bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2095: Role Of Intraplaque Lipids In Atherosclerotic Plaque Vulnerability
Authors: Moly Pricila K , Haider Nezam , Zayed Mohamed , Black Joshua , Succar Bahaa , Saad Emile , Zhou Wei
Published: November 2022

Background:
Hyperlipidemia plays a pivotal role in the pathogenesis of diabetes, atherosclerosis, and cardiovascular diseases. Specific lipids of atherosclerotic plaques are potentially one of the local factors which render them susceptible to rupture. Their underlying unresolved inflammation and oxidative stress contribute to the transition from stable to unstable plaques. Therefore, we aimed to identify, characterize, and compare the intraplaque lipids composition among diabetic and non-diabetic patients and to ascertain their role in inflammation, and atherosclerotic plaque destabilization and rupture.

Methods:
Approved by the IRB, University of Arizona, Tucson Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS) from 40 surgically removed endarterectomy specimens that include
Asymptomatic & Non-diabetic
(n=13),
Symptomatic & Non-diabetic
(n=17),
Asymptomatic & Diabetic
(n=5) iv.
Symptomatic & Diabetic
(n=5) patients. Seventy nine lipid species from the main lipid classes phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cholesteryl ester (CE) phosphatidylglycerol (PG), sphingomyelin (SM), and ceramide (Cer) were included in this lipidomic profiling.

Results:
LC-MS data analysis identified a significant difference (p<0.05) in 2 individual lipid species – a long chain polyunsaturated PC(40:7) and SM(18:1) between diabetic and non-diabetic (Table:1). A total of 23 lipid species were significantly (p<0.05 to p<0.005) differentially present between symptomatic and asymptomatic samples. Symptomatic diabetic and nondiabetic samples showed a significant (p<0.05 to p<0.005) difference in 13 lipids belonging to PC, SM and PE. Our studies also showed a higher peroxidation of intraplaque lipid from diabetic patients compared to non-diabetics, and a significant increase in reactive oxygen species (ROS) production in human coronary artery smooth muscle cells treated with lipids from diabetic plaques.

Conclusions:
This exploratory analysis showed distinct patterns of intraplaque lipid composition. Identification of these high-risk plaque lipid biomarkers will help to delineate the cellular and molecular mechanism responsible for stable to vulnerable plaques transition.
Abstract P2096: Long Qt Syndrome Type 3 Phenotype Dependence On Extracellular Sodium And The Perinexus In A Genetic Mouse Model
Authors: Wu Xiaobo , Hoeker Gregory , Gourdie Robert G , Weinberg Seth , Poelzing Steven
Published: November 2022

Background:
Long QT Syndrome Type 3 (LQT3) is caused by cardiac voltage-gated sodium channel (Nav1.5) gain of function and characterized by action potential duration (APD) prolongation. A guinea pig model of drug-induced LQT3 demonstrated that elevating sodium and widening the perinexus, an intercalated disc cleft adjacent to gap junctions, individually and synergistically exacerbate APD prolongation. The synergistic effect suggests that high sodium with cardiac edema is a risk factor for LQT3 patients. However, it remains unclear if this synergistic effect occurs in a genetic model of LQT3.

Purpose:
To investigate individual and combined effects of sodium concentration and perinexal widening in a genetic mouse model of LQT3.

Methods:
Ventricular action potential duration at 30 (APD30) and 90 (APD90) percent of repolarization was measured from optically mapped, Langendorff-perfused wild type (WT) and ΔKPQ mouse hearts. Hearts were perfused with sodium concentrations of 145 or 160 mM, in the absence or presence of an intercalated disc adhesion inhibitor (βadp1) inducing perinexal widening.

Results:
At baseline with 145 mM sodium, APD30 and APD90 were significantly greater in ΔKPQ than WT hearts. Surprisingly, increased sodium (160 mM), significantly decreased APD90 in WT but not ΔKPQ hearts, which is inconsistent with results from the drug-induced LQT3 guinea pig model. In contrast, 160mM sodium significantly decreased APD30 in both genotypes suggesting elevated sodium increases the transient outward potassium current (Ito). Interestingly, with 145 mM sodium, βadp1 treatment did not significantly change APD30 or APD90 in either genotype. However, increasing sodium from 145 to 160 mM with βadp1 significantly increased APD90 in ΔKPQ hearts but not WT.

Conclusions:
Synergistic APD prolongation was modest in a genetic LQT3 mouse when elevating sodium and widening the perinexus relative to drug-induced LQT3 in guinea pig. A significant difference in action potential morphology between mice and guinea pig is the over-expression of Ito in mice and its absence in guinea pig. Future studies will investigate whether transient outward potassium channels contribute to the effect of high sodium on APD shortening during early repolarization.
Abstract P2100: Scalable Ultra-purification Of Adeno-associated Viral Vectors - A Novel Standard To Boost Transduction Efficacy And Potency For Cardiac Gene Therapy Development
Authors: Jungmann Andreas , Simon Stephanie , Schlegel Philipp , Meinhardt Eric , Steeg Charlotte , Kroemer Jasmin , Ruppert Thomas , Richter Karsten , Nessling Michelle , Raake Philip W , Frey Norbert , Most Patrick , Katus Hugo A , Busch Martin
Published: November 2022

Introduction:
A high transduction efficacy and potency of AAV-based cardiac gene therapies is key for the clinical translation in which hereditary and acquired cardiac disorders will be targeted. As such, ultrapure vectors with superior biological and therapeutic capabilities are a must for these therapies.

