bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3005: Cannabinoid Receptor 1 Antagonist Genistein Attenuates Marijuana-Induced Vascular Inflammation
Authors: Chandy Mark , Wei Tzu-tang T , Nishiga Masataka , Zhang Angela , Kumar Kaavya K , Thomas Dilip , Manhas Amit , Rhee Siyeon , Justesen Johanne M , Chen Ian Y , Wo Hung-Ta , Yang Johnson Y , Khanamiri Saereh , Seidl Frederick , Burns Noah , Liu Chun , Sayed Nazish , Shie Jiun-Jie , Yeh Chih Fan , YANG Kai-chien , Lau Edward , Lynch Kara , Rivas Manuel , Kobilka Brian , Wu Joseph C
Published: November 2022

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ
9
-tetrahydrocannabinol (Δ
9
-THC), the psychoactive component of marijuana, binds cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis is an independent risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD.
In silico
virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) were used to model Δ
9
-THC induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference (CRISPRi), and genistein attenuate the effects of Δ
9
-THC. In mice, genistein blocked Δ
9
-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system (CNS). Genistein is a peripherally restricted CB1 antagonist that attenuates Δ
9
-THC-induced atherosclerosis.
Abstract P3006: A Human Arterial Cell Atlas
Authors: Cheng Paul , Pedroza Albert J , Sharma Disha , Weldy Chad S , Nguyen Trieu , Dalal Alex R , Shad Rohan , Kim Juyong B , Fischbein Michael P , Wirka Robert , Quertermous Thomas
Published: November 2022

Background:
Human vascular diseases are the worldwide leading causes of morbidity and mortality. Nearly all human vascular diseases have arterial segment-specific tropisms despite identical exposures to genetic and environmental risk factors. Understanding the cellular and transcriptomic determinants of arterial identities may hold the key to identifying novel pathophysiology and potential therapies.

Methods:
To specifically determine arterial site-specific differences independent of inter-individual variation, we have generated a human arterial cellular atlas by simultaneously collecting and analyzing up to 8 arterial sites from multiple healthy transplant donors. We performed single cell transcriptomic analysis on arterial segments to determine the differences in cellular composition and transcriptomic programs. We subsequently integrated human genetic data with cell-type specific transcriptomic differences across vascular beds to identify probable causal cells and causal genes associated with human vascular phenotypes.

Results/Conclusions:
Single cell transcriptomic analysis of >150,000 cells sequenced at >50,000 reads per cell revealed that the dominant cellular drivers of transcriptomic differences between distinct arterial segments, i.e. determinants of arterial identity, are fibroblasts and smooth muscle cells, not endothelial cells or macrophages. Adult vascular cells transcriptomes from different segments are most influenced by their embryonic origins but not by anatomical proximity. Differentially regulated genes in fibroblast across different vascular beds were particularly enriched for vascular disease associated genetic signals, suggesting a prominent role for these cells in human disease. While the majority of endothelial cells were transcriptionally similar across vascular beds, a rare, previously undescribed, cluster of endothelial cells were identified who expressed segment-specific transcriptomic signatures. Differentially expressed genes in these cells were enriched for vascular disease signals, suggesting a possible role of these rare cells in human disease.
Abstract P3010: Angptl4 Reduces Atherosclerosis And Promotes Fibrous Cap Stability By Targeting Klf4 In Vascular Smooth Muscle Cells
Authors: Kim Yong-sook , Cho Dong-im , Cho Hyang H , Cho Meeyoung , Ahn Young Keun
Published: November 2022

Background:
Atherosclerosis is vascular disease of chronic inflammation, and we recently showed that angiopoietin-like 4 (ANGPTL4) promote cardiac repair by suppressing pathological inflammation. Given the contribution of inflammation to atherosclerosis, we assessed the effect of ANGPTL4 on atherosclerosis development.

Methods:
We injected recombinant ANGPTL4 protein or PBS to atherosclerotic Apoe-/- mice twice a week fed a high-fat diet for 8 weeks, and analyzed atherosclerotic lesion size, inflammation, and plaque stability. The effect of ANGPTL4 on vascular smooth muscle cells (VSMCs) was studied, and plasma levels of ANGPTL4 in patients were measured.

