bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 576: Phosphodiesterase 2 in Cardiac Arrhythmias and Heart Failure
Authors: Fahmi Mirna S , Günscht Mario , Siegert Johannna , Dutt Fabian , Künzel Stephan , Lorenz Kristina , Wagner Michael , Kämmerer Susanne , El-Armouche Ali
Published: October 2019

Heart failure (HF) patients often suffer from lethal ventricular arrhythmias leading to sudden cardiac arrests. Phosphodiesterase 2 (PDE2), a cGMP activated enzyme hydrolyzing cAMP, is upregulated in HF. Cardiac-specific PDE2 overexpression revealed strong anti-arrhythmic and heart rate lowering effects of PDE2. Here, we validated the cardioprotective role of PDE2 upon modulating cardiac PDE2 activity, first, via assessing cardiac function of cardiac-specific PDE2-KO mice and second, via potentiating PDE2 mediated cGMP/cAMP crosstalk using natriuretic peptide type C (CNP), which are upstream of the cGMP/PDE2 axis. Cardiac function and arrhythmia susceptibility of PDE2-KO were assessed via echocardiography and ECG telemetry under basal conditions and after myocardial infarction (MI). Patch clamp technique was used to investigate CNP effects on action potentials and ion currents in WT isolated cardiomyocytes and in-vivo via ECG-telemetry. Already under basal conditions, PDE2-KO displayed severe irregularities in RR-intervals. 14 days post MI, PDE2-KO showed lower cardiac function and delayed increase in heart rate after arrhythmia provocation (double isoprenaline (ISO) injection (2 mg/kg i.p.) compared to WT controls, indicating diminished cardiac responsiveness. On the other hand, CNP treatment in WT displayed a PDE2-dependent antiarrhythmogenic effect. CNP (1 μM) significantly reduced the total number of delayed afterdepolarizations and spontaneous action potentials upon acute ISO (10 nM) stimulation. Furthermore, CNP significantly reduced the ISO-mediated increase of the L-type Ca
2+
and late Na
+
currents as well as SR Ca
2+
waves. These effects were reversed upon specific PDE2 inhibition with BAY 60-7550. Finally, in vivo, CNP (33 μg/g i.p.) significantly reduced the number of ventricular extrasystoles after arrhythmia provocation. This effect was significantly attenuated after PDE2 inhibition with BAY 60-7550. Here we provide evidence for acute antiarrhythmic effects of CNP upon downstream stimulation of cGMP-stimulated PDE2 and a critical role of PDE2 on heart rate regulation. Therefore, pharmacologically enhancing myocardial PDE2 activity may represent a novel cardioprotective strategy in cardiac arrhythmia and HF.
Abstract 577: Matricryptin p1158/59 Improves Cardiac Function Post-myocardial Infarction by Reducing Adverse Remodeling
Authors: Grilo Gabriel A , Shaver Patti R , Iyer Rugmani P , de Castro Brás Lisandra E
Published: October 2019

Matricryptins are biologically active peptides, generated from extracellular matrix proteolysis, able to regulate cell function and survival. We recently identified a collagen-derived matricryptin (p1158/59) that gradually forms post-myocardial infarction (MI). In humans, p1158/59 plasma levels negatively correlate with left ventricle (LV) filling pressure, indicating that therapeutic elevation of this matricryptin could benefit functional remodeling. Previously, we showed p1158/59 binds to cardiac fibroblasts in vitro to stimulate migration and mice treated with p1158/59 for 7 days post-MI display reduced fibrosis, suggestive of reduced LV remodeling. Our goals were to establish the therapeutic effects of p1158/59 in long term cardiac remodeling and function using a rodent MI model. We used a murine permanent occlusion MI model (male and female C57Bl/6 mice, 4-6 months old, n=6/group) to assess the effects of p1158/59 treatment. Three hours after MI, mice received matricryptin (950 μg/day) or saline solution via an osmotic mini-pump for 28 days (D28). Parameters of cardiac remodeling and function were analyzed by serial echocardiography (D0, D14, D28) and LV remodeling gene arrays. As measured by ejection fraction (EF), matricryptin p1158/59 significantly blunted the reduction in cardiac function observed post-MI (saline 12% EF, p1158/59 31% EF, p=0.004). Additionally, p1158/59 reduced LV dilation displaying decreased end-systolic and end-diastolic volumes compared to controls (ESV p=0.014, EDV p=0.037). Similarly, internal diameter (ID) in diastole and systole were also reduced versus saline (IDd p=0.010, IDs p=0.014). Finally, we observed less tinning of the posterior wall (PW) in p1158/59-treated mice compared to vehicle controls (LVPWd p=0.018, LVPWs p=0.047). These results indicate that p1158/59 attenuated cardiac dysfunction and remodeling post-MI by preserving cardiac compliance and structure. Our data suggest that matricryptin p11/58/59 treatment reduces adverse remodeling post-MI resulting in improved cardiac function.
Abstract 578: Brg1 Protects Cardiomyocytes Against Oxidative Damage Through Activation of the Nrf2 Signaling Pathway in Acute Myocardial Infarction
Authors: Hou Ning , Liu Xiaoping , Liang Guanfeng , Luo Jiandong
Published: October 2019

Background:
Brahma-related gene 1 (Brg1), the core ATPase subunit of a large chromatin remodeling complex, plays critical role in the regulation of gene expression during cardiac growth, differentiation. In adults, Brg1 is turned off in cardiomyocytes and reactivated by cardiac stress. How Brg1 in myocardial infarction (MI) is poorly understood. The Keap1-Nrf2-ARE pathway plays an important role in the development of MI. However, by which Nrf2 activation is mediated in MI still remains to be determined.

