bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 371: Cardiac Endocrinology: Biology of a Heart-derived Hormone That Regulates Body Growth
Authors: Pei Liming
Published: February 2019

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than ANP and BNP discovered over 30 years ago. We recently identified growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. Pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. Blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive. In addition, we used massively parallel single-nucleus RNA-Seq to decipher cardiac cell type specific gene regulatory network that induces GDF15 transcription in heart disease condition. We further determined how GDF15 precursor is processed into mature hormone. Together these studies reveal a new endocrine mechanism (cardiac endocrinology) by which the heart coordinates cardiac function and body growth.
Abstract 379: Effects of Microgravity on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function
Authors: Wnorowski Alexa , Sharma Arun , Chen Haodong , Wu Haodi , Shao Ning-Yi , Churko Jared M , Matsa Elena , Countryman Stefanie , Rubins Kathleen , Wu Sean M , Lee Peter H , Wu Joseph C
Published: February 2019

With extended stays aboard the International Space Station (ISS) becoming commonplace as humanity prepares for exploration-class space missions, the need to better understand the effects of cardiac function during spaceflight is critical. However, primary human heart tissues, which would be useful for
in vitro
studies on heart function, are difficult to obtain and maintain. As a model system, we utilized cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) to study the effects of microgravity on human cardiac function and gene expression at the cellular level. We derived hiPSCs from three healthy volunteers and produced hiPSC-CMs using a high-efficiency hiPSC-CM differentiation protocol. We cultured hiPSC-CMs in a microgravity environment aboard the ISS for approximately one month, during which weekly media changes were conducted. We analyzed the gene expression, structure, and function of flight, post-flight, and ground control hiPSC-CM samples using RNA-sequencing, immunofluorescence, calcium imaging, and contractility assessment. Exposure to microgravity on the ISS resulted in decreased contractile velocity and calcium recycling in the hiPSC-CM, along with increased beating irregularities. RNA-sequencing analysis demonstrated that there were 2635 genes differentially expressed with p≤0.05 between flight, post-flight, and ground control samples. These included genes involved in metabolism, DNA/RNA modification, and molecular transport, indicating that these pathways may be permanently altered by long-term space flight
.
This study represents the first time that hiPSC technology has been used to study the effects of microgravity on human cardiac function and is the first to demonstrate that microgravity affects human heart function on the cellular level.
Abstract 384: Pirfenidone Protects the Heart Through Immune Modulation of Subsets of Cardiac B Lymphocytes
Authors: Adamo Luigi
Published: February 2019

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target inflammation clinically. Using two different
in vivo
models of acute myocardial injury, we show that Pirfenidone confers beneficial effects in the murine heart through a new mechanism that depends upon cardiac B lymphocytes. Naïve hearts contained a large novel population of CD19
+
CD11b
-
CD23
-
CD21
-
IgD
+
IgM
low
lymphocytes, and two smaller populations of CD19
+
CD11b
+
B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19
+
CD11b
-
B lymphocytes. Treatment with Pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19
+
CD11b
-
lymphocytes. B cell depletion abrogated the beneficial effects of Pirfenidone.
In vitro
studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19
+
lymphocytes through a TIRAP dependent pathway. Treatment with Pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that Pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes.
Abstract 392: Chronic Dantrolene Treatment Attenuates Cardiac Dysfunction and Reduces Atrial Fibrillation Inducibility in a Rat Myocardial Infarction Heart Failure Model
Authors: Nofi Colleen , Zhang Kuo , Li Ying , Migirov Allan , Ojamaa Kaie , Gerdes Martin
Published: February 2019

Background:
Cardiac ryanodine receptor (RyR2) dysfunction and diastolic Ca
2+
leak have been linked to arrhythmogenesis not only in inherited arrhythmia syndromes but also in acquired forms of heart disease including heart failure (HF) and atrial fibrillation (AF). Thus, stabilizing RyR2 may confer therapeutic effects in these conditions.

Objective:
This study is to investigate the effects of stabilizing RyR2 with chronic dantrolene treatment on HF development and AF inducibility in a myocardial infarction (MI) induced HF model in rats.

Methods:
MI was induced in adult Sprague-Dawley rats by ligation of the left anterior descending coronary artery. Two weeks following MI surgery, rats with large MI (
>
40%) were randomly assigned into MI+vehicle (n=13) and MI+dantrolene (10mg/kg/d, n=13) groups. Sham-surgery rats (n=7) served as controls.

Results:
Compared to vehicle-treated MI group, 4-week dantrolene treatment significantly improved cardiac function with increased left ventricle (LV) fractional shortening (19.48±3.61 vs 15.43±2.65, p<0.01), decreased LV end-diastolic pressure (12.58±8.52 vs 21.91±7.25, p<0.01), left atrial diameter (4.97±0.75 vs 6.09±1.53 p<0.05) and left atrial fibrosis content. Dantrolene significantly decreased AF inducibility (69% (9 of 13) in MI-vehicle vs 23% (3 of 13) in MI-dantrolene group,
p
<0.05). Dantrolene treatment was associated with reduced phosphorylation of RyR2, and favorably altered gene expressions involving ionic channels, sympathetic signaling, oxidative stress, and inflammatory markers.