Results:
ANGPTL4 treatment reduced the atherosclerotic plaque size and vascular inflammation. In the atherosclerotic lesions, the number of α-SMA(+)MYH11(+) cells was higher, while the number of CD68(+)cells was lower in the ANGPTL4 group than in the vehicle-treated group. Moreover, α-SMA-expressing fibrous cap was significantly thicker in the ANGPTL4 group than in the PBS group. Then, we isolated VSMCs from atherosclerotic aortas, and found that CD68 and Krüppel-like factor 4 (KLF4) were significantly upregulated in the PBS group. As KLF4 is a phenotypic modulator and an inducer of NADP oxidase 1 (Nox1) in VSMCs, the effect of ANGPTL4 on VSMCs was assessed. Cholesterol-induced upregulations of Nox1 and KLF4 were substantially inhibited by ANGPTL4 treatment. Then, we analyzed the circulating ANGPTL4 levels of the cardiovascular disease patients, and found the positive correlation between high ANGPTL4 and low incidence of vascular events.

Conclusions:
Our results showed that ANGPTL4 inhibited atherogenesis by increasing the fibrous cap thickness. KLF4 and Nox1 were suggested as targets of ANGPTL4 in VSMCs to confer plaque stability by attenuating phenotypic changes of VSMCs toward inflammatory macrophage-like cells. Taken together, circulating ANGPTL4 may be associated with regulation of vascular events.
Abstract P3014: Dislocation Of Hexokinase1 From Mitochondria Results In Heart Failure With Preserved Ejection Fraction Through Hyper O-glcnacylation In Endothelial Cells.
Authors: Tatekoshi Yuki , Shapiro Jason S , Nejad Farnaz K , Liu Mingyang , Nayudu Krithika , Chen Chunlei , Ardehali Hossein
Published: November 2022

Introduction:
It is reported that endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF). EC are known to highly rely on glycolysis to keep their function, but the metabolism of EC under the situation with HFpEF is poorly understood.

Objective:
We sought to elucidate the role of hexokinase 1 (HK1) in ECs for the development of HFpEF.

Results:
Isolated ECs from mouse hearts showed higher protein expression of HK1 than isolated cardiomyocytes and fibroblasts, suggesting HK1 has an important role in EC function. Immunogold-staining of HK1 in hearts from C57BL6 mice treated with high fat diet and LNAME, a mouse model of HFpEF, showed increased dislocation of HK1 from the mitochondria in ECs. To study the role of HK1 dislocation, we generated ΔE1HK1 mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag. Subcellular fractionation confirmed that HK1 was dislocated from mitochondria in these mice. Echocardiography showed that the mice developed impaired cardiac relaxation at 20 weeks of age and HFpEF at 40 weeks of age. Significant increased fibrosis and microvascular rarefaction (MR) were observed at 40 weeks of age, but only MR was observed at 20 weeks of age, suggesting EC dysfunction likely precedes and promotes the development of HFpEF in these mice. To study the angiogenic ability of ECs with HK1 dislocation, we isolated ECs from ΔE1HK1 hearts and performed a tubing assay, and observed significantly less tubing in ΔE1HK1 ECs. To elucidate the mechanism by which angiogenesis is reduced in ΔE1HK1 ECs, we next performed metabolomics analysis in these cells. Our data indicated that the levels of metabolites in hexosamine-biosynthetic pathway (HBP) were altered between wild-type (WT) and ΔE1HK1 EC. We next analyzed the levels of O-GlcNAcylation, and showed that ECs from ΔE1HK1 hearts have higher O-GlcNAcylation than those from WT. Finally, treatment with ST045849, an inhibitor of O-GlcNAc transferase (OGT), rescued the less angiogenic ability in ECs from ΔE1HK1 hearts.

Conclusion:
Our studies demonstrate that dislocation of HK1 plays an important role in the development of HFpEF through hyper-O-GlcNAcylation. Drugs that inhibit OGT may provide a therapeutic option for HFpEF.
Abstract P3028: Animal In Vivo: The Proof Of Flow Choking And Bulging Of The Downstream Region Of The Stenosis Artery Due To Air Embolism
Authors: J Anbu , R Deveswaran , S Bharath , V R Sanal Kumar , Choudhary Shiv Kumar , Radhakrishnan Pradeep Kumar , Rajaghatta Sundararam Bharath , Oommen Charlie
Published: November 2022

Background:
Evidences are escalating on the asymptomatic cardiovascular diseases and disorders associated with air embolism due to the flow choking (biofluid/Sanal flow choking) [PMID: 32837737 & PMID: 34326352]. Flow choking occurs anywhere in the circulatory system at a critical systolic to diastolic blood pressure ratio (BPR) . Flow choking leads to the shock-wave generation followed by pressure overshoots if the downstream region of the choked artery is having divergence/bifurcation regions.

