bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 414: A Canine Myocardial Slice Model of Doxorubicin Induced Cardiotoxicity to Study Autophagy Modulation and Its Effect on Extracellular Vesicle Associated Mirna
Authors: Robinson Sally R , Robinson Nicholas A , Boukhalfa Asma , Meola Dawn M , Chen Howard H , Yang Vicky K
Published: October 2020

Dogs with cancer treated with chemotherapy agents such as doxorubicin (DOX) develop cardiovascular toxicity, providing an opportunity to evaluate cardioprotective strategies in the setting of cancer treatment translatable to human disease. However, due to the lack of a suitable approach to culture primary adult canine cardiomyocytes, mechanistic interrogation of cardiotoxicity after cancer therapy remains a challenge. Our study thus aims to validate a canine myocardial slice culture model to study autophagy modulation and the role of extracellular vesicle (EV) associated miRNA in the setting of DOX induced cardiotoxicity. We hypothesize that induction of autophagy in canine myocardial tissue will reduce apoptosis, exert early changes to EVs, and ameliorate DOX cardiotoxicity. Left ventricular tissue from client-owned donated adult dog hearts was sectioned with a vibratome and viability of the cultured myocardial slices was evaluated by histology and MTT assay. Apoptosis was quantified by TUNEL, and autophagy by fluorescent LC3 protein puncta. Secreted EVs were isolated from cultured tissue by size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblot. Canine myocardial slices are consistently viable for 7 days in culture - cardiomyocyte morphology is maintained with low levels of apoptosis, and baseline autophagy is observed. Induction of autophagy with rapamycin treatment results in reduced apoptosis. Cardiac tissue derived extracellular vesicles showed typical size, morphology and enrichment of proteins including tetraspanin CD9 and will be evaluated for changes to EV miRNA profile with autophagy modulation. The canine myocardial slice model allows, for the first-time, the elucidation of complex cross-talk among apoptosis, autophagy, and EVs with molecular and cellular resolutions. Detecting early changes in canine cardiomyocyte autophagy to halt cardiac damage rather than managing its consequences is a paradigm shift translatable towards preventing chemotherapy associated cardiotoxicity.
Abstract 421: Norepinephrine Catabolism Drives Cardiac Fibroblast Activation via Monoamine Oxidase Activity and Rage/β-adrenergic Signaling
Authors: Monroe Blake , Anderson Ethan J
Published: October 2020

The mitochondrial enzyme monoamine oxidase A (MAO-A) plays an increasingly appreciated role in cardiac remodeling induced by diabetes and ischemic injury. Oxidative deamination of norepinephrine (NE) by MAO-A generates 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and H
2
O
2
. Isolation and quantification of catechol-modified proteins from cardiac fibroblast lysate using an aminophenylboronic acid resin showed an MAO-dependent accumulation of catechol adducts in NE-treated cells (P<0.05). Our lab has previously observed increased expression and activity of MAO in myocardium of diabetes patients compared with age-matched nondiabetic patients. Moreover, preliminary data suggest that catecholaldehydes and other biogenic aldehydes might contribute to the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy via pro-fibrotic signaling mechanisms. We hypothesize that NE activates fibroblasts by both canonical pathways (i.e, adrenergic receptors) and by monoamine oxidase-mediated catabolism and activation of the receptor for advanced glycation endproducts (RAGE). Treatment of cardiac fibroblasts with NE (1 μM) resulted in accelerated proliferation, enhanced collagen I & III secretion, robust increases in mitochondrial and total cellular ROS, and upregulated pro-fibrotic gene expression. These effects were abrogated by co-administration of RAGE antagonist FPS-ZM1, MAO inhibitors, β-blocker propranolol, and the aldehyde scavenger carnosine (P<0.05). These findings suggest that NE (and other catecholamines) may influence extracellular matrix remodeling via multiple pathways, including adrenergic and also RAGE, via MAO-mediated catabolism.
Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk
Authors: SAHU Anita , Mukherjee Sromona , Stenson Kate , Mohan Maradumane L , Prasad Sathyamangla V
Published: October 2020

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β
2
AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.
Abstract 427: A Patient-Specific 3D Bioprinted Platform for
in vitro
Disease Modeling and Treatment Planning in Pulmonary Vein Stenosis
Authors: Serpooshan Vahid , Tomov Martin L , Kumar Akaash , Jing Bowen , Bhamidipati Sai Raviteja , Panoskaltsis Nicki , Avaz Reza , SLESNICK Tim C , MANTALARIS Athanasios , Lindsey Brooks , Bauser-Heaton Holly
Published: October 2020

Pulmonary vein stenosis (PVS) is an acute pediatric cardiovascular disease that is always lethal if not treated early. While current clinical interventions (stenting and angioplasties) have shown promising results in treating PVS, they require multiple re-interventions that can lead to re-stenosis and diminished long-term efficacy. Thus, there is an

unmet need

to develop functional
in vitro
models of PVS that can serve as a platform to study clinical interventions.

