bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P407: Role Of Cyclic AMP During The Intracellular Infection Cycle Of
Trypanosoma Cruzi
Authors: Velez-Ramirez Daniel E , Shimogawa Michelle , Hill Kent
Published: November 2021

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. In the 1990s the distribution of the vector, a hematophagous triatomine, and consequently the parasite, was from the southeast tropical regions of Mexico to South America. Now, global warming is causing this distribution to expand to northern territories in Mexico, reaching southern parts of US, in which up to 300,000 people are affected. Furthermore, an increase in chronically-infected immigrants to the US makes Chagas disease a matter of Pan-American public health that it should be addressed by all the America countries. Chagas disease manifests clinically as cardiovascular disease, characterized by hypertrophy of heart, esophagus and colon. Congestive heart failure is the main cause of death (58%) in Chagas patients, whereas cardiac arrhythmias and unexpected deaths add another 36%. A major cause of heart pathology in Chagas disease damage is caused by the host immune system, as it attacks chronically infected tissue. Therefore, pathology of the disease is a direct consequence of the ability of the parasite to invade host cells, so it can establish chronic infection. To achieve this, T. cruzi must sense and adapt to the host environment, but the underlying mechanisms are poorly understood. In particular, parasite signaling pathways used to sense and transduce signals from the host environment are most completely unknown. Our lab studies cAMP signaling in trypanosome parasites and several lines of evidence suggest T. cruzi cAMP signaling is important for host cell invasion, differentiation and persistent infection, which in turn underlies heart tissue pathology of Chagas disease. A transcriptome analysis revealed that mRNA of proteins involved in cAMP metabolism, i.e. adenylate cyclases and phosphodiesterases, are either upregulated or downregulated during the intracellular infection cycle. In fact, the phosphodiesterases have flagellar homologs with known cAMP signaling functions in a related parasite. This suggests that cAMP might fluctuate during as T. cruzi invades, differentiates, and multiplies inside the host cells. We have implemented a cAMP FRET sensor to monitor cAMP levels in trypanosomes to understand the role of cAMP in T. cruzi pathogenesis.
Abstract P408: Mesenchymal Stem Cell-derived Angptl4 Improves Endothelial Permeability And Resolution Of Inflammation In Atherosclerosis
Authors: Cho Dong-im , Kim Yong Sook , Ahn Youngkeun
Published: November 2021

Objective:
Given the fundamental contribution of inflammation to atherosclerosis, we studied the effect of ANGPTL4 in regulating vascular lesions during atherosclerosis.

Methods and Results:
We analyzed plasma levels of ANGPTL4 and found that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than did patients with lower ANGPTL4 levels (
p
<0.05). Moreover, in AMI patients with heart failure (HF) at admission, the recurrence of HF was lower in patients with a higher level of ANGPTL4. We then investigated the therapeutic application of ANGPTL4 in an atherosclerosis model. Apoe-/- mice fed a high-fat diet were injected with PBS or ANGPTL4 protein (2 μg per mouse,
i.p.
) three times per week for 7 weeks. En face staining and mRNA analysis showed that plaque size, necrotic core area, lipid accumulation, and inflammatory molecules were greatly reduced in the ANGPTL4 group. Aortic permeability, measured by leakage of Evans blue dye, was significantly decreased in the ANGPTL4 group. The induction of pro-inflammatory mediators was significantly inhibited in endothelial cells, vascular smooth muscle cells, and macrophages by ANGPTL4 treatment. Endothelial Krüppel-like factor 2 (KLF2) and VE-cadherin were restored to contribute to maintenance of vascular integrity by ANGPTL4 treatment. Elevated levels of circulating leptin, interleukin-6, and interleukin-1β were profoundly reduced. Most importantly, the fibrous cap was significant thicker in the ANGPTL4 group than in the PBS group.

Conclusions:
ANGPTL4 treatment attenuated atherogenesis, which suggests that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. More importantly, ANGPTL4 treatment inhibits necrotic core formation in lesions, leading to the formation of more stable plaques as evidenced by increased fibrous cap thickness.
Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca
2+
Mishandling In Mouse Atrial Myocytes
Authors: Medvedev Roman Y , DeGuire Frank C , Glukhov Alexey
Published: November 2021

Background:
Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca
2+
mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca
2+
leak via augmentation of RyRs phosphorylation.

Methods and Results:
Cell size analysis and Ca
2+
dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p<0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca
2+
spark activity: a fast (<10 min of exposure) ~50% increase (p<0.001) followed by a slow decrease to the level observed in isotonic conditions (>30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca
2+
sparks frequency (p<0.001). Swelling- and MβCD-induced increases in atrial Ca
2+
spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca
2+
sparks frequency than wild-type myocytes (p<0.01), which was significantly reduced (p<0.01) to wild-type level after incubation with SQ22536.