Methods:
Animal
in vivo
study has been carried out on

6
th
December 2021, IST: 2:00-4:00 PM

(the FIRST in the World)
for establishing the phenomenon of flow choking in a stenosis artery at a critical BPR due to air embolism. The high pressure air is injected into the upstream region of the stenosis using a syringe (see Fig.1(a)). Stenosis is created using a knot. Syringe infusion pump is used for altering the flow rate and upstream pressure. The multiple animal
in vivo
studies were carried out on

31
st
December 2021

. It aims for demonstrating the phenomenon of flow choking followed by bulging and/or tearing of the downstream region of the stenosis artery of a small animal.

Results:
Figure 1(b) shows the animal
in vivo
(with the approval of the ethics committee) for the live demonstration of flow choking and bulging in a stenosis artery due to air embolism. Herein, we established the proof of the concept of flow choking due to air embolism in the stenosis-artery at a critical BPR (SBP/DBP) causing the risk of asymptomatic cardiovascular diseases and disorders.

Conclusions:
We concluded that at a critical BPR, the flow choking occurs in any artery. The shock wave generation due to flow choking leads to cardiovascular risk. Frequent flow choking creates
memory effects
(stroke history) in the downstream region due to the enhanced wall stiffness as a result of pressure-overshoot due to multiple shock waves. We further concluded that the critical BPR is a risk factor of flow choking causing asymptomatic cardiovascular disease, if the downstream region of the choked artery is having divergent shape.
Abstract P3030: Fundc1 Mediated Mitochondrial Protein Import As A Novel Mechanism Exacerbating Dilated Cardiomyopathy
Authors: Zhao Fujie , Satyanarayana Ganesh , Cai Zhaohua , Li Jian , Wu Yang , Zhao Jianli , Zhang Zheng , Ma Xin-liang , Wang Yajing , Zou Ming-Hui
Published: November 2022

Background:
Dilated cardiomyopathy (DCM) is a common cause of heart failure and an indication for heart transplantation worldwide. FUN14 Domain Containing 1 (FUNDC1), a canonical mitophagy receptor and mitochondria-associated ER membranes (MAMs) protein, plays an essential role in ischemic heart disease. However, the role of FUNDC1 in DCM remains unidentified. The current study attempted to clarify whether and how FUNDC1 regulates DCM development.

Methods:
Cardiomyocyte-specific
F
UNDC
1
gene knockout (CM-FUNDC1 KO) mice were generated to study the impact and potential mechanisms of FUNDC1 in the heart. Mitochondria were isolated from heart tissues, primary mouse neonatal/adult cardiomyocytes, or H9C2 cell lines to probe the role of FUNDC1 in mitochondrial protein import (MPI).

Results:
All CM-FUNDC1 KO mice developed severe DCM spontaneously and died before 15 months old. The expression of MPI-associated proteins was significantly upregulated in the hearts of CM-FUNDC1 KO mice. Experiments with purified mitochondria demonstrated that MPI efficiency was enhanced by CM-FUNDC1 KO, whereas reduced by FUNDC1 overexpression in an autophagy-independent fashion. We further identified that FUNDC1 selectively downregulated the TIM23 complex without altering other MPI translocases (e.g., TOM20, TIM22 complexes). Downregulation of subunits of TIM23 complex partly reversed the FUNDC1 KO-induced MPI enhancement. Importantly, we demonstrated that FUNDC1 selectively inhibited the import of nuclear gene-encoded respiratory subunits on complex I, IV but not on complex II, III, V or mitochondrial gene-encoded subunits. Finally, FUNDC1 deleted cells exhibited enlarged cell size, retarded cell growth, impaired oxygen consumption rate, and increased mitochondrial superoxide level. All of these phenomena were partly reversed by silence TIM23 complex or reproduced by TIM23 complex overexpression.

