bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 384: Predicting Drugs and Drug Targets Associated With Dilated Cardiomyopathy Using a Gene Expression-based Systems Medicine Platform
Authors: Wathieu Henri , Dakshanamurthy Sivanesan
Published: October 2020

Among the genetic and environmental contributors to dilated cardiomyopathy (DCM), the pathogenesis of drug-induced DCM is heterogeneous, poorly understood, and difficult to predict. In this study, transcriptomic data from two recently published datasets were used to identify genes which are differentially expressed in DCM compared to cardiac tissue from non-failing donors. DCM-associated genes were then mapped using gene set enrichment analysis to associated biochemical pathways, gene ontology functions, and protein-protein interactions, creating a DCM-associated biological perturbation network. Corresponding multi-scale pharmacologic activity networks for over 9000 drugs were produced using public repositories of known drug-protein interactions. Drugs were then rank-ordered based on multi-scale statistical significance of concordance with the DCM perturbation network while accounting for direction of effect using a causal analysis algorithm. A predictive algorithm using overrepresentation analysis was developed using the SIDER database of adverse drug reactions (ADRs) to formulate ADR-protein and ADR-pathway associations, which were then compared to the drug-DCM association networks produced by our model. In addition to a demonstrated concordance between the rank lists for each dataset, this platform prioritized multiple drugs and metabolites which are known to be associated with DCM, such as doxorubicin, risperidone, and 4-hydroxynonenal, as well as associated mechanisms such as bile acid excess and inhibition of DNA topoisomerase II and mitochondrial complex III. Multi-scale concordance of drug and DCM networks highlighted the involvement of heat shock proteins and associated angiotensin II activity, supporting current evidence that its inhibition may be cardioprotective to drug-induced DCM. Our approach also recapitulated the roles of drugs, genes, and pathways that hold prognostic and therapeutic significance for DCM, such as the abilities of sulforaphane and fenofibrate to prevent DCM in animal models of type 2 diabetes. This platform can be used to predict a wide array of adverse drug reactions and their underlying mechanisms based on bioactivity data.
Abstract 392: Angiotensin II TypeI Receptor-mediated Mitochondrial Fission and Suppression of Mitophagy Play Crucial Role in Vascular Senescence and Arteriosclerosis
Authors: Uchikado Yoshihiro , Ikeda Yoshiyuki , Sasaki Yuichi , Akasaki Yuichi , Ohishi Mitsuru
Published: October 2020

Introduction:
Metabolic stress including oxidized low density lipoprotein (ox-LDL) cause mitochondrial dysfunction and evoke vascular senescence and atherosclerosis. Mitochondria are highly dynamic organelles that undergo quality control by mitochondrial dynamics and mitophagy. This study aims to clarify whether and how mitochondrial dynamics and mitophagy are involved in the etiology of vascular senescence and arteriosclerosis.

Methods:
VSMC were stimulated by ox-LDL. We also conducted in vivo experiment using C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE mice and Angiotensin II Type1 Receptor (AT1R).

Results:
Treatment of ox-LDL forced mitochondria to fission through activation of Drp1, induced mitochondrial dysfunction and oxidative stress, and developed cellular senescence. Inhibition of either Drp1, AT1R, MAPK retarded them, suggesting that mitochondrial fission plays key roles to develop premature cellular senescence and is modulated by AT1R/MAPK signal.Administration of ox-LDL decreased the number of mitophagy assessed by electron microscopy and immunohistochemistry of LAMP2 and TOMM20. AT1R signal inhibition increased mitophagy which was not affected by Atg7 knockdown, whereas it was decreased by either Rab9 or Ulk1 knockdown. Immunohistochemistry showed Rab9 dots were co-localized to TOMM20 and LAMP2, whereas LC3 dots were not, suggesting that AT1R signal induces mitophagy through Rab9-dependent alternative autophagy. The degree of vascular senescence was higher, the number of fused mitochondria and mitochondrial function were lower and mitochondrial oxidative stress was higher in ApoE KO than those in WT. DKO attenuated these adverse effect of ApoE KO.

Conclusion:
AT1R regulates vascular senescence and arteriosclerosis via induction of mitochondrial fission and inhibition of mitophagy.
Abstract 397: Differentiation and Maturation of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes in Nanopatterned Cell Culture
Authors: Zhang Yiqiang , Choi Jong-Seob , Smith Alec , MacLellan Robb , Kim Deok-Ho , Zhang Yiqiang
Published: October 2020

Background:
Human induced pluripotent stem cells (hiPSCs) are widely used in studies of developmental and regenerative biomedicine involving various cell types, including cardiomyocytes (CMs). Understanding the cellular and molecular processes during hiPSC-CM differentiation and maturation will be crucial to develop heart regeneration therapies. In addition, while bioengineered cellular cues have been shown to modulate cellular morphology and phenotypes, little is known regarding their effects on molecular and functional maturity of hiPSC-CMs.

Aims:
To determine the expression of cardiac and cell cycle genes and the electrophysiological properties of hiPSC-CMs during their differentiation and maturation on unpatterned (Flat) or isotropic/nanopatterned (Nano) cell culture surface.

Methods and Results:
Using small molecules, WTC-11 hiPSC were differentiated into CMs efficiently (92.5% TNNT2
+
; 95% NKX2-5
+
). The resultant hiPSC-CMs were re-plated in Flat or Nano surfaces and harvested at different time points. Cardiac genes
Myh7, Tnnt2, Serca2a, Ryr2, Cacna1c
, and
Kcnj2
gradually and significantly increased during differentiation; this was accompanied by reduced expression of cell cycle genes. While myofilament genes expressions were similar between Nano and Flat cultured hiPSC-CMs, ion channel genes
Scn5a, Cacna1c
, and
Kcnj2
were significantly higher in Nano group, suggesting that Nano cultured CMs were more matured. In addition, fewer hiPSC-CMs were proliferative (EdU
+
) in 2-week Nano group compared to Flat group. This was associated with decreased expression of active cell cycle genes
Ccne1, Cdk4, Cdk14, Ki67
and
Plk1
in Nano 2-week CMs. Micro-electrode array (MEA) analysis demonstrated that Beat Period, Spike Amplitude, and Field Potential Duration were increased in the Nano group. Fluo-4 Ca
2+
imaging assay revealed improved Ca
2+
transition activities in isotropically cultured hiPSC-CMs.