Conclusions:
Our findings suggest that cell stretch increases spontaneous Ca
2+
spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca
2+
mishandling and AF in pressure overloaded hearts.
Abstract P417: Cardiomyopathy-associated Variant In Troponin T Tail Domain Promotes Disruption Of Both Frank-starling Mechanism And Cardiac Myofilament Performance
Authors: Landim-Vieira Maicon , Ma Weikang , Song Taejeong , Ottenheijm Coen , Hwang Hyun , Knollmann Bjorn C , Sadayappan Sakthivel , Irving Thomas , Chase P. Bryant , Pinto Jose
Published: November 2021

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) tail region has been linked to familial hypertrophic cardiomyopathy (HCM), arrhythmia, and sudden cardiac death. It has been reported that inotropic stimulation with high extracellular Ca
2+
or isoproterenol led to diastolic dysfunction in both isolated and
in vivo
HcTnT-I79N mice hearts. Although HcTnT-I79N effects are acknowledged to be dependent on the inotropic state of the cardiac muscle, little is known about how this pathogenic variant affects the Frank-Starling law of the heart. To further investigate the functional and structural consequences of this deadly variant in a stretch-dependent manner, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements. Sarcomere length (SL 1.9, 2.1 or 2.3 μm) was set at pCa 8 using HeNe laser diffraction and then Ca
2+
-dependence of isometric force, sinusoidal stiffness (SS, 0.2% PTP length oscillation) and rate of tension redevelopment (
k
TR
) were measured. We observed that HcTnT-I79N tissue exhibited increased myofilament Ca
2+
-sensitivity of force, increased SS, slower
k
TR
at all levels of Ca
2+
-activation, and diminished length-dependent activation (LDA). Small-angle X-ray diffraction revealed that HcTnT-I79N permeabilized cardiac muscles exhibit smaller myofilament lattice spacing at longer SLs (2.1 μm and 2.3 μm) compared to NTg. Using 3% Dextran T500 to osmotically compress the myofilament lattice (SL 2.1 μm), HcTnT-I79N showed no change in myofilament lattice spacing and little change in contractile indices associated with LDA. Interestingly, upon osmotic compression, HcTnT-I79N displayed a decrease in disordered relaxed state (DRX, ON state) of myosin and an increase in super-relaxed state (SRX, OFF state) of myosin. We conclude that altered cardiac myofilament performance, lack of responsiveness to osmotic compression, and reduced LDA observed with HcTnT-I79N are partially due to a combination of smaller myofilament lattice and disturbed ON and OFF states of myosin.
Abstract P425: Sirtuin 3 Attenuates Doxorubicin Induced Cardiac Dysfunction By Regulating The Mitochondrial Acetylome And Alterations Of The Cardiac Lipidome
Authors: Tomczyk Mateusz M , Surendran Arun , Xiang Bo , Agarwal Prasoon , Cheung Kyle G , Kereliuk Stephanie , Tong Qiang , Ravandi Amir , Dolinsky Vernon
Published: November 2021

Doxorubicin (DOX) is a chemotherapeutic with dose-dependent cardiotoxic effects that limits its use in patients. Previously we showed that DOX decreases expression of the mitochondrial lysine deacetylase SIRT3 in the mouse heart. We hypothesize that DOX impairs cardiac function and energy production through reduced SIRT3 and altered mitochondrial acetylation. We further hypothesize that increased SIRT3 expression could attenuate DOX-induced cardiac dysfunction via alterations of protein acetylation to enzymes involved in lipid remodeling and metabolic processes. Mice expressing cardiac restricted full length M1-SIRT3 (mitochondrial localized), and short M3-SIRT3 (lacking localization signal) received saline or DOX injections of 8 mg/kg body weight for 4 weeks and compared to non-transgenic (Non-Tg) littermates. Transthoracic echocardiography was performed on all mice (n=10). Total cardiac lipids were isolated from DOX treated cardiac tissue by chloroform:methanol extraction and global lipid analysis was performed by QTRAP LC-MS/MS (n=6). Cardiac mitochondria were and an anti-acetylated lysine antibody was used to enrich for tryptic digested peptides containing Acetyl-K and analyzed by QTRAP LC-MS/MS (n=6). In non-Tg mice, DOX caused cardiac dysfunction and expression of M1-SIRT3 and M3-SIRT3 transgenes in the heart preserved left ventricular posterior wall thickness (P<0.05) and ejection fraction (P<0.05) in DOX treated mice. Triglycerides and phospholipids (PE, PI, PC) were decreased in DOX treated mouse hearts while phosphatidylserine (PS), sphingomyelin and ganglioside (GM3) lipid species were increased (p<0.05). A negative correlation between decreased cardiac output and increased GM3 24:1 (R=-0.62, P<0.05), PS 38:4 levels (R=-0.81, P<0.005) and SM 35:1 (R=-0.65, P<0.05) was identified. 36 acetylated peptides involved in metabolic processes, oxidative stress resistance and lipid remodeling (eg. IDH2, SOD2, HADHA, P<0.05) were significantly altered in DOX-treated mice. Increased SIRT3 expression in the heart rescues DOX-induced cardiac dysfunction. DOX-induced cardiac dysfunction involved alterations in cardiac lipids and acetylated proteins that could be rescued by increased SIRT3 expression in the heart.
Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation
Authors: Marzano Federica , Rapacciuolo Antonio , Koch Walter J , Cannavo Alessandro
Published: November 2021

Introduction:
G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent.

Hypothesis:
The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress.

Methods:
We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies.

Results:
Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels.