Methods and results:
In vivo, adult male C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery for MI model. Our data demonstrated that in peri-infarct zone, the protein of Brg1 was significantly increased 7 days after MI compared with the sham group, accompanied by Nrf2 nuclear translocation, and upregulation of the expressions of NQO1, GSTP1, HO1. We further revealed that with adenoviral intramyocardial injection, the Brg1 overexpression reduced the percentage myocardial infarct, improved cardiac dysfunction, decreased the relative fluorescence intensity of ROS with fluorescent probe DHE in MI mice. Conversely, shRNA-mediated knockdown of Brg1 enlarged the percentage myocardial infarct, exacerbated cardiac dysfunction, increased the relative fluorescence intensity of ROS. More importantly, the Brg1 overexpression significantly induced Nrf2 translocation from cytoplasm to nuclear and upregulated NQO1, GSTP1 and HO1 expressions. Whereas Brg1 knockdown decreased the level of Nrf2 protein in the nucleus, suppressed NQO1,GSTP1 and HO1 expressions. In vitro, Oxygen-Glucose deprivation(OGD) on neonatal cardiomyocytes was established. The effects of Brg1 on OGD and Nrf2 activation were observed with gain- and loss-of-function approaches to regulate Brg1 expression. The results were consistent with
in vivo
study. Moreover, the down-regulation of Nrf2 by brusatol inhibited the increase of these antioxidative genes induced by Brg1 overexpression.

Conclusions:
This study indicated that Brg1-induced cardiac protection was partially mediated through transcription activator Nrf2. The Brg1-Nrf2-ARE pathway may represent a novel therapeutic target for preventing cardiac dysfunction in patients with MI.
Abstract 581: Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species (ROS) via O-linked GlcNAcylation Activation of CaMKII in Mouse Ventricular Myocytes
Authors: Lu Shan , Liao Zhandi , Bossuyt Julie , Bers Donald
Published: October 2019

Diabetes mellitus (DM) is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII activation in autonomous states can occur in DM and contribute to arrhythmogenesis. Diabetic hyperglycemia has been shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a ROS-CaMKII pathway, and these can increase post-infarction mortality. However, the mechanism by which high extracellular [glucose] (Hi-Glu) promotes ROS generation that may synergize with CaMKII activation remains unknown. Here we tested the mechanism by which extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using H
2
DCFDA to measure ROS. Hi-Glu (30 mM) significantly increased the rate of ROS generation, which was prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from both global CaMKIIδ-KO and cardiac specific CaMKIIδ-KO. CaMKII-KO or inhibition prevented Hi-Glu induced sarcoplasmic reticulum (SR) Ca
2+
release events (Ca
2+
sparks and waves). Thus, CaMKII activation is required for Hi-Glu induced ROS generation in cardiomyocytes.

Next, we tested the involvement of O-GlcNAc-CaMKII pathway. Inhibition of de- GlcNAcylation by 100nM Thiamet G (Thm-G) mimicked the effect of Hi-Glu, enhancing the rate of ROS. Also, inhibition of GlcNAcylation (OSMI-1) blocked the ROS induction triggered by either Hi-Glu or Thm-G. Moreover, in new CRSPR-based knock-in mouse in which the functional GlcNAc site on CaMKIIδ was ablated (S280A) neither Hi-Glu nor Thm-G induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc-dependent) ROS production. To identify the ROS source(s), we used different inhibitors: NOX2 inhibitor (Gp91-ds-tat peptide), NOX4 inhibitors (GKT137831), Mito Tempo and NOS pathway inhibitors (L-NAME, L-NIO and L-NPA). Only NOX2 inhibition prevented Hi-Glu/Thm-G induced ROS generation. In summary, our data suggests that diabetic hyperglycemia acutely induces ROS production via an O-GlcNAc-CaMKII dependent pathway and the ROS induced by hyperglycemia is mainly from cytosolic NOX2 complex.
Abstract 582: Obg-like Atpase 1 (OLA1) Regulates Cardiomyocyte Hypertrophic Response via Gsk3β Signaling
Authors: Luong Hien T , Narasimhan Gayathri , Henderson John , Quach Phuong , Annamalai Divya , Krishnamurthy Prasanna
Published: October 2019

Background:
OLA1 that possesses both GTP and ATP hydrolyzing activities has been reported as a translational regulator of cell growth and survival. However, its role in cardiac hypertrophy, a process involving enhanced protein synthesis and increased cell size has not been studied. Hence, we hypothesized that OLA1 expression is regulated by oxidative stress and inflammatory responses in angiotensin-II induced cardiac hypertrophy and its aberrant expression mediates altered GSK3β-β-Catenin signaling.

Methods:
Protein expression of OLA1, GSK3β/P-GSK3β, β-Catenin/P-β-Catenin in total protein extracts of human cardiomyocytes with and without angiotensin II (500nM) treatment were studied. Knockdown of OLA1 expression in cardiomyocytes was achieved by SiRNA transfection and total protein extracts were isolated. In addition, phosphokinase array in OLA1 knockdown cells with and without angiotensin II was performed. H
2
O
2
and LPS treatment in cardiomyocytes for 24h was performed in culture and their total protein extracts were isolated to study the expression of OLA1, GSK3β/P-GSK3β and β-Catenin/P-β-Catenin.