Hypothesis:
We hypothesized that our developed affinity chromatography (AC) based purification system will increase AAV recovery and - potency.

Methods:
AAV vector production, AC based - or iodixanol density gradient (DG) based purification, Q-PCR, WB, EM, LC-MS/MS, P-loop

Results:
Using the same vector input quantity, the AC-based purification enabled an approximately 13-fold greater vector genome copy (vgc) recovery than the DG-based purification. Mass spectrometry analysis demonstrated ultrapure AAV9 vectors as a result of an AC purification (AAV9 93.24% vs. 6,76% contaminants) whereas corresponding DG preparations resulted in highly contaminated vectors (AAV9 5.49% vs. 94,51% contaminants). Biological potency of AC- and DG-purified AAV9 vectors towards cardiac transduction were determined by systemic injections of 1·10
10
, 1·10
11
or 1·10
12
vgc of AAV9-EGFP in C57BL/6 mice (n=8 each group). AC-purified AAVs achieved a significantly higher cardiac transduction efficacy for every dosage assessed by comparative bulk myocardial DNA, RNA and protein level analysis after 2 weeks. Therapeutic potency was examined for a recently published novel target for chronic heart failure namely the RFXP1-RLN system. To this end, a dosage of 5·10
11
vgcs of either AC- or DG purified AAV9-RXFP1 vectors were systemically injected and the cardiac contractile performance increase was captured after 2 weeks in mice of both groups. Of note, 10 minutes after RLN administration, the rise in LV +dp/dt
max.
was already significantly greater in the AC- than the DG-vector treated group (AC: 13594+/-1972 vs. DC: 9822+/-801 mmHg/s; n=8 per group, p<0.01).

Conclusion:
The data clearly promote AC-based AAV purification as a novel standard for cardiovascular basic and translational research. Higher consistency in results, higher therapeutic effects and superior biological potency can be expected from higher production yields of ultrapure AAVs even at lower vector dosages.
Abstract P2102: EB3 Inhibitor VT-109 Activated FOXM1 Pathways To Promote Resolution Of Acute Lung Injury
Authors: Kwok Man Long , Qu Xinyan , Chan Wan Ching , Zhao Shuangping , Le Jonathan , Maienschein-Cline Mark , Komarova Yulia A
Published: November 2022

Acute respiratory distress syndrome (ARDS) is the acute onset of non-cardiogenic pulmonary edema, hypoxemia, and respiratory failure, conditions associated with a high mortality rate. ARDS is frequently caused by lung infections including SARS-CoV-2. In light of the COVID-19 pandemic, development of therapeutic approaches to combat ARDS have become an urgent unmet medical need. As increased vascular leakage is a culprit of alveolar damage and lung inflammation, we hypothesize that an effective therapy against vascular leakage should improve the clinical outcome of ARDS patients. We have identified a drug candidate Vascular Therapeutics (VT)-109, an allosteric inhibitor of End Binding protein 3, which contributes to vascular leakage by amplifying pathological calcium signaling in endothelial cells. Treatment of mice with VT-109 reduced the leakage of proteinous fluids in lungs to 55.4% (n=7, p<0.05) after endotoxin challenge, 77.7% (n=6, p<0.01) after exposure to high volume mechanical ventilation, and 42.5% (n=7, p<0.05) in the two-hit model, a combination of polymicrobial sepsis with normal tidal volume mechanical ventilation. VT-109 reduced the mortality of both septic (n=10, p<0.01) and endotoxin-challenged (n=10, p<0.05) mice. Furthermore, treatment with VT-109 in mice expressing human ACE2 receptor blocked diffuse alveolar damage (n=5, p<0.0001) caused by SARS-CoV-2 infection. In this COVID-19 model, the therapeutic benefits of VT-109 were observed when treatment was initiated at either day 1 or day 3 post-viral infection. Transcriptome analysis of lung endothelial cells in mice challenged with endotoxin showed that VT-109 upregulated 23 FOXM1 target genes (>2-fold) suggesting that it promotes endothelial cell regeneration by activating the FOXM1 pathway. Our data demonstrate that VT-109 successfully treats vascular leakage and is a promising drug candidate for future clinical studies. Funding for this work is provided by the Congressionally Directed Medical Research Programs through the Peer Reviewed Medical Research Program (PRMRP) under award No. W81XWH-21-1-0639. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.
Abstract P2105: Precision Medicine In Pediatric Calmodulinopathy
Authors: Fetterman K. Ashley , Blancard Malorie , Jouni Mariam , Potet Franck , Webster Gregory G , George Alfred L , Burridge Paul W
Published: November 2022