Patient-inspired 3D bioprinted tissue models

provide a unique model to recapitulate and analyze the complex tissue microenvironment impacted by PVS. Here, we developed perfusable
in vitro
models of healthy and stenotic pulmonary vein by 3D reconstruction and bioprinting inspired by patient CT data (
Figure 1
). Models were seeded with human endothelial (ECs) and smooth muscle cells (SMCs) to form a bilayer structure and perfused using a bioreactor to study cell response to stenotic geometry, and to the stent-based treatment. Flow hemodynamics through printed veins were quantified via Computational Fluid Dynamics (CFD) modeling, 4D MRI and 3D Ultrasound Particle Imaging Velocimetry (echo PIV). Cell growth and endothelialization were analyzed. Our work demonstrates the feasibility of bioprinting various cardiovascular cells, to create perfusable, patient-specific vascular constructs that mimic complex
in vivo
geometries. Deeper understanding of EC-SMC crosstalk mechanisms in
in vitro
biomimetic models that incorporate tissue-like geometrical, chemical, and biomechanical ques could offer substantial insights for prevention and treatment of PVS, as well as other cardiovascular disease.
Abstract 430: Immature Cardiac Fibroblasts With Upregulated P53 Contributed to Fibrosis in Ikke Deficient Mouse Myocardial Infarction Model
Authors: Kim Yong Sook , Cho Hyang Hee , Jun Ju Hee , Cho Dong Im , Cho Meeyoung , Ahn Youngkeun
Published: October 2020

Background:
Inhibitor of NF-κB kinase (IKK), an upstream of nuclear factor-kappa B (NF-κB), is a critical modulator for pathophysiological inflammation. IKKε is a non-classical IKK and has been studied in infectious diseases and cancers. However, the role of IKKε in a myocardial infarction (MI) has not been addressed.

Methods and Results:
In this study, we used IKKε knockout (KO) mice to induce MI by coronary artery ligation. The IKKε KO group showed poor early survival rate, large cardiac fibrosis (14.7±4.8% in KO vs. 31.1±10.2% in WT,
p
<0.05), and low fractional shortening (13.47±1.21% in KO vs. 16.36±4.46% in WT,
p
<0.05) compared with WT group. Next, we investigated the inflammatory responses and found that inflammatory markers such as inducible nitric oxide synthase (iNOS) and CD80 were much higher in both cardiac macrophages and bone marrow-derived macrophages (BMDM) in the IKKε KO group than in the wild type (WT) group. To explore the responsible mediator, we performed phosphorylated protein array and found phosphorylated p38 was significantly downregulated in the IKKε knockout BMDM. Conversely, both knockdown of p38 by siRNA and inhibition of p38 by SB203580 treatment in RAW264.7 cells upregulated iNOS. More interestingly, IKKε deficient cardiac fibroblasts showed highly accumulated nuclear p53 and exhibited immature differentiation. The levels of myofibroblast markers containing α-smooth muscle actin, periostin, and transforming growth factor-β1 were lower, and functional contractility was substantially impaired in the cardiac fibroblasts isolated from IKKε KO mice.

Conclusion:
Our data showed excessive inflammation was associated with p38 inactivation in macrophages and pathological fibrosis was resulted from immature myofibroblast phenotype with p53 upregulation. Collectively, IKKε is involved in the control of inflammation resolution and wound healing process in the infarcted myocardium.
Abstract 433: Regulatory and Elastic Protein Modifications in the Myocardium of Hibernating 13-lined Ground Squirrels
Authors: Patterson Maighdlin R , Groening Jonathan R , Hiske Mark A , Carey Hannah V , Ball Karen L , Chung Charles S
Published: October 2020

The 13-lined ground squirrel exhibits remarkable cardiac adaptation during hibernation, periods of torpor being interrupted by repeated inter-bout arousal (IBA). During IBA heart rate rises from 2-4 bpm to as great as 300 bpm. This rise in heart rate occurs over just 5 hours and remains elevated for 12-24 hours before returning to the lower heart rate. With such rapid and marked changes in heart rate, and thus filling time, myocardial stiffness and relaxation must be regulated within a short timeframe. The objective of this work was to establish post-translational modifications (PTMs) in two proteins critical to myocardial stiffness and relaxation; titin and cardiac troponin I (TnI). It was specifically hypothesized that phosphorylation during IBA would be similar to that of summer tissue and distinct from tissue isolated during torpor. Left ventricular tissue (summer, n=9; torpor, n=10; IBA, n=7) was solubilized and separated by either 2-12% gradient SDS-Page (titin) or 12.5% SDS-PAGE (TnI). Total titin phosphorylation was measured via Pro-Q Diamond Phosphoprotein/SYPRO Ruby staining. PKA-specific TnI phosphorylation was quantified via western blotting, using a primary antibody specific to Ser 23,24. While there was a tendency toward decreased total titin phosphorylation in IBA, the difference was not significant. In contrast, there were significant group effects in PKA-specific TnI phosphorylation. Phospho-TnI/Total TnI ratios were lower in torpor when compared to summer (0.22±0.12 vs. 0.68±0.,34, p<0.05). While there was no significant difference between summer and IBA (0.62±0.35), the difference between torpor and IBA failed to reach significance. This data supports the hypothesis that rapid changes in heart rates are associated with changes in cardiac troponin-I phosphorylation, a modification that contributes to rapid rates of relaxation. Further dissection of site specific titin phosphorylation will be required to assess the extent to which PTM modify titin associated stiffness. Results from this work may have significant implications in our understanding of altered compliance in human heart failure.
Abstract 441: Neurofibromin 2 Regulates Metabolism in the Heart
Authors: Zhang Yu , Mizushima Wataru , Oka Shinichi , Yang Zhi , Zhai Peiyong , Abdellatif Maha , Del Re Dominic
Published: October 2020