Conclusion:
Chronic dantrolene treatment attenuated LV dysfunction and reduced AF inducibility, which were associated with decreased phosphorylation of RyR2 and restoration of many adverse changes in gene expression. Stabilizing RyR2 with dantrolene is a promising treatment in HF and AF.
Abstract 404: Sphingosine-1-phosphate Receptor 1 Inhibits Pathological Cardiac Remodeling Through Histone Deacetylases
Authors: Zhou Bisheng , Wolfe Jayne , Lavelle Bianca , Arora Rishi , Wasserstrom J. Andrew , Chun Jerold , Wilsbacher Lisa D
Published: February 2019

Sphingosine-1-phosphate receptor 1 (S1P
1
, encoded by
S1pr1
) is a G protein-coupled receptor that signals in multiple cell types including endothelium and cardiomyocytes. We observed decreased cardiac
S1PR1
mRNA in humans with nonischemic cardiomyopathy; these patterns were also observed in mice after cardiac stress. To assess the importance of reduced
S1pr1
expression, we exposed
S1pr1
heterozygous mice to isoproterenol or angiotensin II. Subsequently, hearts from
S1pr1+/-
mice displayed significantly higher markers of hypertrophy, inflammation, fibrosis, and oxidative stress than wild-type littermates. Cardiomyocyte-specific deletion of
S1pr1
caused pronounced increases in the same markers. In wild-type mice, co-administration of the S1P
1
-specific agonist SEW2871 rescued the pathologic effects of angiotensin II. Because class I histone deacetylases (HDACs) contribute to pathological remodeling, we investigated interactions between S1P
1
and HDAC activities as a potential mechanism. In cultured mouse cardiomyocytes, angiotensin II increased expression of histone deacetylases 1 and 2, as well as other markers of pathological remodeling; co-treatment with SEW2871 inhibited these responses. Moreover, HDAC inhibition in
S1pr1+/-
mice blunted the pathological response to angiotensin II. These results indicate that S1P
1
protects by modulating HDAC1/2 during cardiac stress and support S1P
1
agonism as a therapeutic strategy for pathological cardiac remodeling in humans.
Abstract 412: The Role of ETC Complexes I and II in the Respirasome Assembly in Heart Mitochondria
Authors: Jang Sehwan , Javadov Sabzali
Published: February 2019

Mitochondrial electron transport chain (ETC) plays an important role in ATP synthesis and its dysfunction is associated with human diseases. Recent cryo-electron microscopy studies revealed that individual ETC complexes are assembled into supercomplexes (SCs). Structural organization and physiological role as well as mechanisms of assembling and maintenance of the SCs are yet not completely understood. ETC complexes contribute unequally to structural organization of SCs, particularly the respirasome, the main SC which contains complexes I, III, and IV in various stoichiometries. However, many questions related to structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we focus on the role of complexes I and II in structural organization of SCs using both genetic and chemical inhibition of complex I and complex II in H9c2 cardiac myoblast cells and isolated cardiac mitochondria. Respiratory SCs were analyzed by blue native polyacrylamide gel electrophoresis. Pharmacological inhibition of complex I by rotenone and complex II by malonate stimulated dissociation of respirasome. Rotenone inhibited whereas malonate had no effect on calcium-induced mitochondrial swelling, a marker of permeability transition. On the other hand, the rate of mitochondrial ROS production was remarkably (>5 fold) stimulated by rotenone but not by malonate. Knockdown of the complex I subunit NDUFA11, which according to cryo-electron microscopy studies interacts with complex III in respirasome formation, stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV but not complex II. Downregulation of the SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. In addition, knockdown of NDUFA11 or SDHC significantly reduced ATP levels in the cells. Overall, these studies for the first time provide biochemical evidence that NDUFA11 subunit of complex I is important for structural integrity of respirasome whereas complex II is less likely contribute to the integrity of respirasome.
Abstract 428: Cytoglobin: A Novel Regulator of the DNA Damage Response Within Cardiomyocytes
Authors: Singh Sarvjeet , Rivas Dylan , Canseco Diana , Weiler Melanie , Gemelli Terry , Huang Jian , Tassin Tara , Shelton John , Richardson James A , Sadek Hesham , Mammen Pradeep P
Published: February 2019

Rationale:
Oxidative stress is a major determinant of DNA damage within cardiomyocytes both at baseline and following cardiac injury. Cytoglobin (Cygb), a stress responsive hemoprotein, serves as a redox modulator and imparts cytoprotection in a variety of cell types. In addition, Cygb is involved in regulation of myocyte proliferation and differentiation due to its ability to scavenge free radicals and maintain redox equilibrium within the myocytes.

Objective:
The goal of this study was to investigate the role of Cygb in cardiomyocytes and identify the molecular pathways underlying its cytoprotective function.

Method and Results:
Utilizing transgenic mice with cardiac specific overexpression of Cygb (TgCygb), we demonstrated that Cygb overexpression promoted cardiomyocyte proliferation as compared to the control hearts (NTg) (phospho-Histone 3 IHC: 17 ± 0.7 vs 7 ± 0.5 positive cells/mm
2
TgCygb vs NTg; p<0.05, n=3) . The enhanced cellular proliferation in TgCygb hearts was associated with larger hearts at baseline. However, with exposure to acute cardiac stress (subcutaneous Angiotensin II infusion), TgCygb hearts showed significantly reduced cell death, oxidative stress and an overall lower induction of the DNA damage response pathway as compared to the NTg hearts [TUNEL: 7 ± 0.8 vs 10 ± 1.2 TUNEL positive nuclei/mm
2
, p<0.05, n=3; phospho-ATM IHC: 11 ± 2 vs 22 ± 1.5 phospho-ATM positive cells/mm
2
, p<0.05, n=5, dihydroethidium (DHE) staining: 78 ± 0.6% vs 98 ± 0.7% DHE positive nuclei/mm
2
, p<0.05, n=5]. Using single cell electrophoresis (Comet assay), we demonstrated that Cygb overexpressing cardiomyocytes underwent significantly lower DNA damage due to single and double stranded DNA breaks following exposure to oxidative stress as compared to controls cardiomyocytes (36 ± 2.1 vs 48 ± 3.1; p<0.05, n=6). Finally, chromatin immunoprecipitation, quantitative PCR and immunoblotting indicated that Cygb activates the expression of apurinic/apyrimidinic endonuclease 1 (
Ape1
) and thus promotes DNA base excision repair following acute cardiac injury.