Results:
A significant increase in OLA1 expression from 1 to 3.86±0.72 in angiotensin II treated cells was observed. GSK3β phosphorylation was also increased to 1.27±.10 in angiotensin II treated cells. While, β-Catenin phosphorylation was reduced, total β-Catenin level was significantly increased to 2.26±.34 in angiotensin II treated cells. OLA1 knockdown cells showed a significant reduction (0.12±0.0006) in GSK3β phosphorylation both with and without angiotensin II treatment. Also, phosphorylation of β-Catenin (1.28±.16) was induced in OLA1 knockdown cells. OLA1 expression was up-regulated in both H
2
O
2
and LPS treated cardiomyocytes after 24h. GSK3β phosphorylation and total β-Catenin levels were increased in these cells similar to angiotensin II treated cells. We also found cell proliferation and survival-related phosphoproteins were up-regulated in OLA1 knockdown cells with and without angiotensin II treatment.

Conclusion:
OLA1 was up-regulated during inflammation and reactive oxygen species production which then lead to GSK3β phosphorylation and β-Catenin accumulation in angiotensin II treated cardiomyocytes.
Abstract 584: DJ-1 Confers Protection Against Ischemic Injury by Preserving the Activity of Thioredoxin
Authors: Pantner Yvanna , Shimizu Yuuki , Polavarapu Rohini , Li Lian , Chin Lih-Shen , Calvert John
Published: October 2019

Background:
DJ-1 is a cytoprotective protein expressed in the heart. It can act as an antioxidant in oxidative stress conditions where it is cleaved and converted into an active protease. DJ-1 and Thioredoxin (Trx1), another antioxidant, are known to act on related targets, both playing a role in opposing the apoptosis signaling kinase 1 (ASK1) signaling pathway. We recently found that the cleaved form of DJ-1 attenuates ischemia-reperfusion (I/R)-induced heart failure by reducing glycative stress. Thus, the purpose of this study was to examine the role of DJ-1’s antiglycation activity on Trx1 and how this effects the ASK1 signaling pathway after I/R injury.

Methods and Results:
Initial studies found that DJ-1 deficient mice (DJ-1 KO) showed decreased levels of Trx1 activity after I/R injury compared to wild-type control mice. To explore a possible explanation for this decrease, we assessed the glycation of Trx1. Analysis revealed higher levels of carboxymethyl-lysine (CML) glycation of Trx1 in hearts of DJ-1 KO mice after I/R injury. This was accompanied by an increase in ASK1 phosphorylation and activity, as well as an increase in the phosphorylation of JNK, a downstream target of ASK1. To further determine if DJ-1 directly influences this signaling cascade, we employed an adenoviral approach (AAV9-CMV-DJΔc) to over-express the cleaved form of DJ-1. Compared to mice treated with a control virus, the overexpression of cleaved DJ-1 decreased the glycation of Trx1. Further, Trx1 activity was increased and ASK1 signaling was attenuated.

Conclusion:
These data suggest that the antiglycation activity of DJ-1 plays a role in preserving Trx1 activity after I/R injury. By expanding our perspective of how DJ-1 interacts with Trx1, these data bring us closer to uncovering a therapy that targets the ASK1 signaling pathway in the heart.
Abstract 604: Doxorubicin-induced microRNA-377 Alters Cardiomyocyte and Endothelial Cell Function
Authors: Henderson John , Luong Hien T , Verma Suresh K , Krishnamurthy Prasanna
Published: October 2019

Background:
microRNAs (miRNA/miR) dysregulation has been implicated in cardiac remodeling after injury or stress. We have previously shown that miR-377 negatively affects bone marrow progenitor cells function in response to inflammation/myocardial ischemia. However, its role in doxorubicin (DOX)-induced cardiotoxicity, cell death and endothelial dysfunction is not known.

Objective:
The purpose of this study is to evaluate the role of miRNA-377 in DOX-induced cardiomyocytes (CMs) stress and endothelial dysfunction.

Methods:
miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF) and mouse model of ischemia-reperfusion injury.
In vitro
, we determined the effect of pre-miRNA (pre-miR-377, stimulates miRNA) on Dox-induced cardiomyocyte cell death, endothelial cell migration (using modified Boyden chamber), and vascular tube formation (using matrigel assay).

Results:
Human cardiac biopsies from HF patients show significant increase in miR-377 expression in comparison to non-failing control hearts. Cardiac ischemia-reperfusion (I/R) injury in mice increases myocardial miR-377 expression (vs. sham-operated mice). Interestingly, preparation of single cell suspension of mouse ischemic heart and qPCR analysis show a robust increase in miR-377 expression specifically in the cardiomyocyte as compared to other cardiac cell types. Following DOX-induced cellular stress, miR-377 expression in CMs was elevated, and inhibition of miR-377 attenuates DOX-induced cell death in CMs
in vitro
. Furthermore, transfection of miR-377 mimics in HUVECs significantly inhibits VEGF induced migration and vascular tube formation. Intriguingly, proteome profile of human CD34
+
cells transfected with miR-377 mimics show significant decrease in numerous proangiogenic proteins as compared to nonspecific control transfected cells.