Conclusions:
Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.
Abstract P429: Aldehyde Dehydrogenase 2 Regulates Angiogenesis Of Coronary Microvascular Endothelial Cells Via A Novel Signaling Pathway
Authors: Roy Bipradas , Pan Guodong , Palaniyandi Suresh
Published: November 2021

Coronary microvascular endothelial cell (CMECs) damage is implicated in diabetes-mediated heart failure with preserved ejection fraction (HFpEF). 4-hydroxy-2-nonenal (4HNE), a reactive aldehyde that is increased in diabetic heart, decreases angiogenesis in cultured mouse CMECs by decreasing the mRNA and protein levels of vascular endothelial growth factor receptor (VEGFR)2. Nuclear factor-kappa B (NF-kB), a transcription factor, was shown to transcribe VEGFR2. Thus, we presume 4HNE modulates NF-kB-mediated VEGFR2 transcription and regulates angiogenesis in CMECs. Aldehyde dehydrogenase (ALDH) 2, a mitochondrial enzyme that detoxifies 4HNE and confers cryoprotection. However, ALDH2 activity was reduced in the diabetic hearts which results in the augmentation of 4HNE-induced cardiotoxicity. Thus, we hypothesize that ALDH2 in CMECs reduces 4HNE-mediated cell signaling aberrations, and thereby, preserves coronary angiogenesis. We treated the cultured mouse CMECs with disulfiram (DSF) (2.5 μM), an ALDH2 inhibitor, alda1 (10 μM), an ALDH2 activator and prostratin (1 μM), an NF-κB activator prior to challenging the CMECs with 4HNE (75 μM). Our tube-formation angiogenesis assay revealed that pretreatment with DSF exacerbated a 4HNE-induced decrease in CMECs angiogenesis (P<0.0005 vs con and P<0.05 vs both 4HNE & DSF alone) while pretreatments with alda1 and prostratin attenuated a 4HNE-induced decrease in CMEC angiogenesis (P<0.05 vs 4HNE alone). DSF pretreatment exacerbated 4HNE mediated decrease in ALDH2 (P<0.005 vs con), phospho-IKBα (P<0.0005 vs con and P<0.05 vs both 4HNE and DSF alone), NF-κB levels, and nuclear translocation (P<0.0005 vs con and P<0.05 vs both 4HNE & DSF alone) and VEGFR2 (P<0.0005 vs con and P<0.05 vs both 4HNE and DSF alone) levels in cultured CMECs. Pretreatment with both prostratin and alda1 increased ALDH2 (P<0.0005 vs con), VEGFR2 (P<0.05 vs con) and NF-κB (P<0.005 vs con) levels in CMECs. The cardiac tissue samples of db/db mice when they manifest HFpEF showed increased 4HNE adducts, decreased NF-kB and VEGFR2 levels in CD31+ CMECs besides exhibiting low CMEC density. In conclusion, ALDH2 attenuates 4HNE-mediated decrease in coronary angiogenesis by decreasing VEGFR2 levels via low NF-κB mediated transcription.
Abstract P433: A Pharmacological Model Predicts Drug Activity In Cardiomyocyte Hypertrophy
Authors: Eggertsen Taylor , Saucerman Jeffrey J
Published: November 2021

Introduction:
Cardiomyocyte (CM) hypertrophy is predictive of heart failure, however there are no clinical therapies that target its intracellular pathways. Hypertrophy is a complex process involving numerous neurohormonal and cytokine inputs, resulting in context-dependent responses that determine CM growth. In the face of this complexity, it is critical that computational models are developed. Accurate predictions of drug activity in CM hypertrophy will require a pharmacological model that is developed with and validated against experimental data.

Hypothesis:
We test the hypothesis that our
in silico
pharmacological model accurately predicts drugs that inhibit cardiac hypertrophy as well as the context-dependent mechanisms by which they work.

Methods:
Here we employ a previously published computational model of cardiac hypertrophy signaling. This model utilizes logic-based ordinary differential equations to simulate a network of 106 nodes. Using the DrugBank database, we constructed a pipeline for simulating FDA-approved drugs within this hypertrophy network under multiple environmental contexts. The predicted outcomes of the model were then compared to measured phenotypes from experimental findings in literature.

Results:
Predicted outcomes of our model were successfully validated against 29 out of 36 distinct experiments described in literature. These simulations identify the optimal drug types that inhibit hypertrophy for each of 17 different stimuli. Sensitivity analyses performed by simulating knockdowns in our model reveals context-dependent mechanisms predicted for 51 drug types. These predictions confirmed, for example, the role of celecoxib in inhibiting CM hypertrophy induced by isoproterenol. Mechanistic analysis suggests celecoxib prevents protein kinase B (Akt) inhibition of glycogen synthase kinase 3 beta (GSK3β), consistent with literature.