Conclusions:
Our data demonstrate that Cygb promotes cellular proliferation and protects cardiomyocytes against oxidative stress-mediated DNA damage by directly activating
Ape1
expression.
Abstract 435: Post-transcriptional Regulation of Tropomyosin Isoforms Altered in Human Heart Diseases
Authors: Cao Jun , Belanger KarryAnne , Nutter Curtis , Ji Ping , Jaworski Ela , Elrod Nathan , Verma Sunil , Wagner Eric , Routh Andrew , Kuyumcu-Martinez Muge N.
Published: February 2019

Background:
Tropomyosin 1 (TPM1) is a coiled coil protein that forms polymers along with the actin filament, and it is required for myofibril organization, myocardial contraction, and cardiac development. TPM1 has cardiac muscle and non-muscle specific protein isoforms that dictate its function in cardiac muscle contraction or in cytoskeleton. TPM1 mutations in exon 2b, 5, 6b or 8 are associated with cardiovascular diseases including familial hypertrophic cardiomyopathy, dilated cardiomyopathy and left ventricular non-compaction. Thus, it is of great importance to investigate the mechanisms that control tropomyosin isoforms in order to identify novel therapeutic targets for cardiovascular diseases in which TPM1 is mutated.

Method:
We used nanopore sequencing technology using MinION and poly(A)click seq (PAC-seq) to identify different TPM1 transcripts generated in rat heart derived H9c2 cells. RT-PCR and western blotting were conducted to measure mRNA and protein levels of TPM1 respectively.

Results:
We identified 10 different isoforms of TPM1 using nanopore sequencing and determined several exons that are extensively spliced and favored in cardiac cells. We further identified an RNA binding protein that controls cardiac specific isoforms of TPM1 via alternative splicing and polyadenylation. Importantly, we found that this RNA binding protein regulates alternative splicing of exon 6b of TPM1, which is mutated in patients with cardiomyopathies.

Conclusions:
We identified an RNA binding protein, which controls expression of cardiac specific TPM1 isoforms by alternative polyadenylation and alternative splicing.
Abstract 437: Tyrosine Phosphorylation: Regulator of Cardiac Contraction
Authors: Brundage Elizabeth A , Agatisa-Boyle Brendan , Shettigar Vikram , Jeong Mark Y , Salhi Hussam E , Chung Jae-Hoon , Qian Ziqing , Janssen Paul M , Pei Dehua , Davis Jonathan P , Ziolo Mark T , Biesiadecki Brandon J
Published: February 2019

Myocardial contractile function is depressed in heart failure. Contractile function of the normal and failing heart is in part determined by kinase mediated serine/threonine phosphorylation. While non-receptor tyrosine specific kinases are expressed in the heart and their activity is increased in disease, the role of tyrosine kinase signaling to modulate cardiac contraction is unknown. Previous work demonstrated the cardiac contractile regulatory protein troponin I (TnI) is phosphorylated at tyrosine 26 (Tyr26). Subsequently, we demonstrated TnI Tyr26 phosphorylation alters myofilament function thereby identifying the first tyrosine phosphorylation to directly modulate cardiac contraction. The role of TnI Tyr26 phosphorylation in the normal or diseased heart and the signaling pathways responsible for its phosphorylation are unknown. To identify the kinase responsible for TnI Tyr26 phosphorylation and its effect on in vivo heart function, we generated a novel tyrosine kinase activator and a TnI Tyr26 phosphorylation (Tg Y26E) mouse. Biochemical kinase assays and kinase activation in ventricular myocytes identify Src family kinases as sufficient to induce TnI Tyr26 phosphorylation. Tg Y26E mice exhibit decreased myofilament calcium sensitivity and accelerated relaxation. Finally, we demonstrate TnI Tyr26 phosphorylation is increased during in vivo myocardial ischemia when Src family tyrosine kinases are also activated. Together these data support targeting Src family tyrosine kinase and TnI Tyr26 phosphorylation to improve heart function in myocardial ischemia and the failing heart.
Abstract 452: Alpha-Ketoglutarate Dehydrogenase Inhibition by the Oncometabolite D2-HG Causes Proteome and Metabolome Remodeling in Myocytes
Authors: Karlstaedt Anja , Vitrac Heidi , Raedschelders Koen , Spivia Weston R , M Daniel , Khanna Radhika , VanEyk Jennifer , Taegtmeyer Heinrich
Published: February 2019