Conclusions:
We demonstrate for the first time that: 1) MPI system plays an important role in the progress of DCM; 2) FUNDC1 is a novel negative regulator of MPI via TIM23 pathway; and 3) FUNDC1 is a promising therapeutic target for DCM.
Abstract P3041: DWORF Overexpression In The Heart Results In Enhanced Mitochondrial Function
Authors: Brito-Estrada Omar , Gibson Aaron , Makarewich Cat
Published: November 2022

Background:
Calcium (Ca
2+
) plays a pivotal role in regulating physiological processes in the heart including excitation-contraction coupling, Ca
2+
signaling, and mitochondrial function. It is well established that both Ca
2+
dysregulation and altered cardiac energy metabolism are universal features of heart failure (HF). Dwarf open reading frame (DWORF), a newly discovered microprotein, increases SERCA2a activity in part by relieving SERCA2a inhibition by phospholamban (PLN). DWORF overexpression via cardiac specific transgene or gene therapy is cardioprotective in genetic and experimental HF mouse models. In addition to activation of SERCA2a by PLN displacement, preliminary data indicates that enhanced mitochondrial function contributes to the cardioprotective effects of DWORF. Here we tested the effects of DWORF overexpression on cardiac mitochondrial function and Ca
2+
dynamics.

Methods and Results:
We isolated mitochondria from wildtype (WT) and DWORF transgenic (Tg) mouse hearts and measured O
2
consumption rates using a Seahorse mito stress test. We found that DWORF Tg mice have increased respiratory capacity compared to WT (DWORF Tg 1150.0 pmol/min, WT 799.4 pmol/min +/-146.2) and enhanced spare respiratory capacity (DWORF Tg 489.1 pmol/min, WT 229.4 pmol/min +/- 102.1). To measure mitochondrial Ca
2+
dynamics, we performed Ca
2+
retention capacity assays and found that DWORF Tg mice have increased Ca
2+
uptake rates compared to WT (DWORF Tg 32.32 s, WT 46.46 s +/- 4.39). Western blot analysis of the mitochondrial Ca
2+
uniporter (MCU) in mito extracts from WT and DWORF Tg hearts indicated elevated levels of MCU. However, electron transport chain (ETC) protein expression indicated no significant differences, indicating the enhanced mitochondrial activity observed in DWORF Tg mice was not due to increased ETC levels.

Conclusions:
DWORF Tg mouse heart mitochondria exhibit increased maximal respiration rates and enhanced spare respiratory capacity. However, ETC protein levels are unchanged, indicating altered enzyme activity rather than expression. Additionally, mitochondria from DWORF Tg hearts uptake Ca
2+
faster into the mitochondria through the increased levels of MCU, suggesting enhanced mitochondrial Ca
2+
dynamics.
Abstract P3045: The Role Of Mitochondrial Dysfunction In Arrhythmogenesis
Authors: Chakraborty Akash D , Chiao Ying Ann , Lee Chi Fung
Published: November 2022

Cardiac arrhythmia is triggered by deregulated ion channels and abnormal electrical activity, and it increases susceptibility of patients to sudden death. The regulations of ion movement in and out of the myocytes or subcellular compartments are highly energy-dependent. Although impaired energetics has been implicated in arrhythmia, the causal roles and intricate details of how mitochondrial dysfunction contributes to arrhythmogenesis are far from established. We employed a mouse model with cardiac-specific mitochondrial dysfunction by deleting Ndufs4, a protein of mitochondrial complex I (Ndufs4-cKO). Complex I deficiency in Ndufs4-cKO mice lowered cardiac NAD/NADH ratio but did not affect cardiac function and geometry at baseline. Control and Ndufs4-cKO mice showed normal sinus rhythm at baseline. Ndufs4-cKO mice had 3-fold increases non-sinus arrhythmic events upon an arrhythmogenic stress test. 50% of Ndufs4-cKO mice succumbed to sudden cardiac death with displays of pre-ventricular contractions and ventricular fibrillation. We collected cardiac tissues at baseline and post stress. We observed increased ryanodine receptor, RyR2-S2814 phosphorylation, reduced phospholamban PLN S16 phosphorylation and decreased calsequestrin 2 expression in Ndufs4-cKO hearts post stress, but not at baseline. Isolated Ndfus4-cKO cardiomyocytes after stress test show a trend towards prolonged time to peak shortening and time to peak Ca
2+
transient. In addition, elevating cardiac NAD levels lowered arrhythmic events of Ndufs4-cKO mice after stress test. These results revealed direct evidence that mitochondrial dysfunction increases susceptibility of hearts to arrhythmia by regulating NAD redox balance and SR calcium homeostasis.Ndufs4-cKO mice with chronic high fat diet (HFD) had accelerated declines in systolic and diastolic function and increased hypertrophy, compared to control mice. WT mice with HFD showed diastolic dysfunction and increased arrhythmic events after stress test, while diastolic dysfunction was reversed by elevating cardiac NAD levels. Our data suggest an arrhythmogenic mechanism linking mitochondrial dysfunction and NAD redox imbalance with SR calcium homeostasis, which may explain HFD-induced arrhythmogenesis.
Abstract P3050: The Adropin-GPR19 Signaling Axis In Diabetic Cardiomyopathy
Authors: Mushala Bellina , Xie Bingxian , Thapa Dharendra , Jurczak Michael , Scott Iain
Published: November 2022