Conclusions:
Our pharmacological model accurately predicts FDA-approved drugs that show
in vitro
inhibition of CM hypertrophy.
Abstract P434: A Mouse Model To Control Vessel Diameter With Light
Authors: O'Herron Philip , Hartmann David , Shih Andy Y
Published: November 2021

In diseases such as stroke, hypertension, vascular cognitive impairment, and Alzheimer’s disease, defects in the cerebrovascular system lead to reduced blood flow and vasoreactivity to stimuli. Recently, there has been increased appreciation for the role of small vessels in these vascular pathologies. For example, small vessel dysfunction can cause widespread microinfarcts and capillary stalling, which may underlie cognitive impairment in cases where large scale vascular abnormalities are not readily detected. However, vascular function is difficult to dissociate from concurrent neuronal deficits cause by damage to neuronal circuitry in brain pathology. Thus the ability to directly probe smooth muscle contraction of small, individual vessels in the intact brain would be a valuable tool for increasing our understanding of vascular contributions to cognitive impairment. We developed an experimental paradigm to optically probe the contractile function of arterioles in vivo with high spatiotemporal precision. This was done by expressing the excitatory opsin ReaChR in vascular smooth muscle cells and pericytes. Using a 594 nm light-emitting diode we were able to evoke widespread vasoconstriction across the cranial window. With a 1040 nm focused, pulsed laser for two-photon stimulation, we were able to evoke highly localized constrictions targeted to individual pial artery branches or penetrating arterioles. Our dual light-path imaging system allowed the optogenetic stimulation to be performed with simultaneous two-photon imaging to monitor vessel activity. Using a spatial light modulator, we were also able to constrict vessels both above and below the imaging plane. This is a powerful tool to assay vasoconstrictive function of single arterioles across 3-dimensional vascular networks in vivo. It also presents novel opportunities to study conductance of vascular signals and to modulate dynamics of functional hyperemia.
Abstract P436: Modeling Genetic Dilated Cardiomyopathy Using Induced Pluripotent Stem Cell-derived Engineered Heart Tissues For Precision Medicine
Authors: Butler Renee E , Steltzer Stephanie S , Zepeda Bernardo , Tsui Jonathan H , Mandegar Mo A , Hoey Tim , Ho Jaclyn J
Published: November 2021

Recent advancement of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technologies has opened the door to next-generation modeling of human cardiac biology and disease. Not only does this alleviate the need for human primary tissue and compensate for the well documented deficiencies of rodent models for cardiovascular disease, but these technologies may lead to the identification of better candidates for clinical development. Unfortunately, most current 2D hiPSC-CM models lack the biochemical, mechanical, and electrical feedback that cardiomyocytes endure in a multi-cellular aligned tissue, which limits their translatability into clinical settings. To address this, we incorporated hiPSC-CMs modeling various genetic dilated cardiomyopathies (DCM) into 3D engineered heart tissues (EHTs). Here, we show these models develop distinguishable contractile defects recapitulating hallmarks of DCM when compared to biologically relevant controls. Moreover, this distinct phenotype is quantitative, reproducible, and demonstrates utility for drug discovery. As this innovative technology continues to develop, EHTs are on the forefront of emerging biomimetic assays that can be used to prevent drug attrition in the late stages of drug development.
Abstract P439: Harnessing Multiscale Models Of A Dilated Cardiomyopathy Mutation For Precision Medicine
Authors: Greenberg Lina , Stump W. Tom , Bredemeyer Andrea L , Lavine Kory J , Greenberg Michael J
Published: November 2021

Familial dilated cardiomyopathy (DCM) is a leading cause of both adult and pediatric heart failure. Currently, there is no cure for DCM, and the 5-year transplant free survival rate is <50%. There is therefore an outstanding need to develop new therapeutics. Prior studies have established a strong genetic basis for DCM and identified causative genetic mutations. These observations provide unique opportunities to apply precision medicine approaches that target and circumvent the effects of deleterious mutations. Here, we used a multiscale approach to study the consequences of a human mutation in troponin T that causes DCM, ΔK210. We found that at the molecular scale ΔK210 changes the positioning of tropomyosin along the thin filament, leading to molecular hypocontractility. Using genome edited human stem cell derived cardiomyocytes heterozygous for the mutation, we show reduced cellular contractility at the single cell and tissue levels. Importantly, we demonstrate that mutant tissues show a reduced Frank-Starling response, increased stiffness, and misaligned myocytes. Based on our molecular mechanism, we hypothesized that treatment of ΔK210 with Omecamtiv Mecarbil (OM), a thin filament activator in clinical trials for heart failure, would improve the function of mutant tissues. We found that treatment of ΔK210 molecular complexes and tissues with OM causes a dose-dependent increase in cardiac function, reversing the mutation-induced contractile defect. Taken together, our study demonstrates how mechanistic molecular studies can be harnessed to identify precision medicine therapeutics.
Abstract P440: Cardiac Tissue Chip Model Provides A Platform For Studying Age-related Cardiovascular Complications By Recapitulating Important Hallmarks Of Senescence And Myocardial Ischemia
Authors: Budhathoki Sachin , Graham Caleb , Sethu Palaniappan , Kannappan Ramaswamy
Published: November 2021

Introduction:
With the rise in the elderly population, there has been an exponential growth in cardiovascular diseases and age-related complications. This necessitates a platform for studying cardiovascular disease in the context of aging.

Hypothesis:
An engineered cardiac tissue model that can recapitulate critical aspects of aging can be used to study age-related diseases of the cardiovascular system.

