrophic growth. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of cell cycle withdrawal that occurs in cardiomyocytes after birth.

α-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. Cardiac-specific αE- and αT-catenins double knockout (DKO) mice exhibit sustained cardiomyocyte proliferation beyond the first week of life. To investigate the role of cell-matrix interactions in DKO hearts, the spatial distribution of integrin-FN complexes was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin localization accompanied by altered distribution of α5/β1 integrin, the primary FN receptor. Normally found at the lateral membrane, α5 and β1 accumulated at the ID while N-cadherin was reduced at the termini. FN matrix assembly, as monitored by immunofluorescent staining and its insolubility in the detergent deoxycholate, was increased in DKO hearts. Meanwhile, focal adhesion kinase (FAK) was activated in early postnatal DKO hearts. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated proliferation was dependent on FAK activity. Finally, treatment of DKO pups with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), was sufficient to reduce cardiomyocyte proliferation to wild-type levels suggesting that matrix assembly and tissue stiffness feedback to promote cardiomyocyte proliferation in DKO hearts. This data demonstrates that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls cardiomyocyte proliferation, thus providing a molecular explanation for loss of regenerative potential shortly after birth.
Abstract 343: Lin28 Enhances Cardiac Repair by Reprogramming Metabolism of Cardiac Progenitor Cells
Authors: Tan Ai Wen , Kurian Justin , Harlamova Daria , Wang Tao , Mohsin Sadia , Houser Steven R , Khan Mohsin
Published: February 2019

Rationale:
Cardiac progenitor cells (CPCs) possess incredible repair properties in the neonatal heart but are prone to disease and age diminishing their efficacy. Interestingly, CPCs stemness is linked to a unique metabolic phenotype altered once cells undergo proliferation and commitment. Recent data indicates Lin28 as metabolic regulator of pluripotency, enhancing tissue repair after injury. Nevertheless, there are no studies characterizing the effect of Lin28 on CPC biology or cardiac repair.

Objective:
Determine the effect of Lin28 on CPC function and cardiac repair after injury.

Methods and Results:
Lin28a was highly expressed during embryonic mice heart development and disappeared in adult heart, resulting in a concomitant upregulation of Let-7 expression. Next, we engineered CPCs with Lin28a (CPC-Lin) lentivirus along with green fluorescent protein (GFP) while CPCs expressing GFP (CPC-G) were served as controls. Relative to CPC-G, increased proliferation of CPC-Lin associated with concurrent decrease in apoptosis was observed, as measured by CyQuant and annexin-V based FACS analysis, respectively. We found that CPC-Lin enhanced glycolysis together with increased expression of glycolytic enzymes, as measured by seahorse and qRT-PCR, respectively. Interestingly, CPC-Lin also increased oxidative phosphorylation, as evidenced by the increase in oxygen consumption rate and intracellular ATP production. Higher immunostaining expression of stemness markers (Nanog and Sox2) together with decreased senescence-associated-beta-galactosidase (SA-β-gal) activity was observed in CPC-Lin. To determine in vivo efficacy, CPCs were transplanted to the heart after myocardial infarction (MI). Increased proliferation as indicated by co-localized immunostaining expression of p-histone and GFP was observed in CPC-Lin 2 days after transplantation. Echocardiography analysis revealed that CPC-Lin improved cardiac function by increasing ejection shortening percentage and fractional shortening percentage.

Conclusions:
Metabolic reprogramming of CPCs with Lin28a promotes cardiac repair by modifying metabolism of CPCs, together with enhanced cell proliferation, survival, and in vivo cardiac function after MI.
Abstract 344: Proteomic Analysis Revealing Association of hiPSC-exosomal Proteins in Epigenetic Inheritance
Authors: Tang Yan , Yue Yujia , Garikipati Venkata N , Verma Suresh , Cheng Zhongjian , Cimini Maria , Goukassian David , Wang Chunlin , Cao May , Kishore Raj
Published: February 2019

Background:
Exosomes, as a mediator of cell-to-cell communication has regulatory effects on the genetic and epigenetic processes in the recipient cells. Although the ability of human induced pluripotent stem cells (hiPSCs) derived exosomes to participate in tissue repair is being increasingly recognized, the information inside the hiPSC-exosomes especially protein content is not completely identified yet.

Method:
Exosomes derived from human fibroblast cell line IMR90 and IMR90 derived hiPSC cell line iPS (IMR90)-4 were collected using differential ultracentrifugation, then the exosomal proteins were identified by mass spectrometry analysis. The proteomics results were confirmed by western blot. To validate the role hiPSC-exosome in epigenetic processes, the H9C2 cells were treated with both types of exosomes and the histone epigenetic state were analyzed.