Calmodulinopathy is a life-threatening cardiac arrhythmia syndrome resulting from a single heterozygous mutation in any of the three calmodulin (CaM) genes. Most (68%) persons with a pathogenic CaM variant will experience a major arrhythmic event such as aborted cardiac arrest or sudden cardiac death by the age of five years. Calmodulinopathies result in heterogenous clinical features with one of the predominant clinical phenotypes being long QT syndrome (CaM-LQTS). Despite current treatments, 56% of calmodulinopathy patients experience breakthrough cardiac events, underscoring the need for improved therapeutics. A current barrier to effective treatment is our incomplete understanding of how calmodulin genetic variants contribute to pathological phenotypes. Human induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes (hiPSC-CMs) while maintaining the patient’s genetic background. Application of hiPSC-CMs in the context of this disease allows us to better understand patient-specific heterogeneity at the cellular level. We have established a CaM-LQTS hiPSC-CM model using hiPSCs generated from five persons with a pathogenic CaM variant who present with CaM-LQTS. We hypothesize that we can model a CaM-LQTS phenotype using patient-specific hiPSC-CMs, identify clinically appropriate drugs that correct it, and determine underlying mechanisms that drive this phenotype. We have determined the cellular CaM-LQTS phenotype of each patient line using blinded electrophysiological, contractility, and calcium imaging studies and have identified FDA-approved drugs that alleviate these phenotypes. We are also performing experiments, with a focus on calcium cycling dysregulation, to identify underlying mechanisms that drive the cellular CaM-LQTS phenotype. In conclusion, these studies will provide a patient-specific methodology to identify necessary clinical treatments for CaM-LQTS and elucidate the drivers of this rare and severe disease.
Abstract P2109: Elaborating Safety Margins To Predict Drug Proarrhythmia Using Deep Learning And Patient-derived IPSCs.
Authors: Serrano Ricardo , Feyen Dries , Bruyneel Arne A , Hnatiuk Hnatiuk Anna , Vu Michelle , Amatya Prashila , Perea Gil Isaac , Prado Maricela , Seeger Timon , Wu Joseph C , Karakikes Ioannis , Mercola Mark
Published: November 2022

Introduction:
Drug-induced arrhythmias are a common cause for drug attrition during development and for restricted use or withdrawal from the market. Cell based assays to assess arrhythmia risk typically rely on the quantification of waveform features - e.g. action potential prolongation and after depolarizations - in the cells’ action potential. However, the predictive power of these approaches is limited.

Hypothesis:
We hypothesize that deep learning can extract features relevant to discriminating input classes in a systematic and unbiased manner, effectively removing the need for human-defined metrics. This can lead to a new model to estimate torsadogenic risk of drugs and evaluate the influence of myopathic gene variants on drug-induced arrhythmia.

Methods:
We optically recorded action potentials optically recorded for 40 drugs - characterized high, intermediate, and low or no torsadogenic risk in patients- at 8 concentrations in hiPSC-CMs from 3 healthy donors and 5 hiPSC-CMs isogenic lines carrying 5 gene variants that cause dilated and hypertrophic cardiomyopathies. We designed a convolutional neural network (CNN) to classify non-arrhythmic, arrhythmic and asystolic traces in hiPSC-CMs. Using the class probabilities measured by the CNN, we created torsadogenic and asystolic safety margins for each drug and cell line.

Results:
The arrhythmic class probability computed by the CNN, provided a continuous, dose-dependent metric of the proarrhythmic risk of drugs in healthy and cardiomyopathic hiPSC-CMs. We used this metric to estimate safety margins for drug-induced arrhythmia and achieved a 0.942 AUC in classifying drugs of high-intermediate risk from safe ones. We used this approach to discern the contribution of putative genetic risk factors to arrhythmia susceptibility by comparing the risk profiles of the same drugs in healthy and isogenic hiPSC-CMs carrying causal HCM and DCM gene variants that are associated with arrhythmia in patients.

Conclusions:
We conclude that deep learning algorithms can effectively evaluate proarrhythmic risk of small molecules. Moreover, they can also be used to discern heightened arrhythmic risk caused by genetic mutations that increase the propensity for drug-induced arrhythmia in patients.
Abstract P2115: Differential Cardiac Remodeling Profile Of Immunosuppression Drugs
Authors: Sallam Karim , Thomas Dilip , Gaddam Sadhana , Lopez Nicole , Beck Aimee , Dexheimer Ryan , Beach Leila Y , Rogers Albert J , Zhang Hao , Chen Ian Y , Ameen Mo , Hiesinger William , Teuteberg Jeffrey , Rhee June W , Wang Kevin , Sayed Nazish , Wu Joseph C
Published: November 2022

Introduction:
Heart transplantation provides lifesaving therapy for patients with end-stage heart failure. The longevity of the therapy is limited by Cardiac Graft Dysfunction (CGD), which is an acquired cardiomyopathy affecting transplanted hearts associated with diastolic and/or systolic dysfunction. Some clinical risk factors for CGD have been identified, but none of them are easily modifiable. An unexplored potential contributor to CGD is the choice of immunosuppression agent used despite multiple clinical reports suggesting reduced adverse cardiac remodeling with mammalian target of rapamycin (mTOR) inhibitors compared to calcineurin inhibitors (CNI). This study examines mechanisms of differential cardiac remodeling effects of CNI versus mTOR inhibitors in a human cellular cardiac model.

Methods/Results:
We utilized 3D cardiac spheres composed of induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells (cardiac organoids). Cardiac organoids were treated with 5 days of vehicle, tacrolimus (CNI), or sirolimus (mTOR inhibitor). We did not observe a significant difference in surrogates of systolic or diastolic function in treated cardiac organoids. We pursued single cell-RNA sequencing of drug-treated cardiac organoids and identified gene expression changes consistent with increased extracellular matrix deposition and fibroblast activity in response to CNI treatment. In addition, CNI-treated cardiac organoids cellular composition was notable for increased proportion of fibroblasts and less cardiomyocytes compared to mTOR inhibitor-treated cardiac organoids. To validate gene expression changes observed, we treated cardiac fibroblasts with drugs and observed an increase in collagen production in response to CNI treatment and a reduction in fibroblast number and collagen production in response to mTOR inhibitor treatment. Furthermore, we observed increased ATP production in CNI-treated cardiac fibroblasts, but a reduction in mTOR-treated counterparts.