Background:
Neurofibromin 2 (NF2) is a tumor suppressor and signaling platform that can engage the Hippo pathway to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates injury caused by acute myocardial infarction (MI) through inhibition of the Hippo target YAP. However, NF2 function in the heart remains largely uncharacterized. This study sought to determine whether NF2 contributes to cardiac remodeling, dysfunction, and heart failure resulting from chronic stress.

Methods and Results:
We used a transverse aortic constriction (TAC) model to generate pressure overload (PO), which elicits cardiac hypertrophy, remodeling and heart failure. We found that NF2 is transiently upregulated in WT C57Bl/6J myocardium in response to PO. To investigate if increased NF2 contributes to pathology, we used cardiomyocyte-specific (αMHC-Cre) NF2 knockout (CKO) mice. At baseline, adult NF2 CKO mice had normal cardiac morphology and function. However, following TAC, NF2 CKO hearts unexpectedly showed worsened cardiac function compared to littermate controls. No differences in the extent of TAC-induced hypertrophy or apoptosis were observed between genotypes. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in unstressed NF2 CKO hearts. RNAseq identified, and qPCR confirmed, downregulation of ERRβ and ERRγ expression and function in NF2 CKO hearts, as well as ATP content. Critical ETC genes were also suppressed in NF2 CKO hearts after 3 days PO. Experiments in neonatal rat cardiomyocytes (NRCM) confirmed that altered ERRβ and ERRγ expression and function, as well as ATP content, by NF2 is cell autonomous. Mechanistically, we discovered that nuclear NF2 is enriched following TAC, and that NF2 associates with the proximal promoter regions of ERRβ and ERRγ using DNA pulldown assay. We also found that NF2 activates transcription in NRCMs using the Gal4-UAS system. Future studies will identify transcription factors important for mediating this response.

Conclusion:
Myocardial NF2 is transiently upregulated and may be compensatory during PO through preservation of metabolic gene expression and energy content in the heart.
Abstract 443: Blockade of the Thromboxane/prostanoid Receptor Prevents ECG Abnormalities in RV Pressure Overload
Authors: Carrier Erica J , KIM Kyungsoo , Shay Sheila , Moore Christy , Knollmann Bjorn C , West James D
Published: October 2020

The increased afterload of pulmonary arterial hypertension (PAH) impairs right ventricular function and ultimately leads to failure, as the RV struggles to adapt to increased pressure with remodeling and fibrosis. During PAH, cardiomyocytes upregulate cell-surface expression of the G protein-coupled thromboxane/prostanoid receptor (TPr). Increased myofibroblast and immune cell populations may also contribute to the enhanced TPr expression seen in the PAH RV. Activation of the cardiomyocyte TPr increases intracellular calcium via G
αq
/IP
3
; activation of the receptor in other cells leads to fibrosis and vasoconstriction. Preventing signaling through the TPr prevents RV fibrosis in murine models of PAH without affecting arterial pressure. Because infusion of TPr agonist can cause arrhythmia in anesthetized rabbits, and we have previously found that RV pressure overload causes sustained increases in end-diastolic calcium in RV cardiomyocytes that is blocked with TPr antagonist, we hypothesized that endogenous TPr activation can lead to conduction abnormalities in RV pressure overload. Here, we used pulmonary arterial banding (PAB) of female mice to induce fixed pressure overload of the RV. Sham-operated or PAB mice were treated with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban and were evaluated at 4 weeks past PAB. RV ejection fraction was similarly depressed in vehicle- and antagonist-treated mice, although spontaneous running, RV fibrosis, and RV relaxation time were improved in PAB mice given ifetroban. ECG abnormalities in PAB mice confirmed a prolonged relaxation and suggested delays in repolarization. These were abolished with TPr antagonism. PAB altered RV expression and localization of connexin-43 (Cx43) in vehicle-treated, but not ifetroban-treated mice. Cx43 derangement is associated with impaired cell-to-cell electrical conduction and impulse propagation. Compiled, our findings suggest that endogenous TPr activation produces alterations in RV calcium handling, signaling, and cell-cell junctions that contribute to early failure in pressure overload. Therapeutic TPr antagonism may prevent this deleterious remodeling and prolong survival in patients with PAH.
Abstract 445: Myristoylated Protein Kinase C Beta II Peptide Inhibitor Attenuates Hypoxia-Reoxygenation Injury in Human Umbilical Vein Endothelial Cells
Authors: Dean Tameka , Verwoert Alexis , Lloyd Michael , Heller Gordon , Martorana Rose , Andre Claudine , Thompson Chantel , Major Ahsal , Jenney Francis , Chen Qian , Barsotti Robert , Young Lindon
Published: October 2020