Methods:
Senescence was induced in rat cardiomyoblasts using an acute low-dose doxorubicin treatment. The presence of important senescent markers in the cells like enlarged and flattened nuclei, increased ROS activity, elevated p53 production, DNA damage response foci, and increased expression of cell cycle inhibitor p16
INK4a
was evaluated. These senescent cells were then used to engineer cardiac tissue, which was subjected to hemodynamic stresses associated with the pressure-volume changes in the heart. Myocardial ischemia was imposed in the aging cardiac tissue model using hypoxic treatment.

Results:
Under normal hemodynamic loading, the engineered cardiac tissue retained its cardiac cell characteristics and showed cell alignment along with age-related changes in structure and gene expression. The myocardial ischemic model of the tissue revealed major pathological hallmarks of the disease like increased cell death and natriuretic peptide expression.

Conclusion:
Our model and methodology provide an effective platform for studying the cardiovascular disease pathologies associated with aging and screening drugs against age-related complications.
Abstract P444: Inflammation Resolution And Transition Of Macrophages Are Dependent On Ikke
Authors: Cho Hyang Hee , Kim Yong Sook , Cho Dong Im , Cho Meeyoung , Ahn Youngkeun
Published: November 2021

Rationale:
Myocardial infarction (MI) is a major cause of death and inflammation mainly contributes to pathological progress. Expression of IKKe is induced in inflammatory macrophages, but the pathophysiological roles in cardiac injury remain unclear.

Objective:
We aimed to investigate the role of IKKe in macrophages in the IKKe knockout (KO) MI mouse model.

Methods and Results:
MI was induced by coronary artery ligation, then cardiac macrophages and bone marrow-derived macrophages were isolated for further comparison studies.The IKKe KO group showed poor survival rate, high circulating IL-6 level, large cardiac fibrosis (14.7±4.8% vs. 31.1±10.2%, p<0.05), and low ejection fraction (34.02±5.5% vs. 30.33±5.3% vs. p<0.05) compared with the wild type (WT) group.Next, we investigated the inflammatory and fibrotic responses to understand the relevant mechanism. Flow cytometry and phosphorylated protein array showed that IKKe-deficient macrophages exhibited lower phosphorylated p38 and higher inflammatory markers.Moreover, we identified macrophage-myofibroblast transition (MMT) by detecting aSMA expression in macrophages, and found that the number of cells under MMT was 2.0-fold higher in the IKKe KO group than in the WT group.In parallel to the increase in MMT, the transition of CD206(+) anti-inflammatory macrophages to myofibroblasts was 2.16-fold greater in the IKKe KO mice.Macrophages with chemical or genetic interfering of p38 displayed the remarkable increases in both MMT and inflammatory phenotype.

Conclusions:
Our results suggest that IKKe-p38 axis may potentially induce the inflammation resolution and limit MMT in macrophages in response to MI. Mechanistical understanding of IKKe signaling offers novel therapeutic pathways to treat MI.
Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes
Authors: Ly Olivia T , Brown Grace , Chen Hanna , Hong Liang , Wang Xinge , Han Yong Duk , Pavel Mahmud Arif , Sridhar Arvind , Maienschein-Cline Mark , Youn Seock Won , Chalazan Brandon , Massad Malek G , Abdelhady khaled , Wu Joseph C , Rehman Jalees , KHETANI SALMAN , Darbar Dawood
Published: November 2021

Introduction:
The limited success of pharmacological approaches to atrial fibrillation (
AF
) is due to limitations of
in vitro
and
in vivo
models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity.

Objective:
We hypothesized that a combinatorial approach of biochemical (triiodothyronine [
T3
], insulin-like growth factor-1 [
IGF-1
], and dexamethasone; collectively
TID
), bioenergetic (fatty acids [
FA
]), and electrical stimulation (
ES
) will enhance electrophysiological (
EP
), structural, and metabolic maturity of iPSC-
a
CMs.

Methods:
We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient.

Results:
TID+FA+ES significantly improved structural organization and cell morphology (
Fig. 1a
), enhanced membrane potential stability and improved depolarization (
Fig. 1b
), improved Ca
2+
kinetics with faster and increased Ca
2+
release from sarcoplasmic reticulum (
Fig. 1c
), and increased expression of Na
+
, Ca
2+
, and K
+
channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation (
Fig. 1d
). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient.

Conclusion:
Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.
Abstract P451: Depletion Of Rbl2 Exacerbates Cardiomyocyte Apoptosis And Ischemia-reperfusion Injury By Augmenting E2f1-mediated Bnip3 Expression
Authors: Chen Jingrui , Liu Yuening , Xia Peng , Cheng Zhaokang
Published: November 2021

Background:
Reperfusion therapy, an effective treatment for myocardial infarction, triggers ischemia-reperfusion (I/R) injury and eventually may result in heart failure. Retinoblastoma-like 2 (Rbl2), a major retinoblastoma family member expressed in the heart, maintains the postmitotic state of adult cardiac myocytes. However, the role of Rbl2 in myocardial I/R injury remains unclear. We hypothesize that Rbl2 deficiency exacerbates myocardial injury following I/R.