Conclusion:
We identify reduced extracellular matrix deposition and cardiac fibroblast proliferation in response to mTOR inhibitor as a potential mechanism for the more favorable remodeling profile observed clinically.
Abstract P2121: Modeling Genetic Dilated Cardiomyopathies With Engineered Heart Tissues From Patient-derived And Isogenic Mutant Induced Pluripotent Stem Cells
Authors: Tsui Jonathan H , Zepeda Bernardo , Steltzer Stephanie S , Ho Jaclyn J , Priest James , Hoey Timothy
Published: November 2022

The availability of human induced pluripotent stem cells (hiPSCs) has offered the possibility to study human-derived models of different genetic strata of cardiomyopathy for mechanistic discovery and therapeutic development. However, the phenotypes derived from cardiomyocytes differentiated from hiPSCs in conventional 2-dimensional culture systems often fail to reproducibly model clinical presentations, thereby reducing the translatability of readouts from these assays. Here we report 3-dimensional engineered heart tissues (EHTs) produced with a variety of cell lines harboring patient-derived mutations (TTN, RBM20, BAG3) known to cause dilated cardiomyopathy (DCM) which recapitulate contractile deficits associated with DCM. Internal development of EHTs also recapitulate contractile defects with small interfering RNA (siRNA) knockdown models of haploinsufficiency across multiple disease states. These observed findings with EHTs suggests improved maturity in cardiomyocytes through the presentation of microenvironmental cues akin to those seen
in vivo
. With continued advances in EHT technologies and capabilities, this platform may serve to significantly reduce drug candidate attrition, as well as provide new insights into pathology, prototyping of therapeutic approaches, and mechanism of action that were previously difficult to obtain.
Abstract P3001: Hnrnpa2b1 An Rna-binding Protein That Orchestrates Smooth Muscle Cell Phenotype In Pulmonary Arterial Hypertension.
Authors: Ruffenach Gregoire , Medzikovic Lejla , Aryan Laila , Li Min , Eghbali Mansoureh
Published: November 2022

Background:
RNA-binding proteins (RPBs) are master orchestrators of gene expression regulation. RBPs regulate hundreds of transcripts at once by recognizing specific motifs. Characterizing RBP targets is critical to harvest their full therapeutic potential. However, such investigation has often been restricted to a few RBP targets, limiting our understanding of their function. In cancer, the RBP HNRNPA2B1 (A2B1) promotes the pro-proliferative / anti-apoptotic phenotype. The same phenotype in pulmonary arterial smooth muscle cells (PASMC) is responsible for the development of pulmonary arterial hypertension (PAH). However, A2B1 function has never been investigated in PAH.

Methods:
Through the integration of computational and experimental biology, we investigated the role of A2B1 in human PAH-PASMC. Bioinformatics and RNA-sequencing allowed us to investigate the transcriptome-wide function of A2B1, while RNA immunoprecipitation and A2B1 silencing experiments
in vitro
allowed us to decipher the intricate molecular mechanism at play. In addition, we performed a pre-clinical trial in the monocrotaline-induced pulmonary hypertension rat model to investigate the relevance of A2B1 inhibition in mitigating pulmonary hypertension severity.

Results:
In the present study, we found that A2B1 is up-regulated in human PAH-PASMC. We also show that A2B1 localization shifts in PAH-PASMC from cytoplasmic to nuclear mainly due to significantly higher expression of B1 isoform compared to A2 isoform of A2B1. Using bio-informatics, we identified three known motifs of A2B1 and all mRNAs carrying them. In PAH-PASMC, we demonstrated the complementary non-redundant function of A2B1 motifs as all motifs are implicated in different aspect of the cell cycle. In addition, we showed that in PAH-PASMC, A2B1 promotes the expression of its targets. A2B1 silencing in PAH-PASMC lead to a decrease of all tested mRNAs carrying an A2B1 motif, and an according decrease in proliferation and resistance to apoptosis. Finally,
in vivo
A2B1 inhibition in the lungs rescued pulmonary hypertension in rats.

Conclusion:
Through the integration of computational and experimental biology, our study revealed the role of A2B1 as a master orchestrator of the PAH-PASMC phenotype and its relevance as a therapeutic target in PAH.
Abstract P3002: Endothelial
Trpm2
Deletion Attenuates Ischemic Stroke By Preventing Blood-brain Barrier Disruption
Authors: Zong Pengyu , feng jianlin , Yue Zhichao , Yue Zhichao
Published: November 2022

Introduction:
Endothelial hyperpermeability is a hallmark of brain damage at the early stage of ischemic stroke. The subsequent blood-brain barrier (BBB) disruption drives the initial pathological changes and aggregates neuronal death. Ca
2+
overload is an important cause of endothelial dysfunction. TRPM2 is a Ca
2+
-permeable ion channel activated by oxidative stress, and oxidative stress is a critical contributor for endothelial hyperpermeability during ischemic stroke.

Hypothesis:
We hypothesized that TRPM2-mediated Ca
2+
influx plays a key role in causing endothelial hyperpermeability during ischemic stroke.