Conclusion:
These results demonstrate a significant upregulation of cardiac genes along with a down-regulation of cell cycle genes during the differentiation and maturation of hiPSC-CM on Nano surfaces. Bioengineered nanotopographically patterned substrates promoted the maturation and electrophysiological functions of hiPSC-CMs.
Abstract 403: Leveraging the Zebrafish to Overcome Barriers in Heart Transplantation
Authors: Tessier Shannon N , Da Silveira Cavalcante Luciana , Pendexter Casie A , Cronin Stephanie E , de Vries Reinier J , Ellett Felix , Marques Beatriz , Gopinathan Kaustav , To Samantha , Gonzalez-rosa Juan Manuel , Langenau David M , Uygun Korkut , Toner Mehmet
Published: October 2020

Cardiac transplantation is the only curative therapy for patients with end-stage heart disease; however, there is a severe shortage of viable donor organs. Heart transplantation faces many interwoven challenges, including both biological factors and research limitations. For example, ischemia-reperfusion injury plays a role in early graft dysfunction and is associated with rejection episodes in heart transplantation. Moreover, experimental transplantation relies heavily on animal studies that are laborious and expensive, prohibiting the discovery of novel, bold solutions. We propose that the zebrafish,
Danio rerio
, would be a valuable tool for the field since it’s amenable to high-throughput screens, captures the complex structure of organs, and offers a suite of tools to monitor the biology of cardiac injury. Here, we develop a new subzero heart preservation method by strategically leveraging animal models from zebrafish to mammalian hearts. Using zebrafish larvae, we screened for agents which preserve hearts at -10°C. As a result of these screens, we identified promising preservative cocktails which restored heartbeat in 82% of larvae immediately post-recovery. Next, we excised adult zebrafish hearts and developed methods to mimic the
ex vivo
handling practices of hearts destined for transplant using a heart-on-a-plate assay. Using this assay, we carried forward promising agents identified in our initial zebrafish larvae screen to isolated adult zebrafish hearts that were cooled to -10°C and held for up to 24 hours. After rewarming, heart rate was restored and metabolic rate of zebrafish hearts was like time-matched controls (0.213 ± 0.047 and 0.275 ± 0.060, respectively, p = 0.200). Finally, we report our preliminary scale-up efforts whereby rodent hearts are stored for up to 24 hours at -10°C and viability were assessed by the TUNEL assay. The data shows high viability of cardiomyocytes post-preservation, as compared to controls. In summary, we present data to illustrate our efforts in leveraging the zebrafish to aid new discoveries in subzero heart preservation. Similar efforts to model heart transplantation in zebrafish may provide a different vantage point and enable us to make advances faster.
Abstract 408: An Essential Protective Role for Cardiomyocyte Alpha1a-adrenergic Receptors in a Mouse Model of Myocardial Infarction
Authors: Zhang Jiandong , Ash Tyler , Huang Wei , Smith Alan , Huang Hsiao-ying , Jensen Brian C
Published: October 2020

Our previous work reveals that dabuzalgron, an oral agonist of alpha-1A adrenergic receptors (α1A-ARs) protects rodents from pathological cardiac stress, a finding in agreement with genetic studies of global α1A-AR overexpression/inactivation. However, it remains largely obscure whether cardiomyocyte-specific actions of α1A-ARs alone determines these cardioprotective effects. To test this essential gap in knowledge, we generated a new mouse line lacking α1A-AR only on cardiomyocytes by crossing αMHC-cre mice with floxed α1A-KO mice (CMKO= cre+ fl/fl, CMWT= cre-fl/fl). After validating specific deletion, baseline features were measured by conscious echo. No overt structural or functional anomaly was identified in either group. Fractional Shortening (FS, %) was virtually identical in 2 groups (51.7±0.7 vs. 52.4±5.3, p=NS). Next, both groups were subjected to permanent left anterior descending (LAD) ligation. By day 14, CMKO had significantly higher mortality (9/15 vs 2/13, p=0.025). To determine early changes that might account for this difference, hearts were harvested at day 3 post-ligation from another cohort. HW did not differ between two groups (7.0±1.1 vs. 7.2±1.1 mg/g·BW, p=NS), neither did LW (8.0±2.1 vs. 7.2±1.0 mg/g·BW, p=NS). Serum troponin level was numerically higher in CMKO mice (8.4±12.4 vs 4.8±6.2 ng/mL,p=0.38). CMKO mice had 51% larger injury (infarcted and leukocyte-infiltrating area) compared to CMWT (31.0±10.5 vs. 20.5±4.9% of LV area, p=0.05). Our preliminary postmortem examination suggested a higher trend of cardiac rupture in injured CMKO (3/5 vs 0/5). To test myocardial biomechanical properties between 2 groups, ventricles from mice 3d post ligation were then subjected to biaxial mechanical stress test. Myocardium from infracted CMKO mice exhibited 70% more stiffness (dt Stress/dt Strain) CMWT (190±34.29 vs.111±42 kPa, p=0.004). Collectively, our results demonstrate that mice deficient of α1A-AR on cardiomyocytes had lower late survival, more profound early pathological injury & adverse mechanical properties after LAD ligation, suggesting that activation of cardiomyocyte α1A-ARs plays an essential role in protecting experimental myocadiac infarction, holds the promise of future clinical implications.
Abstract 410: Transthyretin Amyloid Fibrils Deposited in the Microenvironment Alter Cardiac Myocyte and Fibroblast Function and Cytoskeletal Structure
Authors: Dittloff Kyle , Iezzi Antonio , Banach Kathrin , Russell Brenda
Published: October 2020