Methods and results:
Wild type C57BL/6 (8–10-week, male) mice were subjected to 30 min of ischemia followed reperfusion. I/R induced phosphorylation of Rbl2 at Ser952, which has been associated with Rbl2 protein inactivation. To determine the role of Rbl2 in vivo, Rbl2-deficient mice and wild-type littermates were subjected to I/R and infarct size was evaluated by Evans blue/TTC staining. Rbl2 deficiency significantly increased infarct size at 24 h post I/R when compared with wild-type littermate controls. Echocardiography and Masson’s trichrome staining revealed that Rbl2 deficiency exacerbated I/R-induced cardiac dysfunction and fibrosis. Moreover, ablation of Rbl2 exacerbated I/R-induced cardiomyocyte apoptosis, as evidenced by the increased TUNEL positive signal. Consistently, knockdown of Rbl2 augmented H
2
O
2
-induced cleavage of PARP and caspase 3 in neonatal rat cardiomyocytes
,
suggesting that depletion of Rbl2 exacerbated oxidative stress-induced cardiomyocyte apoptosis. Mechanistically, both I/R and H
2
O
2
induced expression of the pro-apoptotic protein BNIP3, which was augmented by depletion of Rbl2. Since the BNIP3 promoter contains an E2F-binding site, we further examined the levels of the transcriptional activator E2F1 and the transcriptional repressor E2F4. Western blotting revealed that disruption of Rbl2 reduced E2F4 but increased E2F1 levels in mouse heart both at baseline and following I/R.

Conclusion:
Our findings suggest that Rbl2 deficiency exacerbates cardiomyocyte apoptosis and ischemia-reperfusion injury by augmenting E2F1-mediated BNIP3 expression.
Abstract P452: Stem Cells Defect And Alarmins S100A8/A9 Overexpression Associate With Early-stage Frailty And Poor Cardiovascular Health In Older Adults
Authors: Bonora Benedetta Maria , Palano Maria Teresa , Testa Gianluca , Fadini Gian Paolo , Sangalli Elena , Madotto Fabiana , Persico Giuseppe , Casciaro Francesca , Vono Rosa , Colpani Ornella , Scavello Francesco , Cappellari Roberta , Orlando Patrizia , Carnelli Franco , Berardi Andrea , De Servi Stefano , Raucci Angela , ABETE PASQUALE , Giorgio Marco , Madeddu Paolo , Spinetti Gaia
Published: November 2021

Importance:
Frailty affects the physical, cognitive, and social domains exposing older adults to an increased risk of cardiovascular diseases and death. The mechanisms linking frailty and cardiovascular outcomes are mostly unknown.

Objective:
Here, we studied the association of hematopoietic stem/progenitor cells (HSPCs) and molecular markers of inflammaging with the cardiopulmonary phenotype and prospective adverse events of individuals classified according to levels of frailty.

Methods:
We conducted one observational study on two patients’ cohorts of older adults characterized for their frailty status examining the abundance (flow cytometry) and gene expression profile (RNAseq) of HSPCs, and the levels of cardiac dysfunction-associated alarmins S100A8/A9 and inflammatory cytokines (ELISA).

Results:
In a cohort of pre-frail 35 individuals (average age: 75 years), a physical frailty score above the median identified subjects with initial alterations in cardiorespiratory function and increased alarmins S100A8/A9 and inflammatory cytokines in peripheral blood. RNA sequencing revealed S100A8/A9 upregulation in HSPCs from the bone marrow (>10-fold) and peripheral blood (>200-fold) of individuals with greater physical frailty. We then interrogated the biomarkers in a cohort of 104 frail individuals (average age: 81 years) with multi-domain health deficits. Reduced levels of circulating HSPCs and increased S100A8/A9 concentrations were independently associated with the frailty index. Remarkably, low HSPCs and high S100A8/A9 simultaneously predicted major adverse cardiovascular events at 1-year follow-up after adjustment for age and frailty index.

Conclusions and Relevance:
Inflammaging resulting from alarmin and pro-inflammatory cytokines in pre-frail individuals is followed by pauperization of HSPCs in frail older people with comorbidities. S100A8/A9 upregulation is activated within HSPCs, identifying a phenotype that drives poor cardiovascular outcomes.
Abstract P456: High Glucose Suppresses Cardiomyocyte Progenitor Cell Regenerative Capacity And The Role Of Mir-195/ezh2 Crosstalk In Gestational Diabetes Mellitus
Authors: Mellacheruvu Pranav , Saha Progyaparamita , Mishra Rachana , Sharma Sudhish , Kaushal Sunjay
Published: November 2021

Introduction:
Gestational diabetes mellitus (GDM) is associated with a five-fold increase in congenital heart defects. It is critical to determine the biological effects of diabetes mellitus (DM)
in vivo
and high glucose
in vitro
on neonatal cardiomyocyte progenitor cells (nCPCs) to maximize their regenerative potential. In the present study we seek to investigate the roles of Mir-195 and its hypothesized target gene, enhancer of zeste homolog 2 (Ezh2), in GDM.

Hypothesis:
We predict that high glucose is associated with decreased cellular proliferation, viability and increased senescence through oxidative stress. We also hypothesize that expression of Mir-195 will be higher in DM-nCPCs and inhibit cell proliferation via Ezh2 silencing.