Results:
Gene ontology (GO) analysis of the exosome proteome showed that the hiPSC-exosome protein content had statistically significant enrichment in epigenetic modification such as acetylation, phosphorylation and methylation. The proteomic analysis indentified 6 epigenetic enzymes (UBE2N, DNMT3B, AURKB, CARM1, HDAC2 and HAT1) which only expressed in hiPSC-exosomes whereas 2 enzymes (PRMT5 and PRMT1) showed increased level in hiPSC-exosomes. The differential expressions of HDAC2 and PRMT5 were validated by western blot. Furthermore, H9C2 cell treated with hiPSC-exosomes showed a significant decrease in pan-acetylation level of H4 compared with IMR90-exosomes.

Conclusion:
In a global analysis presented here, hiPSC-exosomal proteins have been found to be associated with mammalian epigenetic inheritance, which provide important information for the exosome-based therapy.
Abstract 345: Aggregation of Child Cardiac Progenitor Cells into Spheres Activates Notch Signaling and Improves Treatment of Right Ventricular Heart Failure
Authors: Trac David , Xu Chunhui , Davis Michael E
Published: February 2019

Congenital heart disease is rarely cured by surgery and can lead to life-threatening, intractable right ventricular heart failure (HF). Recent stem cell-based clinical trials have been limited by poor differentiation rates and low cell retention. We propose the aggregation of child cardiac progenitor cells (CPCs) into scaffold-free spheres to improve the differentiation of CPCs into mature cardiac phenotypes by enhancing intercellular Notch signaling, a key regulator of CPC fate decisions. Child CPC spheres were produced at a size of 1500 cells per sphere using a microwell array and cultured in suspension for up to 14 days with 70-80% cell viability. By 7 days in culture, aggregation of CPCs increased protein expression of Notch1 intracellular domain (3.8-fold) and transcript expression of Notch1 signaling pathway members, including ADAM10 (3.6-fold), DLL1 (25.0-fold), and JAG1 (3.2-fold), compared to parallel monolayer cultures. Increased Notch signaling activity was accompanied by high transcript levels of cardiac differentiation markers, including MEF2C, MYH7, and cTNNI. In particular, Nkx2.5, an important transcription factor for cardiomyocyte development, was robustly expressed within the nuclei of aggregated CPCs. Inhibition of Notch signaling activity using the small molecule γ-secretase inhibitor DAPT abrogated these effects, including the nuclear translocation of Nkx2.5. To evaluate the therapeutic effects of CPC spheres compared to single CPCs, right ventricular HF was induced in rats by pulmonary artery banding and cells were transplanted into the right ventricular free wall. By 28 days, echocardiographic measurements showed significantly improved right ventricular function with CPC sphere transplantation compared to single CPC transplantation (tricuspid annular plane systolic excursion: 2.03±0.2 vs. 1.61±0.22 mm, P<0.001). Additionally, CPC aggregation improved cell retention at 28 days compared to single CPCs as measured using DiR-labeled CPCs (29.2±4.0 vs. 11.1±7.3% of day 0 fluorescence, P<0.005). The results of our project will facilitate the development of autologous stem-cell based therapies for pediatric HF.
Abstract 354: Accelerated Autophagy is Required for Doxorubicin-induced Oxidative Damage and Cardiomyopathy
Authors: Smuder Ashley J , Kwon Oh-Sung , Talbert Erin E , Christou Demetra D , Yoo Jeung-Ki , Hwang Moon-Hyon , Kavazis Andreas N
Published: February 2019

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is limited by the risk of severe cardiotoxicity. In patients, the acute onset of cardiomyopathy often precludes chemotherapy treatment, resulting in reduced quality of life and increased morbidity. While the mechanisms by which DOX promotes cardiac dysfunction are not well understood, our previous findings show that intracellular metabolism of DOX elicits mitochondrial reactive oxygen species (ROS) generation, and subsequent activation of the autophagy/lysosomal system. Equally, evidence suggests that elimination of vital cellular organelles and proteins within the cell as a result of pathologically elevated autophagy may in turn stimulate an increase in ROS generation. Therefore, we hypothesize that DOX-induced autophagy promotes cardiac dysfunction as a result of an autophagy-induced increase in oxidative stress in the heart. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV-dnATG5) to female Sprague-Dawley rats via tail vein administration four weeks prior to either DOX (20 mg/kg ip) or saline treatment. Two days following treatment, assessment of cardiac function showed significant alterations to the myocardial performance index and fractional shortening percentage in control, DOX treated animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and increased appearance of autophagic vacuoles within the heart. In contrast, rAAV-dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 to ATG5 thereby reducing autophagosome formation and attenuating cardiac dysfunction. Interestingly, protein expression of the antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. These findings indicate that accelerated autophagy promotes DOX-induced cardiac dysfunction and is an underlying cause of chemotherapy-induced oxidative damage to the heart.
Abstract 377: Modeling Cadmium-induced Cardiotoxicity Using Human Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Shen Jiaxi , Wang Xiaochen , Zhou Danni , Li Tongyu , Tang Ling , Gong Tingyu , Su Jun , Liang Ping
Published: February 2019