Conclusions:
These findings suggest that doxorubicin-induced cardiotoxicity is associated with an increase in miR-377 expression in the myocardium and that miR-377 overexpression is detrimental to cardiomyocyte viability and endothelial cell function. Therefore, we anticipate that anti-miR-377 treatment might have a beneficial effect against DOX-induced cardiotoxicity.
Abstract 630: Txlnb-Deficient Mice Reveal That Ubiquitin-Proteosome-System Dysfunction is Not Sufficient to Impair Cardiac Performance
Authors: McLendon Jared M , Zhang Xiaoming , Stein Colleen S , Witmer Nathan H , Abouassaly Gabrielle M , Boudreau Ryan L
Published: October 2019

Prior reports show that impaired cardiac function is associated with, if not caused by, Ubiquitin-Proteasome-System (UPS) dysfunction. Rodent loss-of-function studies for proteins involved in protein folding/ER stress responses, ubiquitin ligation, and proteasomal degradation are frequently associated with accumulation of misfolded/ubiquitinated proteins, decreased proteasomal activities, worse outcomes to cardiac stress, and premature death. By contrast, gain of function models that improve protein folding/trafficking or activate the proteasome prevent these molecular phenotypes and improve cardiac function. We have recently found an exception to this association while characterizing beta-taxilin (TXLNB), an understudied muscle- and heart-enriched protein with unknown function. In cardiomyocytes, TXLNB overexpression decreased ubiquitinated proteins and increased proteasome activity, whereas knockdown promotes proteasomal insufficiency. Similarly, hearts from TXLNB knockout (TXLNB-KO) mice show increased ubiquitinated proteins (>2-fold, n=8, p=.007) and decreased 26SB5 proteasome activity (~40%, n=8, p=.025), starting by 12 weeks of age and persisting through life. Despite robust cardiac proteasomal insufficiency, TXLNB-KO mice surprisingly exhibit normal cardiac function [echo/ekg measures done in several independent cohorts (n>7 per sex/genotype) up to ~24 months of age]. In addition, these mice do not show worse responses to mild pressure-overload induced by thoracic aortic constriction (n>13 per sex/genotype). While their cardiac function is normal, TXLNB-KO males do display slight reductions in heart growth with age (10 months, p=.023, n=7; 18 months, p=.07, n=11-17; 24 months, p=.026, n=3-4), suggesting that TXLNB function may interface with hypertrophic signaling. Together, our findings indicate that TXLNB serves to bolster cardiac UPS, and more importantly, that
UPS dysfunction and proteasomal insufficiency is not sufficient to cause cardiac dysfunction in mice.
Future studies will need to examine if the incongruency with prior reports relates to distinct proteosomal pools (e.g. sarcomeric vs. nuclear vs. ER-associated) and/or the specific identities of the accumulated ubiquitinated proteins.
Abstract 631: Complementary Roles for 3-mercaptopyruvate Sulfurtransferase and Cystathionine γ-lyase in Angiogenesis
Authors: Pavlidou Athanasia , Bibli Sofia-Iris , Modis Katalin , Nagahara Noriyuki , Szabo Csaba , Fleming Ingrid , Papapetropoulos Andreas
Published: October 2019

Introduction:
Hydrogen sulfide (H
2
S) is a signaling molecule with important actions in the cardiovascular system. The major endogenous sources for H
2
S in the endothelium are cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfotransferase (3MST). We have previously shown the CSE-derived H
2
S enhances angiogenesis, however, the role of 3MST in new blood vessel formation remains largely unexplored.

Aim:
To evaluate the impact of 3MST on angiogenesis and to study the potential mechanisms involved.

Methods & Results:
3MST deficient EC exhibited attenuated cell growth, reduced mobility in the scratch wound assay and less sprouting in the fibrin gel bead assay. All of the above-mentioned deficits were less prominent than in cells lacking CSE. In an ex vivo sprouting assay, fewer microvessel-like structures were generated by aortic rings treated with a siRNA directed against 3MST compared to a control oligonucleotide. Similarly, sprouting from 3MST KO aortic rings was less than from wild-type mice. In line with the in vitro responses, angiogenesis in vivo in the Matrigel plug assay and in the retina was reduced in 3MST KO mice, but the decrease was of smaller magnitude compared to that seen with CSE KO mice. The angiogenic switch of EC relies mainly on glycolysis, therefore glycolytic intermediates and TCA metabolites were assessed. Glucose-6P, pyruvate, lactate, α-ketoglutarate and citrate were reduced in CSE KO cells, but not in 3MST KO cells, while fumarate and malate were reduced in 3MST KO, but not CSE KO cells. Moreover, extracellular acidification was reduced in EC lacking CSE, but was only minimally affected in 3MST KO EC. In contrast, oxygen consumption rate was more affected in 3MST KO compared to CSE KO cells. In vivo, 3MST expression was mainly observed in the stalk cells, while CSE was highly expressed in the tip cells. Interestingly, only 3MST ablation resulted in reduced pericyte coverage of retinal vessels.

Conclusion:
We conclude that 3MST and CSE are both required for angiogenesis. 3MST preserves the quiescence of stalk cells by targeting oxidative phosphorylation, while CSE supports tip cell behavior by enhancing their glycolytic capacity.
Abstract 642: Single-Cell RNA-seq Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells
Authors: Paik David T , Tian Lei , Williams Ian M , Liu Chun , Zhang Hao , Williams Damon , Mishra Ridhima , Wu Sean M , Wu Joseph C
Published: October 2019

Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. While these functional differences are presumably imprinted in the transcriptome, the pathways and networks which sustain EC heterogeneity have not been fully delineated. To investigate the transcriptional control of EC specification, we analyzed single-cell RNA-sequencing (scRNA-Seq) data from tissue-specific mouse ECs generated by the
Tabula Muris
consortium. We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either scRNA-Seq or bulk RNA-Seq, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (e.g. liver, brain) whereas others (i.e. adipose, heart) have considerable transcriptional overlap with ECs from other tissue. Using gene set enrichment analysis, we identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. By performing pseudotime trajectory analysis, we found that ECs from endoderm-derived tissues appear to be more developmentally immature when compared with the highly specialized ECs of ectoderm-derived tissues such as brain. In addition, we compared these data from mouse with human fetal heart scRNA-seq data for interspecies correlation in organ-specific EC gene expression. Finally, we identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. In summary, we have utilized scRNA-Seq to uncover transcriptional networks which maintain EC identity and identify novel developmental and angiocrine relationships between tissue-specific ECs.
Abstract 643: Integrated Systems Genetics Analysis of Tmem43 Mouse Model and Murine Genetic Reference Population of Bxd Strains Defines Novel Genetic Modifiers and Pathogenic Mechanisms in Arrhythmogenic Cardiomyopathy
Authors: Munkhsaikhan Undral , Gu Qingqing , Sabau Ramona , Pierre Joseph F , Pierre Joseph F , Dong Deli , Towbin Jeffrey A , Lu Lu , Purevjav Enkhsaikhan
Published: October 2019

Background:
Arrhythmogenic cardiomyopathies (ACMs) are heritable diseases characterized by arrhythmias, related sudden death, fibro-fatty infiltration and progressive heart failure. Although the pathogenic variant p.S358L in TMEM43/
LUMA
causes fully penetrant ACM (ARVC5) in patients, little
in vivo
evidence has been reported and the roles of Tmem43 in normal cardiac function and disease remain obscure and controversial. This study explored Tmem43-associated genetic modifiers using forward, reverse, and systems genetics analyses
in vivo
.

Methods:
Microarray of cardiac and jejunal tissues were performed in a newly created Tmem43
S358L
knock-in mouse and in forty BXD strains, used as a murine genetic reference population (GRP). Levels of plasma and fecal lipids and Pparγ activities were defined in heart and jejunum of Tmem43 animals. Immunohistochemistry was performed in the myocardium of patients with non-TMEM43 origin ACM.

Results:
BXD strain cardiac tissues demonstrated high levels of
Tmem43
expression that was significantly negatively correlated with heart mass and heart rate, while positively correlated with plasma HDL and LDL levels. Expression of
Tmem43
was also significantly (r>0.5, p<0.00075) correlated with
Ppargc1a
(
Pcg1a
) in the heart and intestine of BXDs and Tmem43 mutants. In the Tmem43
S358L
mouse heart,
Tmem43
and
Ppargc1a
were downregulated resulting in reduced Pparγ activities. Conversely,
Tmem43
and
Ppars-
regulated genes, including
Mogat2,
responsible for cholesterol, bile acid, and lipid absorption and re-esterification, were upregulated in the jejunum, resulting in elevated lipid absorption in the gut lumen and hyperlipidemia in Tmem43
S358L
mutants. Expression of TMEM43 was disrupted in cardiomyocyte intercalated disks in ACM patients’ myocardium in contrast to normal controls.

Conclusions:
Tmem43
is an essential gene for cardiac and small intestine signaling and function. The S358L-Tmem43 pathogenic variant is significantly associated with downregulation of
Tmem43
,
Ppagc1a
and
Ppar
γ in the myocardium and upregulation of Pparγ and Pparα signaling in small intestine
in vivo
. Testing plasma lipid levels and TMEM43 expression in ACM patients may provide critical information for personalized predictive care.
Abstract 652: Increased Gα
o
Expression Plays a Pivotal Role in the Progression of Heart Failure by Impairing Ca
2+
Handling
Authors: Inazumi Hideaki , Kuwahara Koichiro , Kuwabara Yoshihiro , Nakagawa Yasuaki , Kinoshita Hideyuki , Moriuchi Kenji , Yanagisawa Hiromu , Nishikimi Toshio , Nakao Kazuwa , Kimura Takeshi
Published: October 2019

Background:
A transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction and premature death due to lethal arrhythmias like human dilated cardiomyopathy(DCM). Underlining mechanisms remain to be elucidated, however.

Purpose:
To clarify how NRSF maintains normal cardiac homeostasis.

Methods and Results:
cDNA microarray analysis of dnNRSF-Tg and newly generated cardiac-specific NRSF knockout mice (NRSFcKO), which show similar cardiac phenotypes to those of dnNRSF-Tg, revealed that cardiac gene expression of GNAO1 that encodes Gα
o
, a member of inhibitory G protein Gα
i
family, is increased in both dnNRSF-Tg and NRSFcKO ventricles.

ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that GNAO1 is a direct target of NRSF.

In dnNRSF-Tg, pharmacological inhibition of Gα
o
by pertussis toxin improved systolic dysfunction and knockdown of Gα
o
by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates.

Electrophysiological analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gα
o
ameliorated abnormalities in Ca
2
+ handling, which include increased current density in surface sarcolemmal L-type Ca
2
+ channel, reduced content of sarcoplasmic reticulum Ca
2+
and lowered peak of Ca
2+
transient.

In addition, genetic reduction of Gα
o
attenuated increased phosphorylation levels of CAMKII and RyR2 in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca
2
+ handling.

Furthermore, we identified increased Gα
o
expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα
o
ameliorated cardiac dysfunction.