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury contributes to local organ damage in heart attack patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are initiating sources of reactive oxygen species (ROS) during I/R injury. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its phosphorylation of p66Shc to enhance mitochondrial-derived ROS production and p47 phox to promote ROS release from NOX. In previous studies, a cell-permeable myristoylated PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-) has been shown to improve post-reperfusion cardiac function and reduce infarct size in rat myocardial I/R injury. The decrease in myocardial I/R injury with myr-PKCβII- may in part be attributed to improved vascular endothelial function. Due to myr-PKCβII- peptide sequence being highly conserved among mammalian species, we hypothesize that myr-PKCβII- will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) mediated injury. HUVECs, cultured in gelatin-coated 96-well plates, were subjected to 24h hypoxia and 24h reoxygenation in a Billups-Rothenburg chamber with 1% O
2
, 5% CO
2
, and balance nitrogen. Myr-PKCβII- (20 μM) was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium-salt (WST-8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student-Newman-Keuls post-hoc analysis. At the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (n=5; p<0.05) in the non-treated H/R group compared to normoxia controls (n=5). Myr-PKCβII- significantly improved HUVEC survival (95±4%; n=5) compared to non-treated H/R controls (n=5; p<0.01) which were not significantly different from normoxia controls. The data suggest that PKCβII inhibition promotes cell survival possibly due to directly attenuating NOX and mitochondrial derived ROS in cells subjected to H/R conditions. Further studies are needed to determine cell survival potential under more severe H/R mediated injury.
Abstract 448: Strain Rate Dependent Myosin Head Position Changes During Relaxation
Authors: Ma Weikang , Bukowski Melissa J , Matus Alexandra R , Irving Thomas C , Chung Charles S
Published: October 2020

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I
1,1
/I
1,0
ratio). During stretch, there was a transient decline of the I
1,1
/I
1,0
ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.
Abstract 461: Use of Cardiac Magnetic Resonance Imaging for the Early Detection of Programmed Cell Death-1 Blockade-induced Cardiotoxicity in a Mouse Model
Authors: Xu Shirley , Karthikeyan Badri , Chen Min , Sonkawade Swati D , Spernyak Joseph , Turowski Steven , Sharma Umesh C , Pokharel Saraswati
Published: October 2020

Introduction:
Immunotherapy has revolutionized the treatment of advanced stage lung cancers and melanoma. However, serious cardiotoxic side effects such as acute myocarditis, heart block, and sudden cardiac arrest have been reported. There is an urgent need to develop accurate diagnostic modalities for the early detection of this condition, such that toxic therapies can be adjudicated based on the anticipated risks vs. benefits.

Hypothesis:
Programmed cell death-1 (PD-1) blockade-induced cardiotoxicity, presenting as myocarditis or contractile dysfunction, can be detected at an early stage by cardiac magnetic resonance imaging (MRI).

Methods:
To develop a murine model of PD-1 blockade-induced cardiotoxicity, we intraperitoneally administered 200 μg (10 mg/kg) of PD-1 antibody (Ab) or IgG isotype control every 3 days for a total of 14 doses. Cardiac function was monitored by MRI with gadolinium infusion. Serum was collected at 27 days and at the time of sacrifice. Immunohistochemical stains were performed on formalin-fixed paraffin embedded tissues.

Results:
Blockade in mice trended towards reduced myocardial signal intensity (SI) after the gadolinium injection, one month after the last dose of PD-1 antibody (myocardial SI: control 2.55 ± 0.168, PD-1 Ab 2.02 ± 0.164, p=0.063, n=4-7). Three months after the last injection, late gadolinium myocardial SI was significantly reduced in treated mice (myocardial SI: control 2.84 ± 0.352, PD-1 Ab 1.50 ± 0.308, p=0.024, n=4-5). There were also significant decreases in stroke volume (mL: control 10.4 ± 0.355, PD-1 Ab 8.36 ± 0.599, p=0.030, n=4-5), radial strain (%: control 49.9 ± 3.91, PD-1 Ab 33.5 ± 3.00, p=0.012, n=4-5), peak acceleration (cm/s
2
: control 7.24 ± 1.03, PD-1 Ab 3.51 ± 1.08, p=0.044, n=4-5), and peak deceleration (cm/s
2
: control -8.03 ± 0.78, PD-1 Ab -4.64 ± 1.04, p=0.042, n=4-5) times. Interestingly, a subset of animals also had serum antibodies directed against cardiac troponin I peptide sequence.