Age-related wild type transthyretin amyloidosis (ATTRwt) is a systemic condition that is characterized by deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in various organ systems. This disease manifests as an infiltrative cardiomyopathy hallmarked by extracellular deposition of misfolded TTR fibrils in the cardiac tissue, leading to cardiac dysfunction and heart failure with preserved ejection fraction (HFpEF); this disease is underdiagnosed and may affect 25% of elderly men and women. We hypothesize that deposition of amyloidogenic TTR fibrils alters cardiomyocyte contractility and sarcomere structure via modulation of cell growth due to altered microenvironmental features such as substrate stiffness. Recombinant TTR fibrils were deposited on glass substrates or substrates mimicking the stiffness of the healthy myocardium (10kPa). Neonatal rat ventricular myocytes exhibited decreased rate of linear contractility via line scan kymography and altered cytoskeletal structure when exposed to deposited TTR fibrils, with greater maladaptive effects seen on 10kPa substrates. Isolated adult mouse atrial cardiomyocytes plated on TTR fibril-containing substrates had attenuated calcium transient amplitude and a prolonged decay constant, consistent with lower amplitude. We also hypothesize that deposited TTR fibrils induce cardiac fibroblast activation and promote progression of fibrosis. Isolated cardiac fibroblasts grown on TTR-coated glass showed a reorganization of the F-actin cytoskeleton, with preferential distribution to the cell periphery. Barrier removal tests after confluence resulted in significantly faster migration velocity of fibroblasts cultured on TTR fibrils. Increased reduction of MTS tetrazolium by a colorimetric assay suggests a significant TTR-related increase in the proliferation rate of cells. Together, results suggest that amyloidogenic TTR fibrils may affect cardiac myocyte and fibroblast structure and function through both chemical and mechanical pathways. As recently approved therapies for ATTRwt are cost-prohibitive and are intended to slow disease progression only, better understanding of disease mechanisms may elucidate novel therapeutic targets.
Abstract 411: Absence of Sexual Dimorphism in Isoproterenol-induced Cardiac Dysfunction in C57BL/6 Mice
Authors: Grant Marianne K , Seelig Davis , Abdelgawad Ibrahim , Zordoky Beshay
Published: October 2020

Sex-related differences in cardiovascular diseases are complex and highly context-dependent. The objective of this work was to comprehensively determine key sex differences in the response to acute and chronic adrenergic stimulation in C57Bl/6 mice. In the current work, there was no statistically significant difference in key echocardiographic parameters between male and female C57Bl/6 mice in response to acute adrenergic stimulation (a single sub-cutaneous dose of isoproterenol 10 mg/kg). After chronic adrenergic administration (sub-cutaneous injections of isoproterenol 10 mg/kg/day for 14 days), there was similar degree of cardiac dysfunction, cardiac hypertrophy, and myocardial fibrosis in male and female mice. Similarly, chronic isoproterenol administration induced hypertrophic and fibrotic genes in hearts of male and female mice to the same extent. Intriguingly, gonadectomy of male and female mice did not have a significant impact on isoproterenol-induced cardiac dysfunction as compared to sham-operated animals. In conclusion, the current work demonstrated lack of sex-related differences in isoproterenol-induced cardiac hypertrophy, dysfunction, and fibrosis in C57Bl/6 mice. This study challenges the conventional dogma of the detrimental cardiovascular effects of male sex hormones and the beneficial effects of female sex hormones.
Abstract 415: Asporin Contributes to Reactive Interstitial Cardiac Fibrosis by Regulating Cardiac Fibroblast Activation
Authors: Medzikovic Lejla , Aryan Laila , Ruffenach Gregoire , Eghbali Mansoureh
Published: October 2020

Cardiac fibrosis critically contributes to heart failure progression. Depending on the pathological insult, cardiac fibrosis either replaces necrotic cardiomyocytes or is reactive to cardiac fibroblast (CF) activation. The extracellular matrix (ECM) consists of various proteins and the role of fibrillar collagen has been well studied. However, the role of non-fibrillar ECM proteins in cardiac fibrosis is less clear. To explore the role of ECM in reactive cardiac fibrosis, we performed bioinformatic analysis on online available microarray GEO datasets from hearts of human hypertrophic cardiomyopathy patients and two mouse models of transverse aortic constriction and Angiotensin II (AngII) infusion. We found that 27 differentially expressed genes were common between the three datasets. Among these genes was the small leucine-rich proteoglycan Asporin (ASPN). ASPN was previously shown to be upregulated in the ECM of replacement fibrosis in porcine ischemia/reperfusion injury. However, not much is known about the role of ASPN in reactive interstitial fibrosis. We show that cardiac ASPN expression is enhanced in mice after short- and long-term AngII infusion compared to saline infusion. In resident CF isolated from adult mice, ASPN expression is upregulated by both AngII and TGF-β stimulation. Here, ASPN expression correlates with a gene signature of activated CFs including periostin (
postn
), α-smooth muscle actin (
acta2
) and collagens I and III (
col1a1, col3a1
), and with functional characteristics of activated CFs including proliferation, migration and collagen production. Modulating ASPN via siRNA in mouse resident CFs inhibits
postn, acta2, col1a1
and
col3a1
expression and total collagen production, indicating repressed CF activation upon ASPN knockdown. Taken together, ASPN may be an attractive novel target against reactive interstitial fibrosis.
Abstract 416: Sarcomere Disassembly After Unloading is Regulated by Ubiquitination and Acetylation of Capz and α-actinin
Authors: Solís Christopher , Solaro R John , Warren Chad M , Russell Brenda
Published: October 2020