Methods:
Specific deletion of
Trpm2
in endothelial cells was achieved by crossing
Cdh5-cre
mice with

Trpm2
fl/fl

mice. Middle cerebral artery occlusion (MCAO) was performed to simulate ischemic stroke. Tetrazolium chloride and Evans blue assays were used to evaluate infarct size and BBB leakage. Occludin and F4-80 / myeloperoxidase staining were used to detect tight junction degradation and immune cell invasion. Cerebral endothelial cells (CECs) were isolated and cultured. Oxygen-glucose deprivation (OGD) was used to simulate
in vitro
ischemic conditions.
In vitro
endothelial leakage assays were used to examine CEC permeability. Rhodamine-123 imaging and Ca
2+
imaging were used to evaluate oxidative stress and Ca
2+
overload in CECs. WB was used to examine CD36 signaling activation and whole-cell current recording was used to detect TRPM2 activation.

Results:
Specific deletion of
Trpm2
in endothelial cells protects mice against MCAO-induced brain injury, which is characterized by reduced infarct size, mitigated plasma extravasation, reduced neutrophil / macrophage invasion and inhibited oxidative stress in the brain.
In vitro
experiments demonstrated that
Trpm2
deletion alleviates ROS production, Ca
2+
overload and endothelial hyperpermeability induced by OGD and CD36 ligand thrombospondin-1 (TSP1). In CECs, activation of CD36 signaling induced by OGD and TSP1 was impaired by TRPM2 knockout. In transfected HEK293T cells, OGD and TSP1 induce the activation of TRPM2 currents in a CD36-dependent manner.

Conclusion:
Endothelial specific TRPM2 knockout attenuates ischemic stroke by preserving BBB integrity.
Abstract P3011: Nrf2 Transcriptional Upregulation Of Idh2 To Tune Mitochondrial Dynamics And Rescue Angiogenic Function Of Diabetic Epcs
Authors: Dai Xiaozhen , Wang Kai , Lin Qian , Oscar Chen , Conklin Danile J , Wintergerst Kupper A , Cai Lu , Deng Zhongbin , Tan Yi
Published: November 2022

Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and
db/db
mice. Survival ability and angiogenic function of EPCs from diabetic patients and
db/db
mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and
db/db
mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in
db/db
mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in
db/db
mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulation of IDH2 expression and improving mitochondrial function of diabetic EPCs.
Abstract P3015: Cite-seq Identifies A Novel Role For Macrophage Trem2 In A Murine Model Of Hfpef
Authors: Smart Charles , Fehrenbach Daniel , Wassenaar Jean J , Agrawal Vineet , Hemnes Anna R , Doran Amanda C , Madhur Meena
Published: November 2022

Deoxycorticosterone acetate (DOCA)-salt is a common model of hypertension and has been used to study heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and to identify immune mediators of cardiac remodeling using unbiased methods. DOCA-salt mice underwent uninephrectomy, implantation of a DOCA pellet, and supplementation of the drinking water with 1%NaCl for three weeks. Control mice underwent a sham procedure and received normal water. Compared to control mice, DOCA-salt mice exhibited increased systolic blood pressure, cardiac hypertrophy, and decreased time to exhaustion upon treadmill exercise testing. In addition, DOCA-salt mice exhibited albuminuria, indicative of renal glomerular injury. On echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.70±.24, 8.22±.45; n=8,5) in DOCA-salt mice, consistent with diastolic dysfunction. CITE-seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, was performed on a total of 4,359 and 7,600 sorted leukocytes from four sham and four DOCA-salt left ventricles, respectively. Differential gene expression analysis revealed major transcriptional changes in cardiac macrophages between groups and identified
Trem2
as being upregulated in cardiac macrophages. We validated a two-fold increase in Trem2 gene expression in left ventricles of DOCA-salt mice and observed a two-fold increase in cardiac Trem2+Mac2+ cells in DOCA-salt by immunofluorescence. To test whether Trem2 has a causal role in HFpEF, wild type and Trem2 deficient animals were subjected to DOCA-salt. After DOCA-salt, Trem2-deficient mice displayed significantly increased heart weight to tibia length ratios (9.39±0.20, 11.21±0.80; n=6-7) and albumin to creatinine ratios (5.5±1.2, 14.2±2.9; n=6-7) compared to wild-type controls. No difference in albuminuria was observed at baseline (0.36±0.05, 0.46±0.10; n=10). In conclusion, the DOCA-salt mouse model induces key features of HFpEF and identifies Trem2 as a potential novel modulator of cardiac remodeling and renal injury.
Abstract P3017: Aldehyde Dehydrogenase 2 Augments Adiponectin Signaling In Coronary Angiogenesis In Hfpef Associated With Diabetes
Authors: Roy Bipradas , Pan Guodong , Giri Shailendra , Amirthalingam Thandavarayan Rajarajan , Palaniyandi Suresh S
Published: November 2022