Autophagy “scavenges” proteins and yields amino acids under conditions of metabolic stress to support cell survival and growth. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including dilated cardiomyopathy. Our recent in vivo work showed that increased D2-HG supply by IDH2-mutant hematopoetic stem cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. While heart failure in cancer is commonly associated with chemotherapy, cancer survivors have a five-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic changes and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase inhibition in myocytes results in metabolomic pertubations, proteomic remodeling, and increased autophagy. We measured autophagic flux and remodeling of the stable proteome upon D2-HG treatment in vivo using wild-type C57BL/J6 mice, and in vitro using both cultured L6 myocytes and adult mouse ventricular cardiomyocytes. We observed increases in the LC3-II/LC3-I ratio and p62 expression in heart and skeletal muscle from mice treated with D2-HG, indicating activation of autophagy. Live cell imaging with GFP-tagged LC3 indicated that D2-HG (1 mM) increased LC3-II lipidation and flux within 24 h. Furthermore, we observed increased phosphorylation and activation of AMPK, while phosphorylation of mTOR and p70S6K were decreased in presence of D2-HG. In vitro exposure to D2-HG resulted in the formation of a molecular complex between Sirt1 and LC3, indicating that increased NAD+ in presence of D2-HG promotes Sirt1 activation in myocytes. Finally, we used LC-MS/MS to assess the effect of D2-HG on the stable proteome and metabolome in heart and skeletal muscle. Myocytes exposed to D2-HG showed proteomic remodeling and metabolomic changes within 24 h. Integrating multi-omics data in a network-level context revealed upregulation of glycolysis and the pentose phosphate pathway. In short, autophagy activation may support proteome remodeling in muscle cells during IDH-mutant leukemia.
Abstract 455: Role of Transcription Co-Factor Friend of GATA 2 (FOG2) in a Hypertensive-Diabetic Mouse Model of Coronary Microvascular Disease
Authors: Guerraty Marie A , Szapary Hannah J , Arany Zoltan , Rader Daniel J
Published: February 2019

Coronary microvascular disease (CMVD) - disease of the coronary pre-arterioles, arterioles, and capillaries – has become an increasingly well-recognized cardiac pathology. Characterized by microvascular remodeling, wall thickening, lumen obliteration, endothelial dysfunction, capillary rarefaction, and perivascular immune cell infiltration, it can lead to inadequate myocardial perfusion, angina, heart failure, or myocardial infarction. Despite these clinical ramifications, our understanding of the pathophysiology of CMVD remains nascent. Friend of GATA 2 (FOG2, also called ZFPM2) is a transcriptional co-factor which is crucial in coronary development; and cardiomyocyte expression of FOG2 is required for the maintenance of the coronary microvasculature in adult mice. We hypothesize that FOG2 is activated in CMVD, and that Hypoxia-Inducible Factor 1a (HIF1a) may play a role in FOG2 activation. To test this hypothesis, we developed a hypertensive-diabetic (HTN/DM) mouse model of CMVD. Briefly, adult female C57BL/6 mice were treated with Angiotensin II (1mg/kg/day SQ osmotic pump for 8 weeks) and low-dose streptozotocin (35 mg/kg IP for 4 days) (n=5) or saline (n=4). HTN/DM mice had increased systolic blood pressure (141.7±1.6 vs 109.2±1.1 mmHg, p<0.0001), mild fasting hyperglycemia (181.8±8.5 vs 148.8±9.8 mg/dl, p=0.04), and decreased glucose tolerance (p<0.0003). HTN/DM mice had evidence of mild LV hypertrophy, preserved EF, and diminished coronary hyperemic velocities by echocardiography. Histology revealed microvascular pathology (vessel wall thickening and immune cell infiltration) and decreased cardiac capillary density (148.8±9.8 vs 163.8±4.3 phf, p=0.008). FOG2 protein was increased in HTN/DM mouse hearts (p<0.05). Treatment of AC16 cardiomyocytes with HIF-activator hydroxylase inhibitor DMOG showed a dose-dependent increase in FOG2 expression and protein nuclear translocation. In summary, we modeled CMVD in mice using mild hypertensive-diabetic insults which are relevant to human disease. We found that FOG2 protein is increased in CMVD suggesting that FOG2 may be relevant to adult CMVD pathophysiology
in vivo
.
We additionally provide
in vitro
evidence that FOG2 is induced in HIF-activating conditions
.
Abstract 461: Prospective Isolation of Heart Field Specific Cardiomyocytes from Differentiating Human Embryonic Stem Cells
Authors: Engel James L , Pezhouman Arash , Nguyen Ngoc B , Zhao Peng , Khoja Suhail , Skelton Rhys J , Sahoo Debashis , Elliott David A , Ardehali Reza
Published: February 2019