Cadmium, a highly ubiquitous toxic heavy metal, has been widely recognized as an environmental and industrial pollutant, which confers serious threats to human health. The molecular mechanisms of the cadmium-induced cardiotoxicity (CIC) have not been studied in human cardiomyocytes at the cellular level. Here we showed that human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can recapitulate the CIC at the cellular level. The cadmium-treated hPSC-CMs exhibited cellular phenotype including reduced cell viability, increased apoptosis, cardiac sarcomeric disorganization, elevated reactive oxygen species, altered electrophysiology and cardiac arrhythmias. RNA-sequencing analysis revealed a differential transcriptome profile and activated MAPK signaling pathway in cadmium-treated hPSC-CMs, and suppression of P38 MAPK but not ERK MAPK or JNK MAPK rescued CIC phenotype. We further identified that suppression of PI3K/Akt signaling pathway is sufficient to reverse the CIC phenotype, which may play an important role in CIC. Taken together, our data indicate that hPSC-CMs can serve as a suitable model for the exploration of molecular mechanisms underlying CIC and for the discovery of CIC cardioprotective drugs.
Abstract 387: Lymphatic Endothelial Cells Derived Exosomes Promote Neolymphangiogenesis After Injury
Authors: Cimini Maria , Borghetti Giulia , Houser Steven , Kishore Raj
Published: February 2019

Neolymphangiogenesis after myocardial infarction (MI) has been extensively documented; however the source of new lymphatic vessels (LVs) is still unknown. It has been described that new LVs may derive from resident lymphatic endothelial cells (LECs) or from trans-differentiation of endothelial cells (ECs) and pericytes from veins into LECs. Which signals activate both the spreading and the trans-differentiation needs to be investigated. LECs are known to release transcytotic vesicles and exosomes, thus, we hypnotized that the enriched protein and RNA cargo of LECs derived exosomes may promote the new LVs formation after injury. Exosomes derived from LECs were isolated from LECs cultured conditioned medium and HUVECs were treated with different concentration of exosomes cargo for 10 days. After treatment, q-PCR for the major endothelial and lymphatic endothelial markers was performed on treated HUVECs. In a dose dependent manner we observed a decreased expression of KDR and CD31, endothelial markers that are not expressed by LECs and an increased expression of Prox-1 and VEGFR3. Prox-1 is the major lymphatic endothelial transcription factor that is not activated in ECs and is responsible for the transcription of LYVE-1 and podoplanin. VEGFR3 is the only known vascular endothelial growth factor receptor expressed on LECs which is not displayed by ECs. Next, we evaluated the effect of LECs derived exosomes on in vitro lymphangiogenesis assay by measuring the ability of LECs to organize into capillary-like structures on matrigel. LECs treated with LECs derived exosomes are able to form tubes and branches as early as within 1 hour compared to untreated cells. Our preliminary results show cargo of exosomes from LECs activates the signal transduction and metabolism of LECs and HUVECs promoting the new LVs formation through spreading and trans-differentiation of the targeted cells.
Abstract 390: Mitochondrial ROS Drive Sudden Cardiac Death and Chronic Proteome Remodeling in Heart Failure
Authors: Dey Swati , DeMazumder Deeptankar , O’Rourke Brian
Published: February 2019

Intro:
Sudden cardiac death (SCD) from ventricular arrhythmias (VT/VF) claims more lives each year than all other disease-related causes of death combined. The underlying links between HF and SCD remain unclear. Reactive oxygen species (ROS) are central to SCD pathophysiology, but their effects depend on where and how ROS are generated. Employing innovative approaches, we hypothesized that mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD.

Methods:
In a unique guinea pig model that mimics human non-ischemic HF with a high incidence of SCD, we performed a randomized 2x2 crossover study with MitoTEMPO therapy. We employed novel, genetically encoded ratiometric redox sensors for quantitative measurement of cytosol (cROS) or mROS of cardiomyocytes.

Results:
We show for the first time that mROS is elevated in failing hearts and that a mitochondrially targeted antioxidant (MitoTEMPO) normalizes cellular ROS, while NADPH oxidase inhibition is effective in correcting only the cROS component. in vivo mitoTEMPO treatment can prevent or reverse HF while eliminating SCD by decreasing T-wave dispersion and suppressing premature ventricular complexes (PVCs). Scavenging mROS suppressed chronic HF-induced remodeling of the expression proteome and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that, in HF, increased mROS disrupts the normal coupling between cytosolic signals and the nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca2+ handling and action potential repolarization.

Conclusions:
mROS drive both acute emergent events, such as electrical instability leading to SCD, and mediates chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant and ion transport protein networks. Impaired mROS scavenging cause failing hearts to become more vulnerable to demand-induced oxidative stress and promote arrhythmic SCD. Maintaining global redox balance by enhanced mROS scavenging may be an effective strategy for SCD therapy in HF.