Cardiac function mainly depends on the total myocyte mass in the ventricles. Assembly and disassembly of sarcomeres occurs to adjust this mass to altered mechanical demand. In the heart, hypertrophic cardiomyopathy results from myofibrillar assembly controlled by post-translational modification of proteins directed by signaling pathways. More is known about assembly on loading than disassembly on unloading. Here, the hypothesis tested is that unloading of mechanical forces affects acetylation (Ac) and ubiquitination (Ub) of the actin-binding proteins, α-actinin and CapZ. Omecamtiv mecarbil (0.5 μM) and mavacamten (1 μM) were used to increase (load) and decrease (unload) cardiomyocyte tension, respectively, via their action on myosin ATPase. Mavacamten decreased myocyte contractility in rat ventricular myocytes (NRVMs) and caused significant sarcomere disassembly by 6 h and 70% atrophy by 24 h. Assembly was preserved with omecamtiv mecarbil (0.5 μM) over the 24 h time period. Post-translational modification was determined in loaded and unloaded NRVMs at 6 h of drug treatment. Bottom-up mass spectrometry analysis showed single residues in α-actinin and CapZ that were acetylated or ubiquitinated. Acetylation levels appeared to increase in the mavacamten-treated samples while these levels are preserved in untreated and omecamtiv mecarbil-treated samples. Ac and Ub in the Z-discs were quantified on immunofluorescent images. The Z-discs colocalized oligo-Ub (K-48 oligo-Ub linkage) and Ac in untreated samples; this Z-disc localization of Ub and Ac was diminished with unloading. Fluorescence recovery after photobleaching (FRAP) measurements of the dynamics of α-actinin and CapZ after reduced cell tension with mavacamten (1 μM) and omecamtiv mercabil (0.5 μM) are ongoing. Overall, results suggest sarcomere assembly is regulated by mechanical forces through a mechanism involving Ac and Ub of myofibrillar proteins. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis.
Abstract 428: Cortical Bone Stem Cell-derived Exosomes Reduce Myofibroblast Activation in Rat and Human Cardiac Fibroblasts
Authors: Schena Giana , KUBO Hajime , Feldsott Eric , Headrick Alaina , Koch Keith , Yang Yijun , Mohsin Sadia , McKinsey Timothy , Houser Steve
Published: October 2020

Rationale:
Heart failure is the one of the leading causes of death in the United States. Post-myocardial infarction is followed by cardiac remodeling that involves extensive fibrosis and ultimately progresses into heart failure. We have seen improvements in scar size and cardiac function as a result of administration of cortical bone stem cell-derived (CBSC) exosomes.

Objectives:
We investigated the mechanism through which CBSC-derived exosomes altered wound healing and reduced scar formation through
in vitro
experimentation. We continue to broaden our understanding of how CBSCs exert their anti-fibrotic effects.

Methods and Results:
We cultured adult rat ventricular fibroblasts and adult human cardiac fibroblasts and treated them +/- TGF β with mouse and human CBSC-derived exosomes, respectively. We saw a dose-dependent decrease in myofibroblast activation with increasing concentrations of mCBSC and hCBSC exosomes, with 100 fold decrease compared to baseline fibroblast activation in hCBSC treated cardiac fibroblasts (9.5 x 10
5
of 1.6 x 10
7
intensity), and by 40% in mCBSC treated cardiac fibroblasts (2.4 x 10
5
of 1.0 x 10
6
intensity). In the presence of TGFβ and 2.0 x 10
6
exosome particles/cell treated, the αSMA levels were still reduced by nearly 50% vs TGFβ alone (1.2 x 10
6
of 2.2 x 10
7
intensity). We performed RNA sequencing on both the rat and human cardiac fibroblasts in order to discover which fibrosis-related genes were being altered by CBSC exosomes treatment.

Conclusions:
Our findings show that the wound healing induced by CBSC exosome treatment post-MI involves the reduction of myofibroblast activation and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.
Abstract 432: Loss of Primary Cilia PertErbBs Ventricular Maturation in the Developing Heart
Authors: Fitzsimons Lindsey A , Ryzhov Sergey V , Tucker Kerry L
Published: October 2020

The embryonic heart requires cardiac neural crest cells (CNCC), which elaborate the sensory organelle called the primary cilium and assist in the development of the cardiac valves, the interventricular septum, and the outflow tract. Our laboratory has modeled congenital heart defects (CHD) resulting from CNCC-specific ciliary loss, using a Wnt1:Cre-2,
Ift88
-targeted conditional elimination of primary cilia.
Ift88
-homozygous mutants (MUT) displayed a variety of predicted CNCC-associated CHDs, but also revealed a novel disorganization of the ventricular endocardium, pronounced noncompaction of the ventricular myocardium, and perinatal lethality. Neuregulin-1 / ErbB signaling is a well-known key modulator of myocardial development as well as the compaction process of the ventricular myocardium. Flow cytometric analysis of embryonic day 12.5 (E12.5) whole hearts revealed that loss of cilia in CNCC of MUT hearts led to a 43% reduction in cardiomyocytes (CM), defined as VCAM-1-positive/CD31-negative cells (p<0.01), and a trend towards increased CD31-positive endocardial cell (EC) populations (34% increase; p = 0.09) when compared to control (CON) hearts. Analysis of cell surface expression of ErbB receptors revealed a slight decrease in ErbB2 and ErbB4 expression in VCAM-1-positive CM of MUT with no change in ErbB3 expression, and decreased ErbB4 expression in MUT EC. Immunofluorescence analysis of proliferation was quantified in various cardiac subpopulations, including cardiac Troponin-T-positive (cTnT+/CM) cells in the ventricular myocardium. While overall cell density at E12.5 did not differ between the MUT and CON (p=0.43), CM proliferation increased significantly in MUT (5.50%) when compared to CON (2.70%; p<0.01). Taken together, decreased ErbB4 expression in the expanding MUT EC population, combined with decreased CM numbers, could serve as a novel mechanistic explanation for the pronounced hypertrabeculation and noncompaction of the MUT phenotype seen at E14.5. Our results indicate that loss of CNCC primary cilia leads to ventricular noncompaction, with a shift in the relative proportion and dynamics of both CM and EC subpopulations, which is potentially attributable to impaired ErbB signaling of the developing endocardium.
Abstract 435: P53 Temporally Regulates the Myofibroblast Activation Response in Pressure Overload
Authors: Burke Ryan M , Lighthouse Janet , Quijada Pearl J , Dirkx Ronald , Small Eric M
Published: October 2020