Cardiovascular disease (CVD) is the leading cause of death worldwide. Human embryonic stem cell (hESC) derived cardiovascular progenitor cells (CPCs) or cardiomyocytes (CMs) represent a promising candidate for cell-based therapies to treat CVD. Following a myocardial infarction there is extensive CM death and damage primarily localized to the left ventricle of the heart, which is predominantly derived from first heart field (FHF) CPCs during development. This is important in future developmental and regenerative therapy studies since heart field specific cardiac cells will be desired. Current hESC differentiation protocols as well as the use of cardiac-specific reporter lines, yield a mixture of cardiac cells without the ability to isolate heart field specific cardiac progenitors and their associated progeny. Here we generated and characterized a FHF specific TBX5-
TdTomato/W
hESC reporter line to show that TBX5
+
cells represent an enriched population of FHF CPCs that can give rise to CMs, endothelial and smooth muscle cells
in vitro
. Transplantation of hESC-derived TBX5
+
and TBX5
-
CPCs into the injured left ventricle of mice showed that these cells were able to survive and predominantly gave rise to CMs without functional integration into the host myocardium. Furthermore, RNA-sequencing analysis of hESC-derived cardiac cells at different stages of development suggested that while TBX5 expression leads to an enrichment of FHF- derived CMs, TBX5
-
cells also generate cardiac cells that resemble second heart field (SHF) CMs at the transcriptional level. To enable prospective isolation of FHF and SHF CMs we generated a double transgenic hESC reporter line TBX5
TdTomato/W
/NKX2-5
eGFP/W
. Using immunostaining, qPCR and electrophysiological studies we were able to confirm the isolation of FHF and SHF specific CMs from differentiating hESCs. Finally, we show that using the novel surface marker, CORIN, we can isolate FHF CMs without the use of genetic reporters. Our studies provide valuable information and resources on the production of heart field specific CMs from hESCs for cell-based therapies to treat CVD.
Abstract 475: Human Cardiac Progenitor Cells Derived Exosomes Stimulate Cardiomyocytes Proliferation by MicroRNA-Hippo Pathway
Authors: Sharma Sudhish , Bigham Grace , Ambastha Chetan , Gunasekaran Muthukumar , Brohawn Phillip Z , Karathanasis Sotirios , Kaushal Sunjay
Published: February 2019

Background:
We identified that human neonatal cardiac progenitor cells (CPCs) improve cardiac function and attenuate adverse left ventricular remodeling after myocardial infarction through their exosomes (nEXOs). RNAseq analysis of nEXOs identified miRs which may be responsible for modulation of Hippo signaling, thereby promoting cardiomyocytes proliferation.

Hypothesis:
nEXOs effectively stimulate endogenous cardiomyocyte (CM) proliferation by targeting the Hippo pathway to restore cardiac function in an injured heart.

Methods and results:
nCPCs were conditioned for 48 hours in serum free nutrition mix (Ham’sF12) and nEXOs were purified from supernatant using size exclusion chromatography (SEC; CL2B) coupled with ultracentrifugation. nEXOs were quantified by Nanosight (NS300) and characterized by transmission electron microscopy and flow cytometry for the presence of CD63 and CD9. Our results show recovery of cardiac function (ejection fraction = 63.4% vs 40.5%, n=10, p<0.001)) and generation of myocardial mass in a rodent MI model following nEXOs’ intra-myocardial injection. We further show that nEXOs are preferentially acquired by CMs in the border zone of the infarction (m-cherry-Alix labeled EXOs). Using deep RNAseq (sequencing depth: 25 million reads) coupled with literature based
in silico
analysis, we identified a candidate panel of miRNAs, predicted to promote CM proliferation by targeting the Hippo Pathway. Our In vitro experiments show that miR-528-3p and miR-7641 (25nM) are the most effective miRs to induce proliferation of quiescent cardiomyocytes (40.1% and 48.8%, respectively, n=3). Increased miR-7641 expression led to a profound increase in quiescent CMs proliferation, in part through repression of the Hippo signal transduction pathway (increased YAP/pYAP ratio). By immunoblotting we show that LATs1/2 (a protein in the Hippo pathway) is directly targeted by miR-7641. nEXOs enriched with miR 7641 promote CM proliferation by 3 folds as compared to non-enriched nEXOs.

Conclusion:
Our data for the first time demonstrates the ability of EXOs derived micro-RNA based therapeutic approaches to improve cardiac function through the activation of cardiomyocyte proliferation.
Abstract 477: Using Computational Models to Predict Stem Cell Function
Authors: Shoja-Taheri Farnaz , George Alex , Platt Manu O , Davis Michael E
Published: February 2019

Congenital heart defects (CHD) are among the most common reasons of mortality in newborns and despite advanced surgical treatments, many patients experience heart failure. Currently, transplantation is the most effective cure for heart defects which has its own limitations such as the possibility of organ rejection. Stem cell/progenitor cell therapy is a novel method in treating heart failure in congenital heart patients. However, choosing stem cells with highest regenerative effects has been a challenge and there is tremendous patient-to-patient variability. We previously showed the age-dependent regenerative effects of human cardiac progenitor cells (hCPCs) in a rat model of juvenile heart failure. In the same study, using a small subset of patients, computational modeling analysis showed that regression models could be made linking sequencing data to phenotypic outcomes. In the current study, we used that quantitative model to determine whether predictions can be made in a larger population of patients regarding cell function in neonatal hCPCs. The goal of this study was to validate the functional responses in each neonatal hCPC line and confirm our model. We performed RNAseq from CPCs isolated from 8 different neonatal patients. We tested 2 functional parameters of our model, proliferation and chemotactic potential of conditioned media using Click-it Edu and Boyden chamber assays, respectively. Interestingly, the observed proliferation and migration responses in each of the selected neonatal hCPC lines matched their predicted counterparts. Furthermore,
in vitro
analysis verified our model in that one of the neonate hCPCs performed far worse than other patients as predicted. Altogether, these data show that cell behavior may be predicted using large data like RNAseq, and that we may be able to identify patients that may exceed or underperform expectations. With systems biology approaches, interventions can be tailored to improve cell therapy, or mimic the qualities of reparative cells.
Abstract 494: Comparing Cardiac Dynamics Between Neonatal and Adult Rats
Authors: Swift Luther M , Hiebert James , Burke Morgan , Ramadan Manelle , Jaimes Rafael , Posnack Nikki
Published: February 2019

Background:
In the broad field of cardiology, pediatric research has lagged behind in establishing appropriate models to study the developing heart. Human cardiomyocytes exhibit a limited life span and immortalized cell lines lack physiologically relevant automaticity. Pediatric animal models include caveats related to action potential morphology, ion channel expression, and excitation-contraction coupling. Changes that occur in these parameters as the animal transition from neonate to adult are relatively unknown, especially as it may be translated to a human model. In quantifying the toxicological effect of plasticizer exposure in a pediatric population, we first sought to establish the limits of using hearts from developing neonatal rats as a viable model.