Background:
Diabetic cardiomyopathy (DCM) is a major complication of diabetes and has been recognized as a cause of heart failure independent of other common risk factors for cardiac disease. In DCM, increased free fatty acid availability and decreased myocardial glucose uptake lead to a reliance on fatty acid oxidation for ATP generation. This loss of metabolic flexibility can reduce cardiac plasticity in response to stress. Energetic inefficiency in diabetic hearts may have profound implications for cardiac function under conditions of increased workload, and therefore therapeutic approaches are warranted. Alleviating metabolic inflexibility, by promoting cardiac glucose utilization, has been proposed as a potential strategy to reverse cardiomyopathy in diabetic and obese patients. Adropin is a liver- and brain-secreted peptide hormone shown to regulate fuel metabolism in a number of tissues, including the heart. Adropin levels are significantly reduced in obese and diabetic human subjects, and this decrease is linked to increased adiposity, insulin resistance, and impaired glucose tolerance. Our lab recently showed that adropin treatment restores glucose oxidation activity
in vivo
by reducing the expression of the mitochondrial acetyltransferase enzyme GCN5L1. The reduction in GCN5L1 abundance altered the acetylation and activity of fuel metabolism enzymes to favor glucose utilization; however, the mechanism that underlies this effect remains unknown. Our data suggests that the class A orphan G-coupled protein receptor, GPR19, acts as a putative cellular receptor for adropin in cardiac cells to regulate mitochondrial bioenergetic output.

Hypothesis:
We hypothesize that adropin-GPR19 signaling regulates GCN5L1 expression, which in turn promotes the deacetylation and activity of pyruvate dehydrogenase to restore glucose utilization in the diabetic heart.

Methods:
We used a novel whole-body GPR19 knockout mouse to perform a preliminary characterization of the effects of GPR19 depletion on cardiac and whole-body physiology. Using
in vivo
metabolic approaches, we report that GPR19 KO mice exhibit sex-dependent glucose intolerance, and display markers of altered cardiac energy metabolism.

Conclusions:
These findings further define the fundamental role of the adropin-GPR19 signaling pathway in the regulation of metabolic homeostasis, and thus highlight a potential target for therapeutic interventions in DCM.
Abstract P3054: Alpha-1a Adrenergic Receptor Activation Increases Electron Transport Chain Activity And Preserves Cardiac Contractility Following Myocardial Infarction
Authors: Sandroni Peyton B , Huang Wei , Jensen Brian C
Published: November 2022