Heart disease is invariably accompanied by the accumulation of resident cardiac fibroblasts (CF) that secrete extracellular matrix and drive the progression of fibrosis and cardiac dysfunction in heart failure. We recently reported that CF exhibit a unique transcriptional response in physiological and pathological remodeling that may underlie the cardioprotective benefits of exercise, and that p53 targets represent the most dysregulated family between the two. To further investigate the role of p53 in pathological cardiac remodeling, we deleted floxed p53 alleles in adult CF using the tamoxifen-inducible
Tcf21
MerCreMer
mouse (p53-CFKO). p53-CFKO mice initially retain normal cardiac function through 14 days of pressure overload, but eventually progress to heart failure more rapidly than do p53-CFWT, which show gradual functional decline throughout. CF accumulation and fibrosis at these time points match the functional results closely, with p53-CFKO mice protected from both at 14 days relative to p53-CFWT. Conversely, deletion of p53 in myofibroblasts (MF) using the tamoxifen-inducible
Postn
MerCreMer
mouse (p53-MFKO) resulted in no delay in functional loss nor in CF accumulation and fibrosis, suggesting a distinct temporal window of p53 activity in CF exists in pressure overload.
In vitro
experimentation confirmed that adenovirally-mediated deletion of p53 could delay, but not reverse, myofibroblast activation - and that this irreversible activation is due to induction of
Cdkn2a
-mediated cell cycle arrest. Furthermore, unbiased single-cell transcriptomic analysis of CF from pressure-overloaded
Tcf21
MerCreMer
mice at the 14-day functional inflection point revealed p53-CFKO mice have a unique CF cluster defined primarily by a mitotic signature, and this cluster comes at the direct expense of the myofibroblast population. Clusters representing migrating and quiescent CF were unchanged in response to p53. This work describes an inverse correlation of proliferative capacity and myofibroblast activation that indicates p53 exerts master control of the timing of CF accumulation and pathological fibrosis in heart disease.
Abstract 436: Maternal Pregestational Diabetes Alters Fetal Gene Regulatory Program to Cause Spectrum of Congenital Heart Defects
Authors: Mansfield Corrin , Manivannan Sathiyanarayanan , Zhang Xinmin , Kodigepalli Madhav K , Garg Vidu , Basu Madhumita
Published: October 2020

Maternal pre-gestational diabetes results in range of congenital malformations with significantpreponderance of congenital heart defects (CHD). We and others have previously demonstratedthat maternal high glucose (HG) acts as primary teratogen in diabetes and in combination withgenetic susceptibility loci, it increases the incidence of CHD ranging from mild ventricular septaldefects (VSD) to complex outflow tract (OFT) abnormalities.
However, the underlying cellularand molecular basis of maternal HG dose-response on fetal cardiac development remainselusive.
In this study, we have utilized Streptozotocin-induced murine model of maternal type1diabetes and demonstrated that spectrum of CHD in HG exposed developing embryos
in utero
,is dose-dependent. We have performed histological analysis of hearts at embryonic (E) day 9.5,11.5, 13.5 and 15.5 exposed to different levels of maternal HG and compared them withuntreated normoglycemic controls. Analysis of E9.5, E11.5 and E13.5 embryos displayedtrabeculation and touchdown defects, thinning of right and left ventricular myocardium, VSD,OFT misalignment, double outlet right ventricle with increasing levels of maternal glucose.Whereas, E15.5 embryos exposed to maternal HG exhibited VSD, cardiac hypertrophy andhyper-trabeculation phenotypes in comparison to normoglycemic controls. To test cell-specificgene-regulatory program in response to hyperglycemia stress, we performed
in vivo
10X singlecell transcriptomics and
in vitro
global RNA-sequencing on murine embryonic cell-lines culturedin normal and increasing doses of HG. RNA-seq analysis revealed differential gene expressionchanges associated with metabolic dysfunction, oxidative stress response, endothelial tomesenchymal transition, cardiomyocytes proliferation and impaired neural crest cell migration.Cardiomyocytes and endothelial cell proliferation (PHH3) and apoptosis (TUNEL) weremeasured using immunohistochemical analysis at E9.5-E15.5 embryonic hearts exposed tomaternal HG. In summary, more in-depth analysis of this study is warranted to indicate howmaternal intrauterine hyperglycemic environment disrupts the fine tuning of fetal cardiogenicgene/epigenetic program to cause CHD.
Abstract 437: A Novel Variant Cell Cycle Related Kinase Protect Heart Against Ischemia by Promoting Cardiomyocytes Survival
Authors: Lai Lo , Qiu Hongyu
Published: October 2020

Background and objects:
Our previous studies identified a novel variant of cell cycle-related kinase in heart (cardiac CCRK or cCCRK) which is different from generic CCRK found in other tissues in terms of its amino acid sequence, substrates, protein-protein interaction and tissue distribution. cCCRK has been found dramatically decreased in the failed heart tissues, while overexpress of cCCRK in cardiac myocytes in vitro protected the cells against induced cell death. Therefore, we tested the hypothesis here that cCCRK acts as a potential candidate of cell survival promotor providing cardiaoprotection in vivo upon cardiac ischemic stress.