Conclusions:
We found that increased expression of Gα
o
, induced by attenuation of NRSF-mediated repression, plays a pivotal role in the progression of heart failure by evoking Ca
2+
handling abnormality. Gα
o
is a potential therapeutic target for heart failure.
Abstract 701: Mesenchymal Stem Cells Overexpressing FGF21 Improve Functional Recovery After Traumatic Brain Injury
Authors: Chen Kai-Yun , Shahror Rami A , Wu Chung-Che , Chiang Yung-Hsiao
Published: October 2019

Traumatic Brain Injury (TBI) is a progressive and complex brain injury that results in many adverse and long term neurological consequences. Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator that has emerged as a therapeutic agent for the treatment of neurodegenerative diseases and brain injuries, as it has been shown to exhibit neuroprotective effects, promote remyelination, enhances angiogenesis, and to stimulate the neurite growth of glia-like cells. In this study, MSCs were genetically engineered to overexpress fibroblast growth factor 21 (FGF21) in order to improve their efficacy in TBI. MSCs overexpressing FGF21 (MSC-FGF21) were transplanted to mouse brain by intracerebroventricular (ICV) injection 24 hours after TBI. Spatial learning and memory tests were performed to examine the effects of MSC-FGF21 at 24 hours following controlled cortical impact (CCI) insult in TBI mice model. We found the FGF21 levels were reduced in the hippocampus of vehicle- treated group mice and that MSC-FGF21 treatment restored the FGF21 level significantly. MSC-FGF21 treatment significantly reduced spatial learning/memory decline at 21 days post injury as measured in the Morris water maze test. Both MSC-mCherry (vehicle control) and MSC-FGF21 treatments induced a significant improvement in short term memory formation as determined by the novel object recognition test (NOR) after 14 days of injury. In addition, MSC-FGF21treatment significantly increased the impaired neurogenesis and restored the dendritic process and morphology of immature neurons in the hippocampal dentate gyrus (DG). Taken together, these data provide compelling evidence that MSC-FGF21 treatment promotes neurogenesis and hippocampal neuroplasticity following TBI insult.
Abstract 708: Platelet Derived Growth Factor-Beta Communication Axis is Critical for Mediating Pericyte-Endothelial Cell Interactions and Angiogenic Support in Combination Therapy of Human Umbilical Cord Perivascular Cells and Endothelial Progenitor Cells
Authors: Iqbal Farwah , Szaraz Peter , Librach Clifford
Published: October 2019

Introduction:
First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) have higher cardiovascular regenerative potential compared to older sources of MSC. We designed a cell combination therapy with FTM HUCPVC and endothelial progenitor cells (EPC) to enhance angiogenesis after MI. However, the mechanism of PVC-EPC interaction is unclear.

Hypothesis:
Platelet derived growth factor beta receptor (PDGFB-R) expression of HUCPVC mediates homing and integration with endothelial networks and required for supporting neovasculature.

Methods:
Expression of pericyte markers PDGFB-R and CD146 of xeno-free expanded (HPL 2.5%) FTM and term HUCPVC was analyzed by flow cytometry. PDGFB-driven trans-ECM migration was assessed by Corning Biocoat Invasion assay with 1-100 ng/ml PDGFB. siRNA for PDGFB-R, CD146 and scrambled control was used with lipofectamine for 72 hours. Silencing was confirmed by qPCR and flow cytometry. 10% HPL was used for PDGFB-R desensitization. HUCPVC of all treatment groups were co-cultured with EPC and rat aortic rings on Matrigel
TM
. Cell migration, endothelial adhesion and network properties were quantified. Statistics: one-way ANOVA N=5

Results:
PDGFB-R and CD146 expression and endothelial network augmenting function of FTM HUCPVC was significantly higher than of term HUCPVC. PDGFB dependent migration of FTM HUCPVC was disabled by both ligand (<10%) and siRNA-based (<5%) downregulation of PDGFB-R. Fluorescence microscopy (24 h) showed limited homing and endothelial interactions of PDGFB-R-reduced FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC had significantly decreased network growth and nodes (
p
<0.0001) compared to control groups (day3). In EPC co-cultures, PDGFB-R-reduced FTM HUCPVC had limited endothelial interactions and coverage compared to control FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC treated networks had significantly thinner (
p
<0.001) and shorter tubules (
p
<0.001) compared to control FTM HUCPVC groups. Down-regulation of C146 did not alter FTM HUCPVC pericyte properties.

Conclusions:
PDGFB-R is crucial for the homing and endothelial support of HUCPVC. This interaction is critical in vascular cell combination therapies and PDGFB-R expression can be a novel criterion for cardiovascular cell therapy.
Abstract 710: Quality of Life Assessment in Yucatan Mini Swine after Myocardial Infarction
Authors: Daugherty Sherry L , Spencer-Bearham Danielle , Jauregui Andrew , Winkelman Jeremy , Lancaster Laryenth D , Fox Kenneth A , Avery Ryan J , Koevary Jennifer , Goldman Steven , Lancaster Jordan J
Published: October 2019

Introduction:
Quality of life assessments (QOL) measurements are valuable in assessing functional class in patients with chronic heart failure (CHF). Yet, limited quantifiable QOL measures are available in pre-clinical animal models of CHF. To address this issue, we developed methods of QOL assessments in mini swine after myocardial infarction (MI).

Methods:
Quality of life activity was continuously monitored in Yucatan mini-swine using collar-mounted activity monitors (Fitbark 2) and by performing treadmill exercise tests. Activity treadmill data were obtained for 1 month before and 1 month following MI. Myocardial infarction was created by 90-minute occlusion-reperfusion of the left anterior descending coronary artery. Infarct size and ventricular volumes were measured with Magnetic Resonance Imaging prior to and 1 month after MI.

Results:
The swine had an average MI size of 20±3%, with increases in LV end-diastolic volume from 62.8±7.8 to 72.1±7.4 ml, LV end-systolic volume from 22.1±2.7 to 36.6±4.8 ml and a decrease in EF from 65±3% to 49±4%, N=5-6 (P<0.05). The 24/7 total FitBark Activity Scores using area under the curve decreased post MI from 184.5±16.8 to 159.4±19.5, N=7-8 (P<0.01).