Heart failure features decreased electron transport chain (ETC) activity in cardiomyocyte mitochondria. Previous studies have demonstrated that alpha-1A adrenergic receptor (α1A-AR) activation increases cardiac contractility in the failing heart. However, α1A-AR activation as a method of ETC regulation following cardiac injury has not been studied. Here, we assessed the hypothesis that α1A-AR activation increases ETC activity following myocardial infarction (MI) induced by left coronary artery ligation. We measured cardiac contractility using echocardiography at baseline and 7 days after MI in male CL57Bl/6J mice receiving the selective α1A-AR agonist A61603 (10 ng/kg/d) (n = 8) or vehicle (0.9% saline) (n = 9) via subcutaneous osmotic minipumps (Panel A). We then profiled citrate synthase (CS), a key enzyme in the citric acid cycle and common marker for mitochondrial mass, and ETC Complex I-IV activities in ventricular lysates. ETC enzyme activities were normalized to CS to define ETC capacity on a per mitochondrion basis. On Day 0, there were no differences in fractional shortening between groups (Panel B, 46 ± 3% vs. 49 ± 3%, p = 0.27) (mean ± SEM). On Day 7, fractional shortening was preserved in A6-treated mice compared to vehicle controls (Panel B, 24 ± 3% vs. 14 ± 1%, p = 0.01). A6 decreased CS activity compared to vehicle controls (Panel C, 11,024 ± 1,035 vs. 15,966 ± 1,068 nmol/mg/min, p < 0.01). When normalized to CS activity, A6 increased Complex I (16 ± 2% vs. 7 ± 1%, p < 0.01) (1,584 ± 116 vs. 1,106 ± 154 nmol/min/mg), Complex III (82 ± 8% vs. 56 ± 5%, p = 0.01) (8,435 ± 461 vs. 8,542 ± 327 nmol/min/mg), and Complex IV (121 ± 12% vs. 92 ± 9.%, p = 0.04) (12,523 ± 673 vs. 14,174 ± 1,156) activities (Panel D), suggesting enhanced ETC activity on a per mitochondrion basis. In conclusion, we show that a low dose of a selective α1A-AR agonist enhances cardiac contractility following MI. Increased ETC enzyme activity, particularly Complex I, may contribute to this beneficial effect. Ours is the first study in which selective α1A-AR activation has been linked to enhanced cardiac contractility and mitochondrial metabolism following MI. These findings may outline a novel mechanism through which α1A-AR activation preserves cardiac function in the injured and failing heart.
Abstract P3063: Downregulation Of The T Cell Unfolded Protein Response Precedes T Cell Cardiac Infiltration In Experimental Heart Failure With Preserved Ejection Fraction
Authors: Smolgovsky Sasha , Bayer Abraham , Carrillo-Salinas Francisco J , kaur Kuljeet , Aronovitz Mark , Sanders Erin , Theall Brandon , Blanton Robert M , Alcaide Pilar
Published: November 2022

Introduction:
Diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is associated with T cell systemic inflammation and downregulation of myocardial unfolded protein response (UPR) genes. The T cell UPR can modulate T cell effector function and immune responses, yet this is largely unexplored in HFpEF.

Hypothesis:
We hypothesized T cells contribute to cardiometabolic HFpEF and that metabolic and nitrosative stress-induced alterations of the T cell UPR modulate T cell activation and diastolic function.

Methods:
Male C57/BL6 (wild-type, WT) or T cell receptor alpha-deficient (
Tcra-/-)
were fed a high-fat diet (HFD) and L-NAME for 1, 3, and 5 weeks, or standard chow (STD). We assessed diastolic function by invasive hemodynamic analyses. Immune cells were characterized in the heart and spleen by flow cytometry, and cardiac pathology was assessed by histology. The UPR gene expression was evaluated in splenic CD4
+
T cells over time by qPCR.

Results:
Unlike WT mice,
Tcra-/-
mice fed HFD/L-NAME for 5 weeks did not develop diastolic dysfunction or cardiomyocyte hypertrophy, demonstrating a critical role for T cells in experimental cardiometabolic HFpEF. Indeed, cardiac CD4
+
T cells were increased in WT mice fed HFD/L-NAME for 5 weeks compared to STD, concordant with an expansion of splenic CD44
hi
CD62l
lo
interferon-gamma (IFNγ)
+
effector cells. Strikingly, splenic T cells also experienced downregulation of the UPR proteins activating transcription factor 4 (
Atf4
),
Atf6
, and X box-binding protein 1 (
Xbp1s/u
). We found that the
Atf4
arm of the T cell UPR is the earliest responder to HFD/L-NAME, with observed downregulation of
Atf4
and its downstream effector C/EBP homologous protein (
Chop
) as early as 3 weeks, prior to onset of diastolic dysfunction, followed by the subsequent downregulation of
Atf6
and
Xbp1s/u
.