Methods and results:
A cardiac specific transgenic (TG) mouse was generated with an alpha- MHC premotor in which cCCRK was overexpressed by 3.5 folds compared to wild type mice (WT). Both cCCRK TG and the litter-mated WT mice were subjected to an 45 minutes myocardial ischemia followed by an 24 hours reperfusion (IR), and the infarct size was measured by 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We found that despite the similar risk area, infarct size was significantly reduced by 50% in the TG mice verse WT mice (p<0.05). The apoptosis in the adjacent area of the heart tissues measured by TUNEL was reduced by 30% in TG mice vs WT (p<0.05). Mechanistically, the survival-promoting signaling pathway, including phosphorylated extracellular-signal-regulated kinase (p-ERK), phosphorylated Mitogen-activated protein kinase/ERK kinase (p-MEK), and P21, was activated in the heart tissues from TG vs WT mice (p<0.05 vs WT). Overexpression of cCCRK in cardiac myocytes in vitro reduced chelerythrine-induced apoptosis reflected by a reduction of caspase 3 activity, this protection was abolished by the Inhibition of MEK with the treatment of its inhibitor U1206 vs vehicle controls (p<0.05 vs vehicle).

Conclusion:
This study demonstrated that overexpression of a variant of CCRK in heart provides protection against ischemic stress by activating survival signaling.
Abstract 442: Epigenetic Regulation of Ezh2 in Direct Human Cardiac Reprogramming
Authors: Zhou Yang , Tang Yawen , Zhao Lianzhong , Lu Rui , Zhang Jianyi
Published: October 2020

Cardiovascular disease is still the leading cause of death in the United States. Due to the limited regenerative capacity of adult hearts, the damage caused by heart injury could not be reversed and often progressed into heart failure. In need of cardiovascular disease treatment, many therapies aimed at either cell transplantation or cell regeneration have been proposed. Direct reprogramming of somatic cells into induced cardiomyocytes (iCMs) is considered to be a promising strategy for regenerative medicine. The induction of cardiomyocytes from non-myocytes has been achieved efficiently via ectopic expression of reprogramming factors both in vitro and in vivo with mice models. However, as human cells are more resistant to the reprogramming process, the generation of human iCMs (hiCMs) has been restricted by the factor that using more complex cocktails generated only functionally immature cells with lower efficiency and longer conversion time. The inefficiency of hiCMs production called for the identification and elucidation of underlying species-specific regulatory mechanisms in human, and removal of the additional epigenetic barriers which might be damping the hiCMs reprogramming. Here, we identified a human-specific epigenetic barrier, Enhancer of zesta homolog 2 (EZH2), via an unbiased loss-of-function screening. With the knockdown of EZH2, the hiCM reprogramming efficiency was significantly increased, accompanied with profound repression of collagen and extracellular matrix genes, which are related to the formation of fibrosis. Consistently, Inhibition of EZH2 catalytic activity via small molecules promotes hiCM reprogramming, suggesting that EZH2’s inhibitory effect was mediated by epigenetic regulation of histone modifications. Therefore, our study revealed a previously unrecognized regulatory mechanism of human cardiac reprogramming, which allows us to overcome the fibroblast fate barriers and ease the cardiac cell fate conversion.
Abstract 447: Myristoylation of Protein Kinase C Beta II Peptide Inhibitor Facilitates Rapid Attenuation of Phorbol 12-myristate 13-acetate in Activated Superoxide Release in Isolated Rat Polymorphonuclear Leukocytes
Authors: Singh Sunit G , Dean Tameka , Lloyd Michael , Decker Rourke , Castro Jeremy , Madison Ian T , Suarez Michael , Martorana Rose , Beale Melinda , Gjoka Redona , Metellus Daphne , Humayun Annam , Dang Jennifer , Chen Qian , Barsotti Robert , Young Lindon
Published: October 2020

Protein kinase C beta II (PKCβII) activates polymorphonuclear leukocyte (PMN) superoxide (SO) production via NADPH oxidase (NOX-2) phosphorylation to exacerbate myocardial ischemia/reperfusion (I/R) injury. In prior studies, myristoylation (myr) of PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-), which disrupts PKCβII translocation/phosphorylation of NOX-2, was shown to dose-dependently attenuate PMN SO release induced by phorbol 12-myristate 13-acetate (PMA), a broad-spectrum PKC agonist. However, the role of myr on the inhibitory effects of myr-PKCβII- has yet to be elucidated. We hypothesized that myr-PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) would augment, myr-PKCβII- would attenuate, and scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram), a control for myr, would not affect PMA-induced PMN SO release compared to unconjugated peptides and nontreated controls. Rat PMNs (5х10
6
) were incubated for 15 min at 37
o
C in the presence/absence of SO dismutase (SOD; 10 μg/mL), unconjugated PKCβII+/-, myr-PKCβII+/-, or myr-PKCβII-scram (all 20 μM). SO release was measured by the change in absorbance at 550 nm via ferricytochrome
c
reduction after PMA (100 nM) stimulation for 390 sec. Data were analyzed by ANOVA using Student-Newman-Keuls post hoc analysis. Myr-PKCβII- significantly attenuated SO release (0.30±0.02; n=27; p<0.05) compared to nontreated controls (0.46±0.01; n=73), myr-PKCβII+ (0.46±0.03; n=25), unconjugated PKCβII+ (0.43±0.04; n=15), PKCβII- (0.43±0.02; n=22) and myr-PKCβII-scram (0.65±0.04; n=22). SOD (n=8), which rapidly converts SO to H
2
O
2
, significantly reduced absorbance by 94±7%, indicating that absorbance increased mainly due to PMA stimulation. Cell viability (trypan blue exclusion) was similar in all groups (94±2%). Unexpectedly, myr-PKCβII-scram significantly stimulated the highest increase in absorbance compared to all groups (p<0.01). Future studies will determine whether myr-PKCβII-scram augments absorbance by a different mechanism. Results suggest that myr improves myr-PKCβII- delivery compared to unconjugated PKCβII- but does not affect inhibition of PMA-induced PMN SO release. Myr-PKCβII- may thus effectively limit inflammation-induced I/R injury.
Abstract 452: Role of CD38 in Cardiovascular Performance and Its Use as a Therapeutic Target in Obesity-Induced Diastolic Dysfunction
Authors: Agorrody Guillermo , Peclat Thais , Gomez Lilian S , Kashyap Sonu , Escande Carlos , Contreras Paola , Chini Eduardo N
Published: October 2020