Methods:
We subjected nCPCs
in vitro
to increasing glucose concentrations; cellular proliferation, migration, reactive oxygen species generation, and apoptosis were assessed using cell counting kit-8, wound healing, dihydroethidium, and annexin assays respectively. Our
in vivo
experiments involved injecting four-week old female mice with streptozocin. After pairing diabetic mice with non-diabetic male mice, timed embryos at E14.5 were evaluated for viability, proliferation and characterization. mRNA expression levels of Mir-195 and Ezh2 protein levels were detected using RT-qPCR and western blot analysis respectively. Lipofectamine transfection, with siRNA inhibiting Mir-195, was performed on c-kit+ cardiac stem cells obtained from diabetic mothers.

Results:
We found that subjecting nCPCs
in vitro
to increased glucose concentration led to increased % cell death, decreased proliferation and expression of paracrine factors indicating poorer secretome quality from these cells. Our
in vivo
models showed that maternal diabetes impedes prenatal development as decreased expression of c-kit+/Lin- cells and ISL1+ cells and increased DHE positivity were seen in DM-nCPCs at E14.5. Expression of Mir-195 was higher in DM-nCPCs but Ezh2 mRNA and protein expression levels were significantly decreased. siRNA inhibition of Mir-195 revealed higher EZH2 expression in c-kit+ cells and concomitant increase in regenerative capacity.

Conclusion:
In conclusion, the viability of DM-nCPCs both
in vivo
and
in vitro
is decreased compared to NDM-nCPCs suggesting decreased postnatal regenerative capacity. Mir-195 is associated with increased apoptosis and decreased proliferation of nCPCs via abrogation of the protective effects of EZH2.
Abstract P469: Scale-up And Characterization Of Therapeutic Mesenchymal Stromal Cell-derived Extracellular Vesicles
Authors: Uman Selen , Burdick Jason A
Published: November 2021

Introduction:
Early studies have shown therapeutic benefits of mesenchymal stromal cells (MSCs) in cardioprotection due to their angiogenic, proliferative, anti-apoptotic and anti-inflammatory properties, which are now attributed to secreted factors such as extracellular vesicles (EVs). While MSC-EVs have shown promise in small animals for cardiovascular therapies, large animal studies are required to evaluate the therapeutic benefit of MSC-EVs for clinical translation. One of the biggest challenges for large animal studies is the need to generate clinically-relevant quality and quantity of EVs without batch-to-batch variations that could compromise efficacy. This study aims to explore three different cell culture methods (traditionally-used tissue culture plates (TCP), 3-D printed bioscaffolds in a perfusion system (P), and microcarriers in dynamic spinner flask conditions (M)) to scale-up the production of MSC-EVs across four different biological donors and rigorously investigate EV yield, size, shape, and content.

Methods:
MSCs were isolated from the iliac crest of four different Yucatan minipigs using heparinized syringes, and cells were expanded to passage four, at which point they were seeded onto the respective cell culture methods. EVs were collected from conditioned medium (CM) via differential ultracentrifugation. EV size, distribution, yield, and protein concentration were studied using Nanoparticle Tracking Analysis (NTA) and microBCA assays.

Results:
Both perfusion bioreactor and spinner flask systems enabled sustained maintenance of large numbers of cells. Across biological donors and fabrication methods, modes remained within 50-150 nm and were not statistically different. Microcarrier-based spinner flasks and perfusion bioreactor set-ups both improved EV yield, up to 6 times in efficiency. Ongoing research focuses on examining differences in EV content across biological donors using RNA-sequencing and proteomics.
Abstract P472: Edil3 Autoantibodies Do Not Distinguish Kawasaki Disease (KD) From Febrile Controls, But Are Acutely Lower In Children With Cardiac Involvement.
Authors: Welliver R. Ross , Hicar Mark
Published: November 2021

Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms. Epidemiologic and demographic data support that a single preceding infectious agent may lead to KD, however, the cause remains unknown. The leading theory on pathogenesis is a post-infectious self-limiting autoimmune response, but this is still controversial. A recent study of individuals with KD demonstrated an association with autoantibody response to EDIL3 (also known as DEL-1). EDIL3 is a structural glycoprotein expressed by both macrophages and vascular endothelium. This presents an attractive potential target for investigation, as EDIL3 is known to modulate inflammation via leukocyte binding and infiltration through vessel walls. Additionally, EDIL3 is highly expressed in aortic and coronary artery tissue samples, showing some of the highest non-neural expression. Genetic variants of Del-1 have been associated with risk of intracranial aneurysms. Due to this burgeoning literature, we interrogated our cohort of plasma samples from febrile children, including Kawasaki disease, to confirm specificity of targeting to EDIL3. Plasma samples from febrile children and children with KD, including timecourse samples, were interrogated with ELISA assays using full-length recombinant EDIL3 (LSBio). In contrary to the published report, EDIL3 binding does not appear to be specific to KD when compared to febrile controls inclusive of a variety of disease etiologies (viral, bacterial, etc.). On serial samples (pre-IVIG, post-IVIG and convalescent) from the two different patient cohorts, antibodies binding EDIL3 are elevated in post-IVIG samples. This further supports general autoantibody reactivity against EDIL3 as IVIG is a pooled blood product from thousands of individuals typically. Intriguingly, in comparison of samples from children with or without any coronary artery Z scores above 2.5, EDIL3 autoantibody responses were actually lower in those with dilated coronary arteries. Current efforts are focused on identify specific human autoantibodies against EDIL3. Implications of these findings will be explored.
Abstract P475: Microrna-129-5p Rescues Myocardial Fibrosis And Calcification By Targeting Asporin And Sox9 In Cardiac Fibroblasts
Authors: Medzikovic Lejla , Aryan Laila , Ruffenach Gregoire , Li Min , Savalli Nicoletta , Olcese Riccardo , HONG JASON , Sharma Salil , Fishbein Gregory , Eghbali Mansoureh
Published: November 2021