Conclusions:
Our study showed that cardiac MRI with gadolinium can identify abnormal myocardial perfusion and reduced contractile function in mice treated with a PD-1 blocker. The presence of serum antibodies against cardiac troponin I may have a mechanistic value, which warrants further investigation.
Abstract 462: Cardiac-specific LMCD1/dyxin-knockout in Mice Blunts Pressure Overload-induced Activation of Calcineurin Signaling
Authors: Kilian Lucia S , Voran Jakob , Schmiedel Nesrin , Stiebeling Katharina , Richter Julika , Frey Norbert , Frank Derk
Published: October 2020

We and others have shown that LMCD1 expression levels are upregulated in various
in vitro
and
in vivo
models of hypertrophy and that LMCD1 is necessary and sufficient to induce cardiomyocyte hypertrophy
in vitro
. We successfully generated a new mouse line with a conditional cardiac knockout of LMCD1. We performed echocardiographic, morphometric, and molecular analysis in these LMCD1-deficient and appropriate control-mice under basic conditions as well as 14 days after transverse aortic banding (TAC)-induced left ventricular (LV) pressure overload. Our aim was to investigate the hypothesis of potential beneficial effects of LMCD1-downregulation
in vivo
. These knockout (KO)-mice revealed under basic conditions a significant reduction of LMCD1 in the heart to <10% on protein level compared to control (WT)-mice (females and males n=5 each, p<0.001), while anatomic and functional parameters of the heart as well as LMCD1 levels in all other tested organs remained unchanged. Sham-operated KO-mice also showed significantly reduced level of LMCD1 in the LV compared to Sham-operated WT-mice (protein level <20%, p<0.001, n=8). No significant increase of LMCD1 in TAC- compared to Sham-operated KO-mice was found. TAC-operated KO-mice showed no significant differences in heart anatomy and function when compared to TAC-operated WT-mice. However, we determined a consistent trend toward improved heart function (ejection fraction and fractional shortening). Furthermore, TAC-operated KO-mice showed reduced activation of the fetal gene program in LV-tissue compared to TAC-operated WT-mice: mRNA levels of the hypertrophic markers NppA, NppB, and Rcan1-4 were all decreased (WT-TAC n=8 vs. KO-TAC n=10: NppA 8.5±2.0 vs. 5.1±1.5, p<0.05; NppB 1.9±0.2 vs. 1.7±0.3, p=0.093; Rcan1-4 6.0±0.2 vs. 3.2 vs. 0.7, p<0.05), suggesting a protective role of LMCD1-knockout. The reduction of calcineurin (CnA)-responsive Rcan1-4 specifically suggests a protective role of LMCD1-knockout in CnA-dependent signaling. Taken together, our preliminary data reveals protective effects of LMCD1-knockout against TAC-induced hypertrophic signaling. Ongoing experiments focus on effects of LMCD1-knockout on apoptosis and fibrosis and its role in Angiotensin-induced hypertrophy.
Abstract 469: Thymosin Beta4 Enhancing Therapeutic Efficacy of Human Adipose-derived Stem Cells in Mouse Ischemic Hindlimb Model
Authors: Kim Jong-ho , Joo Hyung Joon , Hong Soon Jun
Published: October 2020

Thymosin Beta4 (Tβ4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tβ4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tβ4 enhanced endogenous
TMSB4X
mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tβ4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tβ4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tβ4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tβ4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tβ4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tβ4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.
Abstract 483: Novel Evaluation Method of Spheroid
Authors: Matsushita Hiroshi , Xu Shanxiu , He Yusheng , Inoue Takahiro , Nguyen Vivian , Nurminsky Katherine , Hibino Narutoshi
Published: October 2020

Introduction:
The 3D culture methods are growing in importance in various fields, such as tissue engineering, cancer research and drug estimation. There are many methods to create 3D cell aggregation, as known as spheroids, e.g. hanging drop method, low attach U-shape plate, microwell low attach plate, spinning flask and magnetic levitation. In either field, researchers require not only high-scale but also high-quality spheroids creation. However, the method for evaluation of spheroids has not yet established. We established an accurate method of quantification of spheroids by measuring cell density and area of them with NIH Image J.

Method:
Spheroids were created with a hanging drop method (HD) and 96 well low attach U-shape plate (LAP) by using H9C2 cells, NIH 3T3 cells, human cardiac fibroblast and human dermal fibroblast cells using 22000 cells, 33000 cells and 44000 cells per spheroids. Spheroids were created five times using one of these systems.These spheroids were taken pictures in 10 times magnification with a microscope under constant condition using one tenth of spheroids solution created with HD and LAP and then quantified mean gray value and area using NIH image J. We saw relationship between its cell diameter and these data and also measured the cost and time for creating one spheroid and compared them among these methods. We also compared these data with histological analysis.