Background:
Cardiac tissue possesses high energetic requirements. During development of heart failure there is a shift from fatty acid oxidation to the utilization of other substrates. This transition leads to the decline in NAD/NADH ratio, which increases susceptibility to stress. CD38 is the main NAD
+
-consuming enzyme responsible for NAD
+
levels regulation in tissue and therefore represents a possible therapeutic target in such conditions.

Objective:
To investigate the role of CD38 in cardiovascular performance and its potential use as a therapeutic target in obesity-induced heart failure.

Results:
When comparing adult Wild-Type (WT) and CD38 Knock-out (KO) mice we observed that the latter had higher exercise performance, increased heart rate variability (HRV) and decreased heart rate (HR). When comparing WT and CD38 catalytically inactive (CI) mice we observed the same results, showing its relation with CD38 catalytic activity. WT mice treated with an antibody which blocks CD38 activity showed improved exercise capacity and HRV. In both genetically modified or antibody treated groups, NAD+ levels were significantly increased in heart. In order to explore the NAD
+
role in these findings, we compared WT control and CD38KO treated with FK866 (Nampt inhibitor) and observed that NAD+ levels in heart decreased as well as exercise capacity. We then aimed to demonstrate CD38 role as a target for therapeutic intervention in a model of cardiac strain induced by obesity in aged mice. We compared WT and CD38 CI aged mice, placed in normal diet (ND) or high fat diet (HFD). WT animals fed HFD showed decreased exercise capacity and this effect is reversed in CD38 CI fed HFD. Similarly, WT placed in HFD developed diastolic dysfunction seen as a significant increase in E/e’ ratio, and CD38 CI placed in HFD were protect against the development of this dysfunction. Mechanistically, we observed that blockage of CD38 resulted in decreased protein acetylation and increased SERCA expression.

Conclusion:
CD38 plays a role in cardiovascular performance by regulation of NAD
+
homeostasis. In a model of obesity-induced diastolic dysfunction NAD+-boosting by blocking CD38 activity led to protection by up-regulating SERCA expression.
Abstract 454: Modeling
FLNC
Mutations Causing Cardiomyopathy
Authors: Ohiri Joyce C
Published: October 2020

Genetic mutations lead to cardiomyopathy, a condition linked to heart failure and arrhythmias.Mutations in the
FLNC
gene have recently been reported in multiple forms of cardiomyopathyincluding dilated, hypertrophic and restrictive forms. The
FLNC
gene encodes filamin C, anactin-binding protein highly expressed in the heart and skeletal muscle. Filamin C is comprisedof three domains: an actin-binding domain at the N-terminus, a rod domain consisting of 24 Igrepeats in the mid region, and a dimerization domain at the C-terminus. Missense mutations in
FLNC
have also been described in myofibrillar myopathy, a disorder in skeletal musclecharacterized by intracellular aggregates within muscle. Where examined,
FLNC
mutant heartsdo not have evident protein aggregation leading to the hypothesis that
FLNC
mutations havedifferent modes of action in skeletal and cardiac muscle. We identified a
FLNC
Val2715fs87Xmutation in a 55 yo M with cardiac fibrosis and a history of arrhythmias. We found a second
FLNC
mutation, Glu2458SerfsX71, which was found in a family with sudden death andhypertrophic cardiomyopathy. A third
FLNC
mutation, Arg650X, was found in a 41 yo F withhistory of postpartum cardiomyopathy; this patient also harbors a second
FLNC
variant ofuncertain significance, intron 5, c.970-4A>G (Intronic), which is predicted to affect splicing onher other FLNC allele. Notably, this patient was diagnosed with a muscle disorder in early life.Finally, a fourth
FLNC
variant, Phe106Leu, found in two brothers with skeletal and cardiacmyopathy was also studied. To examine the mechanisms by which
FLNC
mutations causecardiomyopathy, we generated patient-derived induced pluripotent stem cells (iPSCs) anddifferentiated these cell lines into cardiomyocytes. Cardiomyocyte function was analyzed usingautomated field potential measurements with the Nanion CardioExcyte. We identified a slowerrate of repolarization with
FLNC
truncations compared to control. These data demonstrate thepathogenic findings associated with FLNC mutations and provide mechanistic insight intocardiomyopathy and arrhythmias resulting from these mutations.
Abstract 455: The Roles of VEGF Receptors in Human Cardiac Progenitor Cell Contribution to New Vascular Formation
Authors: Zhao Hanqing , Ponnambalam Sreenivasan , Smith Andrew J
Published: October 2020

Background:
Human cardiac progenitor cells (CPCs) have been shown to play a valuable role in myocardial tissue maintenance, including their ability to develop into endothelial and vascular smooth muscle cells. The vascular endothelial growth factor (VEGF) ligand family has been identified as essential for angiogenesis. VEGF receptors (VEGFRs) comprise three main subtypes, VEGFRs1,2,3; our prior data identified CPC expression of both VEGFRs and of pro-angiogenic secreted growth factors.

Hypothesis:
Human CPCs utilise VEGFR signalling to potentiate CPC-driven angiogenesis, directly via CPC differentiation and indirectly via secrotome.