Myocardial fibrosis promotes heart failure (HF) progression by impairing myocardial compliance, but also may predispose to myocardial calcification, further impairing cardiac function. Transition of resident cardiac fibroblast (CF) to pro-fibrotic myofibroblasts (MF) and osteogenic cell fates (OF) are key events which are partially controlled by microRNAs (miRs). To discover novel miRs involved in myocardial fibrosis and calcification, we compared online-available microarray datasets of left ventricles (LV) from failing human and mouse hearts. Assessing differentially-expressed miRs known to regulate fibrosis and calcification genes revealed that miR-129-5p is significantly downregulated in HF LV. Bioinformatic target analysis revealed small leucin-rich proteoglycan Asporin (Aspn) and SRY-Box Transcription Factor 9 (Sox9) as two novel miR-129-5p targets upregulated in both mouse and human diseased LV. Thus far, nothing is known about miR-129-5p in cardiac fibrosis and calcification. Additionally, the role of Asporin in myocardial fibrosis and the roles of either Asporin or Sox9 in myocardial calcification remain undiscovered. We show that miR-129-5p is expressed in CF in mouse and human hearts and is downregulated in CF of both HF patients and Angiotensin II (AngII)-injured mice, while Asporin and Sox9 are upregulated in CF of HF LV.
In vitro
, AngII or transforming growth factor-β downregulated miR-129-5p expression in primary adult mouse CF. Overexpression of miR-129-5p in CF inhibited expression of MF and OF transition markers, reduced migration, collagen production and calcium deposition. We validated Asporin and Sox9 as direct targets of miR-129-5p. Accordingly, silencing of Asporin and Sox9 in CF attenuated molecular and functional characteristics of MF and OF transition. Strikingly, systemic delivery of miR-129-5p mimics in mice directly targets CF and is sufficient to rescue preexisting AngII-induced myocardial fibrosis, calcification, diastolic- and systolic dysfunction. In conclusion, miR-129-5p rescues myocardial fibrosis and calcification by attenuating MF and OF transition via inhibition of Asporin and Sox9 in CF and is a promising therapeutic target.
Abstract P481: Short-term Synbiotic, B420 And Oligofructose, Treatment Reverse High-fat-diet Related Pathologies In Ischemic Reperfusion Mouse Models
Authors: Hoyer-Kimura Christina H , Behm Christine , Fricks Joshua P , Duca Frank , Konhilas John P
Published: November 2021

The gut microbiome impacts metabolic homeostasis, and several inflammatory pathologies including obesity, glucose intolerance, and worsened cardiovascular disease outcomes are associated with an altered gut microbiome. We have previously demonstrated
Bifidobacterium animalis lactis
420 ([B420], a probiotic) attenuates myocardial infarct injury in mice following ischemic reperfusion (IR) injury. Further, Oligofructose ([OFS], a prebiotic) improves glucose tolerance, and reduces weight gain in high-fat-diet (HFD) mice. However, the combined ability of B420 and OFS (a synbiotic) on cardiovascular and metabolic disease is unknown.

We hypothesized that short-term (2 weeks) synbiotic treatment improves adiposity and glucose tolerance and reduces IR infarct size in HFD fed male mice more than prebiotic or probiotic treatments alone. Adult male mice were fed HFD (45% fat) for 2 weeks and were then treated with OFS (10microliters/gram weight, 200miligram OFS/ 300microliters saline), B420 (10microliters/gram weight, 10
9
CFU/ 300microliters saline), or B420+OFS (synbiotic treatment) via daily gavage for 2 weeks. Following treatment mice underwent an IR protocol, (45-minute ligation followed by reperfusion). Oral glucose tolerance tests were conducted prior to and after treatment to determine blood glucose. Fasted mice were gavaged with glucose (2.5g/kg glucose) and blood glucose was measured over 120 minutes. Body weight, and kcal intake were measured weekly. Upon sacrifice epididymal fat mas was weighed to determine adiposity. Data were analyzed by one-way ANOVA or T-test.

We found that short-term synbiotic treatment with B420+OFS during HFD-feeding reduced adiposity and improved glucose tolerance, while probiotic or prebiotic treatment alone had no effect. However, short-term treatment, including synbiotic, did not significantly reduce heart infarct size as previously demonstrated with longer treatment periods. Taken together, these results suggest that synbiotic treatment improves metabolic impairments more rapidly than B420 or OFS alone, and precedes improvements in infarct size.