4-hydroxy-2-nonenal (4HNE), a reactive aldehyde, is elevated in diabetes that decreases coronary angiogenesis and this was rescued by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme that detoxifies 4HNE. Adiponectin (APN), a pleiotropic hormone, has pro-angiogenic properties which was shown to be critical in diabetes and its complications. However, the interaction between ALDH2 and APN signaling in coronary angiogenesis is unknown. Coronary endothelial cell (CEC) damage is the initiating step of diabetes-mediated heart failure with preserved ejection fraction (HFpEF) pathogenesis. Thus, we hypothesize that ALDH2 restores 4HNE-induced downregulation of APN signaling in CECs and subsequent coronary angiogenesis in diabetic HFpEF. First of all, APN significantly increased coronary angiogenesis. However, 4HNE treatment significantly decreased the levels of APN signaling molecules including adiponectin receptor (AdipoR)1, APPL1, phospho-AMPK, AMPK, phospho-AKT, and AKT in mouse CECs. Treatment with disulfiram (DSF), an ALDH2 inhibitor, exacerbated 4HNE-mediated decreases in APN-induced increased coronary angiogenesis and APN-signaling cascades, whereas pretreatment with alda1, an ALDH2 activator, rescued the effect of 4HNE. We employed control (db/m) mice, spontaneous type-2 diabetic (db/db) mice, ALDH2*2 knock-in mutant mice with intrinsic low ALDH2 activity (AL), and diabetic mice with intrinsic low ALDH2 activity (AF) that were created by crossing db/db and AL mice to test our hypothesis in vivo. AF mice exhibit severe HFpEF at 6 months compared to age-matched db/db mice as well as 3 months-old AF mice. At 6m, db/db, AL & AF mice significantly decreased APN response to coronary angiogenesis along with increased serum APN levels and decreased cardiac tissue levels of AdipoR1, APPL1, P-AMPK, and P-AKT implicating occurrence of APN resistance which was higher in AF mice. Alda-1 treatment improved APN-mediated angiogenesis significantly in db/db and AF mice. In conclusion, 4HNE-induces APN resistance and subsequently decreases coronary angiogenesis in diabetic HFpEF which was rescued by ALDH2 activation.
Abstract P3018: A Requirement For P21 Activated Kinase1 To Preserve Cardiac Function And Metabolic Homeostasis
Authors: Rosas Paola C , Batra Ashley , Rafael-Clyke Koreena , Kurup Shreya , Chowdhury Shamim
Published: November 2022

Background/Hypothesis:
Heart failure with preserved ejection fraction (HFpEF, EF>50%) continues to lack effective treatment. Obesity and diabetes are strong predictors of HF, with >80% of HFpEF patients being obese or overweight and ~40% suffering from diabetes. P21 activated kinase-1 (PAK1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Cdc42/Rac1. We tested the hypothesis that PAK1 is an important element in the regulation of cardiac function and metabolic homeostasis.

Material and methods:
3-6 months male and female global PAK1 knock-out mice (PAK1
-/-
) and wild type (WT) controls were used to measure % of fat mass (by NMR), adipocyte size and glucose tolerance. We used echocardiography to assess cardiac dysfunction and western blots (WB) to examine cardiac protein alterations.

Results (see Table):
PAK1
-/-
hearts exhibited preserved ejection fraction and diastolic dysfunction with slower e’, increased E/e’ ratio and prolonged IVRT. Additionally, PAK1
-/-
hearts showed dilated left atrium and thickening of posterior wall. WB analyses showed decreased phosphorylation of cMyBP-C and dysregulation of calcium handling proteins. Moreover, PAK1
-/-
mice exhibited increased % fat mass, adipocyte hypertrophy and impaired glucose tolerance test.

Conclusion:
Deletion of PAK1 resulted in obesity, impaired glucose homeostasis and HFpEF. Thus, PAK1 is required to support cardiometabolic health.
Abstract P3020: Chronic Heart Failure As A Sequel After Severe Burn Injury - First Insight Into A Novel Pathological Heart-skin Axis
Authors: Hundeshagen Gabriel , Mertin Victoria , Thiele Philipp , Jungmann Andreas , Trogisch Felix A , Drews Oliver , Heineke Joerg , Van Linthout Sophie , Fielitz Jens , Mayr Manuel , Busch Martin , Kneser Ulrich , Most Patrick
Published: November 2022

Background:
We described chronic heart failure with preserved ejection fraction (HFpEF) as a long-term sequel in survivors of severe pediatric burn injury (BI) (Hundeshagen et al., Lancet Child & Adolescent Health, 2017). Applying a widely used standardized scald injury rat model in burn research, we sought to uncover systemic and molecular pathomechanisms that may cause post-BI HFpEF development.

Methods and Results:
Male adolescent SD-rats were subjected to a 60% total body surface area full-thickness BI (B; 100% survival) or sham (S) procedure (each n=10) and characterized them up to 90 days (3, 7, 30 and 90d) by serial echocardiography (E), bulk myocardial NGS and -proteomics, RT-PCR, IB, histology (H) and plasma proteomics for cardiac performance and molecular alterations, respectively. B rats mirrored typical post-burn clinical traits as significant loss in body (-27%*) or skeletal muscle weight (-30%*) e.g., with elevated atrophy markers as Murf1 (5-fold*) throughout the observation period vs S (30d, *P<0.05) Our focus on the heart revealed in vivo heart weight loss (-22%*), cardiomyocyte hypotrophy (-8%*) and diminished mTOR activity in B hearts (p/t-mTORC2 -43%*) vs S as well as significantly diminished left ventricular (LV) GLS with unchanged LV-EF. RT-PCR and H showed significant cardiac accumulation of cardiodepressive factors (i.e., S100A8 and A9) and e.g., granulocyte (MPO, >3-fold*) infiltration as well LV fibrosis (2.2-fold*). Cardiac proteomics yielded e.g., neutrophil degranulation as lead GO-term. Serial blood and plasma proteomic and ELISA analysis indicated elevated WBC (+26%*) and levels e.g., of IL6, S100A8/A9, CH3L1 and other HF markers alike changes in human post-BI plasma samples. WGCNA for bulk myocardial NGS and clinical traits related activated immunological and pro-fibrotic pathways in post-BI hearts to cardiac dysfunction in B.