Objective:
This study aimed to quantify the advantages and limitations associated with using neonatal rat hearts as a model for toxicological screening.

Methods/Design:
The heart from rats ranging in age from 2 days old, up to adult were excised, and the aorta was cannulated. It was placed on a Langendorff system and retrograde perfused. Calcium and voltage sensitive dyes were used to stain the heart for imaging. It was mechanically uncoupled with blebbistatin to eliminate motion artifacts and an ECG was recorded continuously.

Results/Discussion:
Preliminary results showed that compared to adult cohorts, neonatal rats displayed a longer action potential duration (APD80: adult= 85.9ms, neonatal=95.5ms, p=0.026), likely associated with delayed Ito expression. Likewise, calcium handling was also slower in the neonatal hearts as shown by the calcium imaging. (Cad80: Adults: 128.9ms, neonatal=138.8, p=.004) Without autonomic modulation, we observed an increase in heart rate from 320 to 350bpm between neonatal and adult populations which has been shown
in vivo
. The developing excitation contraction coupling machinery will be further probed for the mechanism behind these phenomena.
Abstract 514: Focal Adhesion Kinase Inhibition Prevents Prolonged Nuclear Factor-κB Activation and Reduces Atherosclerosis in ApoE-/- Mice
Authors: Murphy James M , Lim Steve
Published: February 2019

A key event in atherogenesis is the expression of cell adhesion molecules on endothelial cells (ECs) that recruit leukocytes to the vessel wall, where they accumulate over time and form atherosclerotic plaques filled with fatty lipids. Although lipid-lowering drugs have been beneficial in slowing down atherosclerosis, there is currently no treatment available to reduce the underlying triggers that promote EC activation. Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that plays a key role in cytokine-induced pro-inflammatory molecule expression; however, the molecular mechanism has not been elucidated. We have revealed that a small molecule FAK inhibitor (PF-271) in human ECs reduces tumor necrosis factor-α (TNF-α)-induced sustained activation of nuclear factor-κB (NF-kB), a major inflammatory transcription factor. While NF-κB is normally bound to inhibitor of NF-κB α (IκBα) in the cytosol, TNF-α leads to rapid activation and nuclear localization of NF-κB after degradation of IκBα by activation of IκB kinase (IKK). Chronic exposure to cytokines leads to oscillations in NF-κB activation as IκBα is continually made by active NF-κB and degraded by active IKK. Surprisingly, we discovered that FAK inhibition blocks prolonged NF-κB activation through a time-dependent increase in IκBα protein stability, suggesting decreased IKK activity. By performing
in vitro
IKK kinase assays, we found that FAK inhibition did indeed reduce sustained IKK activity as seen by reduced serine phosphorylation of IκBα 3hr after TNF-α stimulation. Since both cell adhesion molecule expression and NF-κB in ECs is important for atherosclerotic lesion development, we tested the efficacy of FAK inhibition on atherogenesis
in vivo
. We found that oral delivery of PF-271 (twice daily, 8 weeks) significantly reduced atherosclerotic lesion area in ApoE-/- mice fed a western diet. Taken together, our data demonstrates that FAK inhibition potentially reduces atherogenesis by blocking sustained NF-κB signaling through decreased IKK activity.
Abstract 516: Pharmacological Inhibition of Phosphatase and Tensin Homolog Ameliorates Adipose Tissue Inflammation and Increases Splenic Regulatory B and T Cells in Rats With Metabolic Syndrome
Authors: Uchinaka Ayako , Kawai Yumeno , Komuro Kanako , Fujieda Aoi , Wataya Minami , Yoneda Mamoru , Komatsu Yuki , Aoyama Kiyoshi , Murohara Toyoaki , Nagata Kohzo
Published: February 2019

Objectives:
B cells have emerged as a critical player in innate and adaptive immune responses. However, little is known about the role of B cells in obesity- and/or stress-induced immune responses. We previously reported that restraint stress exacerbates cardiac and adipose tissue pathology via β-adrenergic signaling in rats with metabolic syndrome (MetS). Phosphatidylinositol 3’-kinase (PI3K)/Akt pathway regulates interleukin-10 production by B cells. Phosphatase and tensin homolog (PTEN) is a lipid/protein phosphatase that can negatively regulate PI3K/Akt pathway. To identify the possible role of PI3K signaling in stress-induced immune responses in metabolic disorders, we now examined the effects of restraint stress and PTEN inhibition on B and T cell subsets in rats with MetS.

Methods:
We used DahlS.Z-

Lepr
fa

/

Lepr
fa

(DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as an animal model of MetS. Rats were exposed to restraint stress (restraint cage, 2 h/day) for 2 wk from 9 wk of age with or without daily administration of the PTEN inhibitor bisperoxovanadium-pic [bpV(pic), 0.2 mg/kg, i.p.]. Age-matched homozygous lean littermates of DS/obese rats (DahlS.Z-

Lepr
+

/

Lepr
+

rats) served as controls.