Conclusions:
These data demonstrate the feasibility of measuring quality of life in mini swine after myocardial infarction defined by activity level and treadmill exercise testing. This work establishes the potential basis for presenting quality of life measurements in animals as adjunctive assessment of new treatments for chronic heart failure after myocardial infarction.
Abstract 712: mRNA Stability in Pluripotency and Differentiation of Induced Pluripotent Stem Cells
Authors: Luong Hien T , Babu Sahana S , Henderson John , Quach Phuong T , Krishnamurthy Prasanna
Published: October 2019

Background:
Induced- pluripotent stem cells (iPSC) have distinct transcriptional machinery for the maintenance of pluripotency and achievement of differentiation with the regulation of pluripotency markers, growth factors, and differentiation factors. The Hu family of RNA-binding proteins, HuR (also known as ELAVL1 or human embryonic lethal abnormal vision-like protein), binds to the 3'-untranslated region of mRNAs and regulates transcript stability and translation. However, the role of HuR in pluripotency or differentiation of iPSC to cardiomyocytes (iCM) remains unclear.

Methods:
HuR knockdown in human dermal fibroblast-derived iPSCs was achieved by CRISPR/Cas9 or lentiviral shRNA transduction and subsequently differentiated into cardiomyocytes (iCM). Then, the expression of HuR, pluripotency and cardiomyocyte markers were evaluated following the initiation of differentiation.

Results:
At basal level, HuR expression was higher in the iPSCs compared to dermal fibroblasts. Upon differentiation of iPSCs into iCM, HuR mRNA expression gradually reduced with significantly lower levels on day 17. As expected, pluripotency markers gradually reduced upon differentiation with significantly lower levels from day 6 onwards. We observed a corresponding increase in ISL1, MESP1 (mesoderm/cardiac progenitor markers) from day 3 through day 8 with a steep fall from day 8 to day 17. This was associated with Myosin light chain-2V and GATA4 expression increases from day 8 through day 17. Interestingly, knockdown of HuR resulted in clumps of colonies with differentiated cells and a corresponding increase in cardiac-troponin positive cells. However, as a general observation, HuR knockdown reduced beating intensity compared to wild type cells.

Conclusions:
Based on these data, we could speculate that HuR might be necessary for maintenance of pluripotency and loss of which renders cells to differentiate in culture. HuR knockdown yields higher number of c-troponin positive cells but its effect on functional maturity of iCM needs to be further evaluated.
Abstract 714: Mapping the Transcriptional Dynamics of Pluripotent Stem Cell-derived Endothelial Cell Differentiation by Single Cell RNA Sequencing
Authors: McCracken Ian R , Taylor Richard S , De La Cuesta Fernando , Dobie Ross , Henderson Beth E , Mountford Joanne C , Caudrillier Axelle , Henderson Neil C , Ponting Chris P , Baker Andrew H
Published: October 2019

Objectives:
Human embryonic stem cell derived endothelial cell (hESC-EC) differentiation offers a model to study the process of endothelial development, commitment and maturation, thus potentially informing future regeneration strategies. We used high throughput single cell RNA sequencing (scRNA-seq) to characterise the cellular heterogeneity and associated transcriptional signatures throughout hESC-EC differentiation.

Methods and Results:
Using an established eight-day hESC-EC (H9) differentiation protocol yielding 66% CD31
+
/CD144
+
cells, we sampled cells for scRNA-seq (10X Chromium) at pluripotent (day 0), mesodermal (day 4), early (day 6) and late (day 8) endothelial stages (total 21,369 cells). The pluripotent and mesodermal population were largely homogeneous prior to the emergence of distinct endothelial and mesenchymal populations. Pseudotime analysis characterised the transcriptional signatures throughout differentiation, revealing a bifurcation point giving rise to both populations. Repeating the study with a second hESC line (RC11) produced equivalent populations and transcriptional signatures. In addition, scRNA-seq analysis of an alternative hESC-EC differentiation protocol revealed substantially more heterogeneity, including nephrogenic and haemogenic populations. However, pseudotime analysis revealed common expression of known and uncharacterised endothelial transcription factors across both protocols. Comparison of hESC-EC to foetal and mature EC revealed their transcriptional distinction, suggesting their non-specified nature.

Conclusions:
We sequenced over 105,000 cells allowing characterisation of hESC-EC differentiation across different hESC lines and protocols. We identified a common bifurcation point and remarkable concordance between the transcriptional signatures across different hESC lines and differentiation protocols. This included identifying novel transcription factors that may drive endothelial commitment and maturation.
Abstract 715: A Metabolic Mechanism for Cardiomyocyte Cell Cycle Arrest
Authors: Mills Richard J
Published: October 2019

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CM), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix, metabolic substrate and growth factor conditions that enhance cardiac tissue viability, function and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions hPSC-CMs were refractory to mitogenic stimuli and we found key proliferation pathways including β-catenin and YAP1 were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock novel therapeutic strategies.
Abstract 725: A Transcriptional Basis for Neonatal Heart Regeneration at Single Cell Resolution
Authors: Wang Zhaoning , Cui Miao , Shah Akansha , Tan Wei , Liu Ning , Bassel-Duby Rhonda , Olson Eric N
Published: October 2019