Conclusion:
The first ever report of the development of HFpEF as a novel systemic consequence of severe burn injury in a rodent model prepares the ground for further mechanistic and translational studies. Cardiac inflammation and fibrosis that negatively impact cardiac performance may be mechanistic key findings guiding further therapeutic studies and validation of post-BI HF biomarkers.
Abstract P3021: Blocking Grk2-induced Adiponectin Receptor 1 Serine
205
Phosphorylation As A Potential Target Against Post-ischemic Pathological Remodeling
Authors: Zhao Jianli
Published: November 2022

Background:
Despite significantly reduced acute MI mortality in recent years, ischemic HF continues to escalate. Therapeutic interventions effectively reversing pathologic remodeling are an urgent unmet medical need. We recently demonstrated that AdipoR1 phosphorylation by GRK2 contributes to maladaptive remodeling in the ischemic heart. The current study clarified the underlying mechanisms leading to AdipoR1 phosphorylative desensitization, and investigated whether blocking AdipoR1 phosphorylation may restore its protective signaling, reversing post-MI remodeling.

Methods and Results:
Specific site(s) and underlying molecular mechanisms responsible for AdipoR1 phosphorylative desensitization were investigated in vitro (neonatal and adult cardiomyocytes). The effects of AdipoR1 phosphorylation inhibition upon adiponectin (APN) post-MI remodeling and HF progression were investigated in vivo. Among 4 previously identified sites sensitive to GRK2 phosphorylation, alanine substitution of Ser
205
(AdipoR1
S205A
), but not other 3 sites, rescued GRK2-suppressed AdipoR1 functions, restoring APN-induced cell salvage kinase activation and reducing oxidative cell death. AdipoR1 phosphorylation promoted clathrin-dependent (not caveolae) endocytosis and lysosomal-mediated (not proteasome) degradation, reducing AdipoR1 protein level and suppressing AdipoR1-mediated cytoprotective action. GRK2-induced AdipoR1 endocytosis and degradation were blocked by AdipoR1
S205A
overexpression. Moreover, AdipoR1
S205E
(pseudo-phosphorylation) phenocopied GRK2 effects, promoted AdipoR1 endocytosis and degradation, and inhibited AdipoR1 biological function. Most importantly, AdipoR1 function was preserved during HF development in AdipoR1
S205A
KI mice. APN administration 7 days after MI reversed post-MI remodeling and improved cardiac function. However, re-expressing hAdipoR1
WT
in AdipoR1-KO mice failed to restore APN cardioprotection.

Conclusion:
Ser
205
is responsible for AdipoR1 phosphorylative desensitization in the failing heart. Blockade of AdipoR1 phosphorylation followed by pharmacologic APN administration is a novel therapy effective in reversing post-MI remodeling and mitigating HF progression.
Abstract P3022: Multiscale Characterization Of Left Ventricular Diastolic Dysfunction In Diabetic And Cardiac Myosin Binding Protein-C Phospho-Ablated Murine Models
Authors: Neelakantan Sunder , Kumar Mohit , Singh Rohit , Koch Sheryl E , Rubinstein Jack , Burton Aaron J , Sadayappan Sakthivel , Avazmohammadi Reza
Published: November 2022

Introduction:
Left ventricular (LV) diastolic dysfunction (LVDD) is often characterized by organ-level (global) metrics whose quantitative connections to mechanical remodeling events at the tissue and fiber levels remain poorly studied.

Methods:
We performed measurements in the LV at three length scales, namely, organ, tissue, and fiber, in two murine models of LVDD: (i) diabetic (db/db) (n=10) versus wild-type (WT; n=10) mice, and (ii) novel knock-in cardiac myosin binding protein-C (cMyBP-C) phospho-ablated (KI-3A; n=8) versus cMyBP-C phospho-mimetic (KI-3D, n=6) and WT (n=8) mice. Echocardiography was done in vivo. Left ventricular free wall (LVFW) specimens were harvested and passively stretched along with circumferential (C) and longitudinal (L) directions (schematically identified in Fig. 1c) in select mice. Passive tests were followed by activation-relaxation tests of the LVFW specimens at resting biaxial stretches. Active force-pCa measurements at resting stretch were carried out on the skinned papillary muscle fibers.

Results:
Both db/db and KI-3A mice demonstrated slow relaxation at the organ level indicated by increased E/e' (Figs. 1a-b). db/db LVFW specimens showed increased circumferential stiffness at the tissue level under biaxial passive stretching (p<0.05 for strains >0.25). Biaxial activation-relaxation tests of LVFW specimens in knock-in mice (Fig. 1c shows the results for KI-3A mice) revealed that KI-3A mice relax significantly slower than KI-3D mice along the longitudinal direction (Fig. 1d). Fiber-level tests demonstrated that active force development was significantly enhanced in db/db mice (Fig. 1e) while it was significantly suppressed in KI-3A mice relative to KI-3D and WT mice (Fig. 1f).

Conclusions:
Although both db/db and KI-3A mice showed LVDD at the organ level, they demonstrated different mechanical alterations at the fiber and tissue fiber levels, suggesting that global metrics should be complemented with metrics characterizing the LV properties at the fiber and tissue levels to fully characterize LVDD.
Abstract P3023: The Regulatory Role Of Mir-21 In Heart Failure With Preserved Ejection Fraction (hfpef)
Authors: Juguilon Cody , Enrick Molly , Gadd James , Wang Yang , Clark Alyssa , Kolz Chris , Ohanyan Vahagn A , Chilian William M , Yin Liya
Published: November 2022

Introduction:
Coronary microvascular dysfunction (CMD) is characterized by impaired endothelial-dependent vasodilation. These impairments are seen in diabetic cardiomyopathy, ischemia with no obstructive coronary artery disease, and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated. Moreover, how microRNA (miR-21) regulates CMD in HFpEF is not entirely understood.