Result:
Spheroids created with HD had no relation between cell number and spheroid quality, whereas those with LAP demonstrated that higher cell numbers created larger and higher density of spheroid consistently. The number of spheroids created with the hanging drop method was higher but more inconstant and the success rate was low. However, Spheroids creation with this method took much less cost and time compared with LAP. We could see a positive correlation between area and cell size and negative correlation between gray value and its cell diameter when we create spheroids with HD with the same kinds of cells, i.e. NIH 3T3 cells, human cardiac fibroblast, and human dermal fibroblast cells.

Conclusion:
Although HD has the potentiality to create a large number of spheroids at once and this method was cost and time effective, the quality of spheroids was not consistent compared to LAP. The cell diameter influenced the quality of spheroids when we created them with HD.
Abstract 485: Mito-Targeted Antioxidant Superior to Non-Targeted Isoform in Modulating Energy Metabolism and Preventing Cardiovascular Remodelling in Spontaneously Hypertensive Rat
Authors: Saheera Sherin , Potnuri Ajay Godwin , Nair Raunak R
Published: October 2020

Hypertension induced left ventricular hypertrophy (LVH) augments the risk of cardiovascular anomalies. Oxidative stress leads to the activation of the hypertrophic program with a metabolic switch from fatty acid to glucose oxidation. Mitochondria, the major source of free radicals, exhibit alterations in hypertensive heart diseases. Targeted antioxidants are expected to reduce mitochondrial reactive oxygen species more effectively than general antioxidants. This study was designed to assess whether mito-targeted antioxidant, MitoTempol is more effective than general oxidant, Tempol on hypertension, hypertrophy and cardiac energy metabolism. Spontaneously Hypertensive Rat were administered either Tempol (20mg/kg/day) or Mito Tempol (2mg/kg/day) orally for 30 days. Post treatment, animals were subjected to 2D-echocardiography. The Myocardial lysates were subjected to Insolution digestion followed by RPLC - LTQ-Orbitrap-MS analysis. Mid-ventricular sections were probed for markers of energy metabolism and fibrosis. The beneficial effect on cardiovascular structure and function was significantly higher for Mito Tempol. Increase in mitochondrial antioxidants and stimulation of fatty acid metabolism with significant improvement in cardiovascular function was apparent in SHR treated with Mito Tempol. The study indicates that Mito Tempol is superior to its non- targeted isoform in preventing hypertension induced LVH, and the beneficial effects on heart are possibly mediated by reversal of metabolic remodelling.
Abstract 487: Dihyrosphingosine 1 Phosphate Mediates Collagen Synthesis in Cardiac Fibroblasts Throught Pi3k/akt- Mtor Signalling Pathway
Authors: Magaye Ruth R , Sevira Feby , Xiong Xin , Flynn Bernard , Wang Bing
Published: October 2020

Background:
Cardiac fibrosis is one of the hallmarks of cardiac remodelling in cardiomyopathies such as heart Failure (HF). Dyslipidemia plays a role in the progression of HF. The sphingolipid, dihydrosphingosine 1 phosphate (dhS1P) has been shown to bind to high density lipids in plasma. Unlike its analog, spingosine 1 phosphate (S1P), the role of dhS1P in cardiac fibrosis is not known. The aim of this study is to determine the role dhS1P plays in cardiac fibrosis through the PI3K/Akt- mTOR pathway.

Method:
Neonatal rat cardiac fibroblasts (NCF) were isolated from 1-2 day old pups with enzymic digestion. After pre-treating with the PI3K inhibitor, Wortmannin (W, 0.1 - 10.0μM), cells were stimulated with dhS1P for 48 hours. NCF collagen synthesis was determined by 3H-proline incorporation. NCF were also treated for protein and gene expression analysis.

Results:
Exogenous addition of 3 μM dhS1P stimulated significant increase in collagen synthesis (p<0.005) which was dose dependently inhibited by W (p < 0.0001, Fig. 1A). Western blot analysis showed that W reduced Akt, mTOR, and S6 activation in the presence of dhS1P. dhS1P also increased protein levels of TGFβ, Coll 1 and TIMP1. W reduced dhS1P elevated TIMP1, and SK1, but not TGFβ1 gene expression (Fig. 1B).

Conclusion:
Our study demonstrates for the first time that dhS1P can cause cardiac cellular fibrosis via PI3K/Akt- mTOR pathway. Its inhibition may represent a novel therapeutic strategy for cardiac fibrosis.
Abstract 493: An Age-associated Reduction in Na
V
1.5 Protein Expression Correlates With an Increase in Na
V
1.8 Protein Expression in the Right Atrial Appendage of the Human Heart
Authors: Isaac Emmanuel , Cooper Stephanie , Lancaster Matthew K , loubani Mahmoud , Jones Sandra A
Published: October 2020

Background:
Na
v
1.5 is the predominantly expressed voltage-gated sodium channel (VGSC) isoform in the heart, responsible for phase 0 of the action potential. Our rodent studies have shown expression of neuronal isoform Na
v
1.8 protein in the adult heart. Others have associated Na
v
1.8 expression with aberrant sodium currents leading to increased frequency of arrhythmogenic episodes.