Methods:
Human adult myocardial tissue samples were collected during cardiac surgery and c-Kit-positive (c-Kit
+
), CD45-negative (CD45
-
) CPCs isolated by immunomagnetic bead sorting, with five CPC lines generated from individual-donor samples. The c-Kit
+
/ CD45
-
CPC population was then characterised by clonogenicity assay, immunocytochemistry (ICC), and real-time RT-qPCR. Human CPC lines were FACS sorted into 3 lineage groups: endothelial (CD31
+
), smooth muscle (CD91
+
/CD140b
+
/CD31
-
) and uncommitted (CD91
-
/CD140b
-
/CD31
-
). Expression of VEGFRs and markers (SDF1; TGF-β) in CPC sub-populations was assessed by: qPCR; Western blot; ICC. Impact on signal transduction by VEGF-A stimulation was identified by Western blot and ICC.

Results:
Human CPCs were sorted into populations of: endothelial linage CD31
+
(1.26% of total cells), smooth muscle lineage CD91
+
/CD140b
+
/CD31
-
(13.77%) and CD91
-
/CD140b
-
/CD31
-
(31.28%) cells. Analysis of gene expression identified VEGFRs 1, 2 and 3 in all three sub-populations, but only VEGFR1 expression was confirmed at protein level, seen in all three sub-populations. High expression levels of growth factors secreted by CPCs (SDF, TGF-β, VEGFs, FGF-2) were identified in human CPCs, also in all three sub-populations.

Conclusion:
Human CPC lines were isolated and analysed in bulk and sub-populations, identifying VEGFR1 expression at both gene and protein levels, but not VEGFR2 and VEGFR3. Our further work will identify signalling pathways in human CPCs linked to VEGF-A stimulation, along with the impact of VEFG-A stimulation on CPC secretome and linked angiogenic potential.
Abstract 457: Outer Membrane Vesicle From Pseudomonas Aeruginosa Causes Inflammation and Cardiac Dysfunction
Authors: Kumar Naresh , Pestrak Matthew , Wu Qian , dellos-nolan Sheri , Yu Lianbo , Lafuse William , Ganesan Latha , Wozniak Daniel , Rajaram Murugesan
Published: October 2020

Rationale:
P. aeruginosa
(
P.a
) infection causes severe pneumonia in ICU patients that lead to cardiac inflammation, eventual tissue remodeling, fibrosis, and cardiac complication. Growing evidence indicates that cardiac resident CCR2
-
, MHC-II
low
macrophages function as important regulators of cardiac inflammation and steady-state heart function. After tissue injury or infection, inflammatory monocytes are recruited to the heart and differentiate into inflammatory macrophages, which contribute to cardiac inflammation and dysfunction. Our preliminary result showed
P.a.
infection causes cardiac inflammation, fibrosis, and cardiac dysfunction without disseminating into the heart tissue. Also, the gram-negative bacteria can release toxins or outer membrane vesicles (OMVs) that target host cells and induces apoptosis. Thus we hypothesize that the cardiac damage caused by
P.a
infection is mainly due to the release of bacterial toxins and OMVs or inflammatory mediators released by immune cells. Understanding the molecular mechanism of cardiac inflammation and dysfunction is critical for the development of host-directed therapy.

Objective:
To identify the mechanism of
P.a
induced inflammation and cardiac dysfunction to develop therapies to prevent cardiac damage during infection.

Methods and Results:
To investigate the role of OMVs in cardiac inflammation and cardiac dysfunction, we have isolated OMVs from
P. aeruginosa
culture supernatant and purified using ultracentrifugation followed by gradient centrifugation. We quantified OMVs and used to stimulate the MDMs, BMDMs and hiPSC cardiomyocytes. Our
in vitro
study showed that OMVs activates Akt and MAPK signaling pathways and induces the production of inflammatory cytokines from macrophages. Additionally, we found that OMVs incubation significantly reduced the hiPSC myocyte contraction.

Conclusion:
These studies demonstrated that OMVs from
P.a
induced inflammatory cytokine production and subsequent myocyte contractile dysfunction. Studies are ongoing to determine the role of OMVs on the action potential of hiPSC-CMs. In future, we will determine the impact of OMVs in cardiac dysfunction in mouse model.
Abstract 458: Chronic Inhibition of Protein Kinase C Improves Coronary Endothelial Function in the Setting of Diabetes and Hypoxia
Authors: Zhang Zhiqi , Liu Yuhong , shi guangbin , Justin Kim , Yi Song , Sellke Frank W , Feng Jun
Published: October 2020

Background:
Both Diabetes (DM) and hypoxia/re-oxygenation (H/R) upregulate protein kinase C (PKC), resulting in coronary endothelial dysfunction. We hypothesized that chronic inhibition of PKC protects coronary endothelial function against DM and H/R injury.

Methods:
Genetically modified obesity and type-2 diabetic mice (female) were orally treated with or without selective PKCα/β inhibitor ruboxistaurin (10μg/g) for 4 weeks (n = 6/group). Coronary small arteries (70-110 μm in diameter) were then dissected from the harvested heart of mice. The isolated vessels were subjected to 60-min of hyperkalemic cardioplegic hypoxia (15°C) and then reperfused with oxygenated Krebs-Henseleit buffer for 60 minutes. At the end of 60-min re-oxygenation, all vessels was pretreated with endothelin-1 and then the responses to endothelium-dependent vasodilators, adenosine-diphosphate (ADP) and substance P were examined.

Results:
PKCα/β inhibition significantly improved the recovery of coronary endothelial function in the small coronary arteries of diabetic mice showing increased relaxation response to ADP and substance P in dose-dependent manner compared with the DM control (no-pretreatment alone) (*p<0.05).

Conclusion:
Chronic inhibition of PKC significantly improved the recovery of coronary endothelial function against a period of cardiolpegic H/R in the diabetic microvasculature. These data suggest that the PKC inhibitor ruboxistaurin represents a novel potential therapeutic for coronary endothelial dysfunctionin patients with DM following cardioplegic ischemia and reperfusion.