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide novel insights for heart repair. Neonatal heart regeneration is orchestrated by multiple cell types including cardiac resident cells and immune cells that infiltrate the heart after injury. To systematically elucidate the transcriptional response after injury at cellular level during heart regeneration, we performed single cell RNA-sequencing on hearts at various time points post P1 or P8 myocardial infarction injury, and coupled that with bulk tissue RNA-sequencing data collected at the same time points. This integrated approach provides detailed transcriptome landscape and dynamics during heart regeneration at single cell resolution. We further depict a ligand-receptor interaction network that potentially reveal cellular signaling and cell-cell cross-talks during heart regeneration in cell autonomous and non-autonomous manners. Furthermore, to uncover the transcriptional dynamics in a single cardiomyocyte (CM), which was technically challenging due to incompatibility of CM diameter with Drop-Seq platforms, we performed single nucleus RNA-sequencing for CM population, and identified a subset of CMs in the regenerative hearts with unique molecular properties. Taken together, our data provide , and suggest numerous inroads that might be therapeutically manipulated to enhance cardiac function in response to injury.
Abstract 735: A Novel Class of Sphingolipids Mediate Autophagy and Apoptosis in Models of Ischemia
Authors: Kovilakath Anna , Cowart A. Lauren
Published: October 2019

Rationale:
Heart failure caused by ischemic cardiomyopathy is the leading cause of death and disability in the USA and world, accounting for 10 million deaths in 2016. Sphingolipids including ceramides and sphingosine-1-phosphate have been demonstrated to play roles in myocardial injury. In general, these lipids are synthesized from serine palmitoyltransferase (SPT) derived using serine and palmitoyl-CoA. While the standard SPT enzyme exists as a heterodimer of two subunits, Sptlc1 and Sptlc2, a new subunit was recently discovered, Sptlc3, which enables the SPT complex to use myristoyl-CoA, thereby synthesizing a previously underappreciated group of atypical sphingolipids. We previously demonstrated that these atypical lipids are a major component of myocardial sphingolipid pools. Furthermore, in contrast to canonical sphingolipids, the atypical lipids promote cardiomyocyte apoptosis. Therefore, the goal of this research is to determine the contribution of these novel lipids to cardiomyocyte apoptosis and therefore whether they may contribute to ischemic injury.

Objectives:
To determine the contribution of Sptlc3 and atypical sphingolipids to cardiomyocyte apoptosis and ischemic injury and to assess the potential relevance of Sptlc3 to human HF.

Methods:
Murine primary cardiac fibroblasts and cardiomyocytes were isolated and subject to ischemic induction. Following this, cells were analyzed with TUNEL staining, lipidomic analysis or treatment with a Sptlc3 mimetic.

Results:
Ischemic cells show significantly increased Sptlc3 expression, non-canonical sphingolipids in the sphingolipid pool and apoptotic cells as compared to their controls. The cells treated with the Sptlc3 mimetic show apoptotic and a non-canonical autophagic pathway being induced in cells.

Conclusions:
We have identified a novel class of sphingolipids enriched in myocardium of mice with cardiomyopathies. Furthermore, the enzyme that produces these, Sptlc3, is robustly increased in human heart failure. Because the Sptlc3-derived lipids promoted cardiomyocyte apoptosis, we propose that Sptlc3 may mediate myocardial injury in ischemia.
Abstract 745: Voluntary Late-life Exercise Attenuates Frailty and Improves Maladaptive Changes Associated With Cardiac Aging in Female Mice
Authors: Bisset Elise S , Howlett Susan E
Published: October 2019

Voluntary aerobic exercise has many positive health benefits and may actually attenuate frailty. Exercise may also improve cardiac function in the setting of aging, although links between exercise, frailty and age-dependent cardiac remodeling are unclear. To investigate age-related effects of exercise, 21-23 month old female C57BL/6 mice were divided into two groups, an exercise group (n=9) and a sedentary group (n=9). Frailty was measured with a clinical frailty index (FI) tool, then mice were randomly assigned to either exercise or sedentary groups matched by their initial FI scores. The exercise group was allowed constant access to a running wheel for 13 weeks, while sedentary animals had no wheel access. Cardiac function was measured with echocardiography at baseline, midpoint (6 weeks) and endpoint (13 weeks) in both groups. FI scores increased with age in sedentary mice (0.18 ± 0.02 to 0.34 ± 0.05; baseline vs endpoint) but not in the exercise group (0.18 ± 0.02 to 0.20 ± 0.01) and this difference was significant (at endpoint; p<0.05). Fractional shortening (FS) decreased with age in sedentary mice (34.81 ± 3.3% to 22.86 ± 5.5%; p<0.05; at endpoint) but exercise protected hearts against this decline (34.64 ± 2.3% to 36.16 ± 3.1%). A similar pattern was seen with ejection fraction (EF) where EF deteriorated with age (63.28 ± 4.2% to 44.70 ± 9.2%; p<0.05 at endpoint) but stayed similar in exercised animals (63.79 ± 3.6% to 65.74 ± 4.1%). Isovolumic relaxation time decreased with age in sedentary mice (20.43 ± 1.1 ms to 16.50 ± 1.7 ms; p<0.05) but not in exercised mice (19.32 ± 1.1 ms to 16.80 ± 1.1 ms). In addition, left ventricular (LV) mass was actually smaller in the exercise group compared to the sedentary group at endpoint (138.4 ± 19 mg vs 211.6 ± 34 mg; p<0.05). Interestingly, age-associated changes in EF, FS and LV mass at the endpoint were correlated with and closely graded by FI score in all mice (e.g. EF: r= 0.67, p<0.05, FS: r=0.68, p<0.05; LV mass: r=0.68, p<0.05). These results show that voluntary exercise attenuates frailty and prevents negative signs of cardiac aging in older female mice. These data suggest that cardiac function is closely linked to overall health, quantified as frailty, and that late-life interventions to reduce frailty can improve heart health.