Aim:
To investigate age-associated changes in the protein expression of VGSCs Na
v
1.5 and Na
v
1.8 in the right atrial appendage of the human heart.

Methods:
Tissues from the right atrial appendage were obtained with NHS ethical approval (IRAS 197493) from patients undergoing routine cardiac surgery, coronary artery bypass or valve repair. The protein expression of VGSC isoforms Na
v
1.5 and Na
v
1.8 were quantified using specific protein-directed primary antibodies (Alomone, Israel) coupled with a secondary antibody conjugated to HRP for western blot (Abcam, UK), or Alexa Fluor 488 (ThermoFisher, UK) for immunohistochemistry with confocal microscopy (LSM 710 Zeiss, UK). Densities were quantified using Image J software.

Results:
Derived from 26 patients, the protein expression of Na
v
1.5 arranged in chronological age order (43-87 years of age) demonstrated a negative correlation between increasing age and Na
v
1.5 protein expression (Pearson correlation coefficient of -0.25). The contrary was shown for Na
v
1.8, which had a positive correlation with increasing age (Pearson correlation coefficient of 0.26), as older patients show increased Na
v
1.8 protein expression. This novel finding was reflected when we compared confocal images from elderly patients with their younger counterparts, as the elderly showed a higher density of labelled Na
v
1.8 protein and a lower density of labelled Na
v
1.5 protein, particularly located at the t-tubules and intercalated disks.

Conclusion:
With increasing chronological age, the human heart reduces expression of the predominant cardiac isoform Na
v
1.5 whilst increasing the expression of Na
v
1.8: This finding has the potential to increase the risk of an elderly person’s heart to an increased susceptibility of arrhythmias.
Abstract 499: Modeling Carfilzomib Induced Cardiotoxicity Using Human Induced Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Forghani Parvin , Rashid Aysha , Li Dong , Mandawat Anant , XU Chunhui
Published: October 2020

Cardiovascular toxicity post Carfilzomib (Cfz/Kyprolis) therapy has been identified in several clinical settings. A prevalent challenge in side effects of anti-cancer drugs is the translation of findings from preclinical research into clinical practice. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as a physiological
in vitro
model to overcome some of these challenges. Here we used both 2D and 3D hiPSC-CMs to elucidate the underlying mechanism of post-Cfz cardiotoxicity. hiPSC-CMs were exposed to clinically relevant doses of Cfz based on C
max
for Cfz (5.88 μM). Data normalization against the control group demonstrates significant reduction in cell viability following two days of treatment with Cfz in 3 different cell lines (IMR-90, SCVI273 and 902). Increased Caspase3/7 activity post Cfz treatment paralleled with a substantial decrease in mitochondrial membrane potential and increase in oxidative stress following Cfz treatment. Also, significant decrease in oxygen consumption rate was observed after one-day exposure. In addition, we observed impaired Ca
2+
handling at the single cell level following Cfz treatment. Using video microscopy with motion vector analysis we also observed significant decrease in contractility of 3D hiPSC-CMs following Cfz treatment. Additionally, we observed disrupted expression of α-actinin, alterations in structural organization of sarcomeres, circularity and aspect ratio. Altogether, these results suggest that Cfz induced cardiotoxicity as indicated by cell viability, oxidative stress, mitochondrial and structural damages along with abnormal Ca
2+
handing and contractility dysfunction.
Abstract 502: Inhibition of Histone Deacetylase 6 Protects Human Induced Pluripotent Stem Cells-derived Cardiomyocytes Through Inhibition of Autophagy
Authors: Gurusamy Narasimman , RAJASINGH SHEEJA , Sigamani Vinoth , Kirankumar Shivaani , Vasanthan Jayavardini , Johnson Rajasingh
Published: October 2020

Introduction:
Autophagy is known to play an important role in mediating cardiac hypertrophy. However, the mechanism is poorly understood. Since the protein histone deacetylase 6 (HDAC6) contributes to cardiac dysfunction in response to angiotensin II (AngII) signaling, we have examined the role of HDAC6 inhibitor tubastatin A (TBA) in AngII-induced remodeling in human induced pluripotent stem cells-derived cardiomyocytes (iCMCs).

Hypothesis:
We hypothesize that the inhibition of HDAC6 protects iCMCs from AngII-induced cardiac hypertrophy through inhibition of autophagy.