Results:
Treatment with bpV (pic) attenuated hypertension in DS/obese rats under restraint stress. This agent also reduced subcutaneous fat mass and alleviated adipocyte hypertrophy and inflammation in subcutaneous adipose tissue as well as tended to suppress epididymal adipose tissue inflammation in such rats. Flow cytometric analysis showed that regulatory B (Breg) cells were increased and B-1 cells, which attenuate inflammation and insulin resistance, tended to be increased by bpV (pic) in the spleen of DS/obese rats subjected to restrain stress. In addition, bpV (pic) treatment led to an increase in splenic regulatory T (Treg) cells and downregulation of CD8
+
T cells in epididymal adipose tissue of stressed rats.

Conclusions:
PTEN inhibition with bpV (pic) attenuated subcutaneous adipose tissue inflammation as well as increased splenic Breg and Treg cells in DS/obese rats under restraint stress. Pharmacological inhibition of negative regulator of PI3K may represent a therapeutic approach for the treatment of MetS.
Abstract 528: Cell Specific Signaling Regulated by PI3Kγ Modulates Myofibroblast Differentiation
Authors: Mohan Maradumane L , Grove Lisa M , Papay Robert S , Olman Mitchell A , Naga Prasad Sathyamangla V
Published: February 2019

Phosphoinositide 3 Kinase γ (PI3Kγ) regulates anti-apoptotic Akt signaling. Previous studies have established a role for PI3Kγ in cardiac fibrosis, a key underlying cause of heart failure. However, less is known about the mechanism by which PI3Kγ regulates cardiac myofibroblast differentiation, hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) overexpression. Measurement of αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ
-/-
) showed significant baseline and pressure overload induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ
-/-
exhibited a myofibroblast phenotype with αSMA in stress fibers. Correspondingly, immunoblotting showed significantly higher αSMA in PI3Kγ
-/-
CF than WT. It has been previously shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). To understand whether PI3Kγ regulates ERK signaling in a cell specific manner and thereby possibly αSMA, the phosphorylation of ERK by insulin stimulation were compared in CF isolated from WT and PI3Kγ
-/-
. Intriguingly, there was significant loss of ERK phosphorylation in CF from PI3Kγ
-/-
when compared to CF from WT. However, ERK phosphorylation was not altered in cardiomyocytes (CM) due to the absence of PI3Kγ. Confirming this differential regulation of ERK in CM and CF, there was no change in the association of ERK and protein phosphatase 2A (PP2A) in CM of WT and PI3Kγ
-/-
. However, there was increased association of PP2A with ERK in CF of PI3Kγ
-/-
when compared to CF from WT. This is because in the CM, ERK is regulated by Dual Specificity Phosphatase (DUSP8). However, in the CF, PP2A is the major regulator of ERK and thus PI3Kγ plays a major role in ERK signaling in CF. Consistent with previous observations, we found that association of DUSP8 with ERK was not observed in CF. Moreover, ERK-DUSP8 interaction was not dependent on the presence or absence of PI3Kγ. These data indicate that PI3Kγ regulates signaling pathways in a cell specific manner with respect to cardiac remodeling.
Abstract 532: Titin’s N2B Element is Critical to Cardiac Mechanotransduction during Volume Overload, but not Pressure Overload
Authors: Strom Joshua , Bull Mathew , Saripalli Chandra , Gohlke Jochen , Gotthardt Michael , Granzier Henk
Published: February 2019

The heart senses and responds to changes in mechanical loading conditions by initiating signaling events to produce structural and functional remodeling through a process known as mechanotransduction. Within cardiac myocytes, the giant protein titin has been implicated as a key player in the conversion of mechanical stimuli into downstream signaling events as part of the adaptive responses of mechanotransduction. The N2B element within the extensible I-band of titin has been proposed to be a stretch sensor and signaling hot spot important to titin’s role as a mechanosensor. To investigate the role of the N2B element in mechanotransduction, N2B knockout (N2BKO) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC) or volume overload by aortocaval fistula (ACF). Following 4 weeks TAC, WT mice respond with 37% LV hypertrophy (4.0 ± 0.1 mg/gBW for TAC vs 2.9 ± 0.1 mg/gBW for sham) compared to 51% (3.8 ± 0.1 mg/gBW for TAC vs 2.5 ± 0.1 mg/gBW for sham) in N2BKO. In contrast, N2BKO mice demonstrated a severe lack of a hypertrophic response following 1-week ACF with 26% (4.0 ± 0.1 mg/gBW for ACF vs 3.1 ± 0.1 mg/gBW for sham) LV hypertrophy in WT compared to -2% (2.8 ± 0.1 mg/gBW for ACF vs 2.8 ± 0.1 mg/gBW for sham) in N2BKO. Additionally, while there was no incidence of mortality in WT mice subjected to ACF, approximately 36% (5 of 14) of N2BKO mice died within 1 week following ACF. Western blot analysis of canonical hypertrophy signaling kinases revealed no differences between WT and N2BKO mice at baseline or 1 week post-ACF. Four-and-a half LIM domains 1 and 2 (FHL1 and FHL2) have previously been shown to localize to the N2B element and FHL1 has been linked to mechanotransduction in TAC. In N2BKO, FHL1 protein is expressed at levels comparable to WT in both TAC and ACF; however, FHL2 protein levels are reduced by ~98% compared to WT (1.00 ± 0.26 for WT vs 0.02 ± 0.01% for N2BKO). These data indicate that while the N2B element is not required for mechanotransduction in pressure overload of the LV, it is critical for mechanosensing and the adaptive hypertrophy response during volume overload. FHL2 is an attractive candidate as a link between titin’s N2B element and the hypertrophy response following ACF and further work will critical examine its involvement.
Abstract 535: Differences in Energetic Remodeling Between Right and Left Atria in Patients With Atrial Fibrillation
Authors: Emelyanova Larisa , Komas Steve , Olet Susan , Ryan Sean , Warner Catherine , Rizvi Farhan , Ross Gracious R. , Kress David C. , O’Hair Daniel P. , Downey Francis , Jahangir Arshad
Published: February 2019