Methods:
miR-21 knockout and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS) for 6 months and blood lipid and glucose were measured. Echocardiography and treadmill exercise exertion tests were performed to assess cardiac function. Myocardial blood flow (MBF) under stress was measured by contrast echocardiography or doppler after the treatment with different dosages of norepinephrine and mean arterial blood pressure was measured simultaneously by femoral pressure catheter. Cardiac fibrosis was detected using trichrome staining and molecular pathways were elucidated via gene and protein analysis.

Results:
Our preliminary data show decreased MBF during stress in WT mice fed a HFHS diet. While ejection fraction was not changed, cardiac index, stroke volume, and running distance were decreased. Furthermore, E/E’ ratio was increased in WT mice fed HFHS diet compared to the WT mice fed a chow diet, suggesting heart failure and impaired diastolic function with normal systolic function. Moreover, perivascular fibrosis was increased in the WT mice fed a HFHS diet compared to WT mice fed a chow diet. However, the ablation of miR-21 reversed all these changes in the mice fed a HFHS diet.

Conclusions:
miR-21 regulates CMD and ameliorated HFpEF. Further investigation will elucidate the pathways and mechanisms converging with miR-21 to regulate HFpEF.
Abstract P3029: Role Of Histone Deacetylase 9 In The Development Of Adipose Tissue Senescence And Mitochondrial Dysfunction
Authors: Goo Brandee M , Ahmadieh Samah , Zarzour Abdalrahman , Veerapaneni Praneet , Kim David , Cave Stephen , Ogbi Mourad , Nguyen Jenny , Lei Yun , Lu Xin-Yun , Kim Ha Won , Weintraub Neal L
Published: November 2022

Background:
The prevalence of obesity increases with age and is a major risk factor for the development of cardiovascular disease. Our previous studies showed that histone deacetylase 9 (HDAC9) gene deletion protected mice against obesity-associated metabolic disorder. Here, we hypothesized that HDAC9 expression during aging plays an important role in the development of adipose tissue senescence and mitochondrial dysfunction.

Methods and Results:
Visceral adipose tissue from 20-month-old wild type (WT) mice showed increased expression of HDAC9 (2-fold), p16 (5-fold) and p21 (2-fold) compared to 3-month-old mice. In addition, HDAC9 expression in human subcutaneous adipose tissue positively correlated with age (r
2
=0.088, p=0.034). Furthermore, 1-year-old HDAC9 knockout (KO) mice were protected against the development of age-associated weight gain compared to age-matched WT, in conjunction with reduced levels of senescence-associated beta-galactosidase (SABG) staining and lower expression of p16 (0.7-fold) and p21 (0.5-fold). Interestingly, preadipocytes, which reside in adipose tissue and replenish aged adipocytes, were protected against H2O2 and UV-light induced senescence as evaluated by SABG staining, p16 and p21 expression. Next, we measured adipose tissue mitochondrial function, which is mechanistically linked to senescence, using the Seahorse analyzer. HDAC9 KO significantly increased adipose tissue mitochondrial oxygen consumption rate (e.g., increased basal respiration, proton leak and ATP production). To gain further mechanistic insight, we performed RNA-sequencing of WT and KO adipose tissues and identified thiosulfate sulfurtransferase (TST), an enzyme that is reduced in human obesity and modulates mitochondrial function, as one of the potential mediators for adipose tissue senescence and metabolism. Interestingly, TST expression was reduced in 1-year-old compared to 3-month-old mice (0.2-fold) and enriched in KO compared to WT (4-fold) adipose tissue by qPCR analysis.

Conclusion:
HDAC9 plays a crucial role in the development of adipose tissue senescence, potentially through regulation of TST and mitochondrial function and may be a promising target to maintain healthy adipose tissue during aging.
Abstract P3031: Highly Sensitive Impedance Microsensors Based On Graphene Quantum Dots For The Detection Of Metabolically Active Plaques In Animal Models
Authors: Wang Kaidong , Hsiai Tzung K
Published: November 2022

Detection of rupture-prone and unstable atherosclerotic lesions is of vital values for patients with acute coronary syndromes or stroke. Despite advances in diagnosis and therapy, sensing of metabolically active but angiographically invisible atherosclerotic lesions remains an unsatisfied clinical challenge but highly desirable. Graphene quantum dots (GQDs) have been widely studied in recent years due to its unique optical and electrical properties, thus providing a huge opportunity for building high-performance sensors. Herein, a highly sensitive impedance microsensor based on platinum nanoparticles (PtNPs) @GQDs was constructed for atherosclerotic lesions assessment, especially for the perception of metabolically active but angiographically invisible atherosclerotic lesions. We designed two-step procedures to prepare PtNPs@GQDs microsensors including electrochemical reduction of GQDs and subsequent electrochemical deposition of ultrafine PtNPs. Atherosclerotic lesions samples at mouse aorta and pig carotid confirm this microsensors with highly sensitive impedance signals and indicated possible atherosclerotic lesions. The equivalent circuits simulation suggested that synergetic PtNPs@GQDs creates more parallel conduction path, thus leading to highly sensitive impedance signals. Our studies offer a universal strategy to prepare high-performance impedance sensors and unique opportunities of translational medicine for early atherosclerotic disease detection and prevention of acute coronary syndromes and stroke.