Methods and Results:
We have generated and characterized induced pluripotent stem cells from human adult skin fibroblasts and subsequently differentiated them into iCMCs. Treatment with 10 μM angiotensin II for 24 hrs increased the HDAC6 activity and lead to hypertrophy in iCMCs. The AngII-induced hypertrophy, and the excessive contractility in iCMCs were reversed by the inhibition of HDAC6 with TBA (1 μM for 24 hours). The number of LC3-positive iCMCs, and the mRNA and the protein expression of autophagic genes Beclin-1, LC3, and p62 were increased by the presence of AngII, and the anti-autophagic gene Bcl2 was decreased by AngII. The inhibition of HDAC6 with TBA reversed the AngII-mediated changes in the autophagic genes expressions in iCMCs. Autophagic vacuoles were identified with monodansylcadaverine (MDC, green) and lysosomes with LysoTracker (red)
(Fig. 1A)
. The number of autophagolysosomes were increased by AngII, and this was decreased with TBA in iCMCs
(Fig. 1B)
.

Conclusions:
Our report indicates for the first time that the AngII-induced cardiac hypertrophy-mediated autophagy is effectively inhibited by the suppression of HDAC6 in human iCMCs.
Abstract 508: Nuclear Lamins At Enhancers: A New Hypothesis for Laminopathies
Authors: Ikegami Kohta , Secchia Stefano , Almakki Omar , Stutzman Alexis V , Ikegami Sachie , Ayres Natalia , Liu Sunny Y , Lieb Jason D , Moskowitz Ivan P
Published: October 2020

The segregation of heterochromatin domains (LADs) at the nuclear periphery by the nuclear lamina, composed by polymerized nuclear Lamin A/C, provides a longstanding paradigm for the control of gene expression and for the mechanisms underlying Lamin-A/C-associated disorders, including progeria and cardiomyopathy. Here, we provide evidence supporting a novel paradigm that Lamin A/C functions as a transcription factor in the nuclear interior. We discovered that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C), required for lamin depolymerization during mitosis, populated the nuclear interior throughout the cell cycle. pS22-Lamin A/C ChIP-deq demonstrated localization at a large subset of putative active enhancers, not LADs. pS22-Lamin A/C-binding sites were co-occupied by the transcriptional activator c-Jun. In progeria patient-derived fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged. New pS22-Lamin A/C binding was accompanied by increased histone acetylation and increased c-Jun binding, whereas loss of pS22-Lamin A/C-binding was accompanied by loss of histone acetylation and c-Jun binding. New pS22-Lamin A/C enhancer binding in progeria was associated with upregulated expression of genes implicated in progeria pathophysiology, including cardiovascular disease. In contrast, alteration of LADs in progeria-patient cells could not explain the observed gene expression changes. These results suggest that Lamin A/C regulates gene expression by enhancer binding in the nuclear interior, independent of its function at the nuclear lamina, providing a new paradigm for the pathogenesis of lamin-associated disorders. pS22-Lamin A/C was also present in the nuclear interior of adult mouse cardiomyocytes. Cardiomyocyte-specific deletion of
Lmna
encoding Lamin A/C in adult mice caused extensive transcriptional changes in the heart and dilated cardiomyopathy, without apparent reduction of nuclear peripheral Lamin A/C. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C may underlie the pathogenesis of disorders caused by
LMNA
mutations, including cardiomyopathy.
Abstract 509: Oxytocin Mediates Neuroendocrine Reprogramming of the Epicardium in Heart Regeneration
Authors: Wasserman Aaron H , Huang Amanda R , Aguirre Aitor
Published: October 2020

Cardiovascular disease (CVD) is the leading cause of mortality both in the United States and worldwide. CVD often results in the massive loss of contractile cardiac cells and tissue. Critical work in the last two decades demonstrates that lost cells can be partially replenished by the epicardium, the outermost mesothelial layer of the heart. Upon cardiac injury, mature epicardial cells activate and undergo epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EPDCs), which are a type of multipotent stem cell that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. This process alone is insufficient for significant regeneration, but its efficiency can be improved by priming with specific factors (e.g., thymosin beta-4). Our group has recently discovered evidence that oxytocin (OT), a hypothalamic neuroendocrine peptide, induces a pro-regenerative phenotype
in vitro
in human induced pluripotent stem cell (iPSC) derived epicardial cells. We hypothesize that upon cardiac injury, oxytocin is released into the bloodstream, causing activation of the epicardium and mobilization of EPDCs to elicit regeneration of damaged tissue and restoration of function. Here, we show that we can differentiate mature, high-quality epicardial cells from iPSCs and that Ki67 levels and cell counts increase after three days of OT exposure. In addition, the peptide alters gene expression levels of several epithelial, mesenchymal, and EMT markers, indicating a transition to a dedifferentiated gene profile characteristic of EPDCs. Finally, when OT is administered intravenously to mice, it accelerates healing from cardiac injury by inducing epicardial activation. Future studies will aim to further reveal the physiological contribution of OT to heart regeneration
in vivo
and determine its molecular mechanism of action. Our findings have the potential to uncover a novel mechanism of neuroendocrine reprogramming of the injured heart and yield significant translational advances in the treatment of CVD.