Introduction:
Right and left atria have different susceptibilities toward developing atrial fibrillation (AF). The molecular bases of these differences are not well characterized. Given the complexity of AF development and progression, understanding AF-associated changes in myocardial energetics between the atria will help improve mechanistic insights and therapeutics for better clinical management of AF. The aim was to compare changes in mitochondrial oxidative phosphorylation system (OXPHOS), glycolysis and Krebs cycle metabolites in right atrial (RAA) and left atrial (LAA) appendage tissue from patients with (AF) and without (non-AF) AF.

Methods:
RAA and LAA from well-matched AF (n=54) and non-AF (n=58) patients undergoing elective open heart surgery was collected. Functional activity of OXPHOS complexes I-V was measured spectrophotometrically. Protein expression level of OXPHOS complexes was determined by Western blot. OXPHOS gene expression level was performed using RT-PCR. Metabolites were profiled using high performance liquid chromatography coupled to tandem mass spectrometry. Comparison between groups was done applying the 2 sample
t
- and Wilcoxon rank sum tests with 5% level of significance.

Results:
The most significant AF-associated alterations in myocardial energetics were observed in RAA. In AF patients, out of 84 OXPHOS genes, expression of 14 genes was significantly reduced in RAA (p<0.05) and 2 genes was reduced in LAA (p<0.05). There was AF-associated reduction in complex I (p=0.01) and IV (p=0.04) protein expression in RAA without changes in LAA. Unlike LAA, mitochondria from RAA revealed decline in complex I (p=0.01) and II (p=0.03) activity in AF compared to non-AF patients. In AF patients, glycolysis metabolites level of glucose-6-phosphate and phosphoenolpyruvate was reduced in RAA (p=0.03), whereas 2-phosphoglycerate was reduced in LAA (p=0.02). AF was related with decrease in NAD
+
(p=0.03), GDP (p=0.05), citrate (p=0.03), total pool of adenine nucleotides (p=0.02) and glutathione (p=0.03) level in RAA without changes in LAA.

Conclusion:
AF is associated with different energetic remodeling in right and left atria, suggesting that dissimilar mechanisms may contribute to development and progression of AF.
Abstract 541: LPP3 Deficiency Impairs Mitochondrial Function and Enhances Myocardial LPA Mediated Signaling
Authors: Miriyala Sumitra , Bhattarai Susmita , Sharma Sudha , LaBrie Timothy M , Tupper Hailey D , Escalante-Alcalde Diana , Bhuiyan Md. Shenuarin , Chandra Mini , Panchatcharam Manikandan
Published: February 2019

The enzyme autotaxin is responsible for the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA), and the enzyme lipid phosphate phosphatase 3 (LPP3) is responsible for the degradation of LPA. Recently, we have shown that LPP3 plays a prominent role in the heart compared to other isoforms of LPP(Redox Biol. 2018 Apr;14:261-271). LPP3 is localized on the Mitochondria-Associated Endoplasmic Reticulum Membranes (MEM), and changes in the activity of LPP3 in the MEM play a critical role in the transport of essential membranous lipids. Mitochondrial membranous lipids regulated by the MEM are necessary for the mitochondria to survive, as they are not produced in the mitochondria. Lack of LPP3 decreased both diacylglycerols (P < 0.001) and phosphatidylglycerol (P < 0.001) in the membrane fractions as assessed by mass spectrometry(MS/MS). Lpp3
Δ
myocardium showed distorted intercalated disc with damaged mitochondria with disorganized cristae and amorphous matrix densities as compared to lpp3
fl
in transmission electron microscopy. Lack of LPP3 resulted in deteriorating cardiac function. To further understand the mitochondrial regulation mediated by LPP3, we studied the effect of LPA on mitochondrial respiration under different conditions using the Seahorse XF24 analyzer to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cardiomyocytes. Our results show that the exposure to LPA significantly decreased OCR (P < 0.001) and increased ECAR (P < 0.001). The addition of autotaxin inhibitors PF8380 had a reverse effect on OCR and ECAR. The increase in OCR and reduction in ECAR was even more pronounced after blocking of LPA signaling using Brp-LPA receptor blocker (P < 0.001). Our real-time observation studies on mitochondrial oxidative phosphorylation status revealed mitochondrial dysfunction with increased production of superoxide (a normal byproduct of oxidative phosphorylation that is formed at increased rates when the electron transport chain is defective) in the cardiomyocytes that lack LPP3. The re-expression of LPP3 mitigated oxidative stress. This study is crucial for the understanding of the importance of LPP3 in the preservation of both cardiac and mitochondrial function.