Conclusions:
T cells contribute to diastolic dysfunction and cardiomyocyte hypertrophy in experimental cardiometabolic HFpEF. Early modulation of T cell
Atf4
precedes diastolic dysfunction, enhanced IFNγ
+
effector T cell activation, and global T cell UPR downregulation. Ongoing studies are focused on understanding the functional consequences of impaired T cell UPR in cardiometabolic HFpEF.
Abstract P3066: Macrophage-derived Ccl24 Worsens Cardiac Dysfunction Following Pressure-overload
Authors: Parthiban Preethy , Barrow Fanta , Yucel Dogacan , Gonigam Richard , Herman Adam , Rhoades Hailey , Demirchian Maya , Wang Haiguang , Van Berlo Jop H , Revelo Xavier
Published: November 2022

Heart failure (HF) is the leading cause of death worldwide. Systemic inflammation is associated with HF and is considered an independent predictor of adverse cardiac outcomes. Macrophages are the most abundant immune cells in the heart and have integral roles in cardiac homeostasis and remodeling. Despite their unique identity and homeostatic functions, the precise role of cardiac macrophages during cardiac remodeling is poorly understood. We have previously reported that cardiac resident macrophages expand early in response to pressure overload injury and provide cardioprotection. Using single-cell mRNA sequencing, we revealed that these macrophages consist of distinct subsets including a population that highly expresses C-C Motif Chemokine Ligand 24 (CCL24). CCL24 is a chemokine that promotes immune cell trafficking and activation through its receptor expressed on immune cells, fibroblasts, and cardiomyocytes. Here, we tested whether CCL24 regulates cardiac adaptation to pressure overload by performing transverse aortic constriction (TAC) in whole-body CCL24 knockout (CCL24KO) mice. Aortic constriction during TAC increases the pressure overload in ventricular chambers leading to an early compensated hypertrophy and diastolic dysfunction followed by systolic dysfunction and decompensated heart failure. Despite no differences at steady state, CCL24KO mice showed an improved cardiac function after TAC-induced pressure overload. Compared with WT controls, CCL24KO mice had a higher ejection fraction and fractional shortening. Furthermore, CCL24KO mice had increased hypertrophy but improved diastolic function. These results suggest that CCL24 worsens cardiac dysfunction leading to decompensated HF in response to pressure overload. Interestingly, CCL24KO mice have higher proportions of neutrophils and eosinophils at steady state, suggesting that the presence of these cells may facilitate cardiac repair. Overall, our findings indicate that macrophage-derived CCL24 is detrimental to cardiac function in response to pressure overload injury. The results of this study uncover CCL24 as a central mechanism by which cardiac resident macrophages promote cardiac inflammation and HF.
Abstract P3067: Sleep Deprivation In Obesogenic Setting Alters Lipidome And Microbiome Toward Suboptimal Inflammation In Acute Heart Failure
Authors: Halade Ganesh V , Mat Yusuf , Gowda Siddabasave Gowda B , Jain Shalini , Hui Shu-Ping , Yadav Hariom , Kain Vasundhara
Published: November 2022

Introduction:
Sleep is fundamental medicine for cardiac homeostasis and the immune fitness of human health. Sleep-deprived individuals are prone to higher incidences of a heart attack. The lipid-dense diet (obesogenic diet-OBD) is a cumulative risk factor for chronic inflammation in cardiovascular disease, thus understanding how sleep fragmentation (SF) in obesity setting impacts immune and cardiac health is an unmet medical need to develop novel therapeutics.
Hypothesis:
We hypothesized whether the co-existence of SF with OBD dysregulates gut homeostasis and leukocyte-derived reparative mediators thereby impairing cardiac repair.

Methods:
Two months old male C57BL/6J mice were randomized into two main groups; control (fed standard lab chow) and OBD (fed 10% w/w safflower oil-enriched diet) for 2 months. For SF, mice were subjected to SF chamber (2-minute interval bar sweep in light period (0700–1900 h) for 3 days. Mice of control, control+SF, OBD control, OBD+SF groups were further subjected to permanent coronary artery ligation to induce acute heart failure.

Results:
OBD fed mice had higher levels of plasma linolenic acid with a decrease in eicosapentaenoic acid and docosahexaenoic acid. The OBD mice had lower levels of
Lactobacillus johnsonii
indicating loss of probiotic microbiota. SF in OBD mice increased
Firmicutes/Bacteroidetes
(F/B) ratio indicative of a detrimental change in SF-directed microbiome signatures. OBD+SF group increased in the neutrophil: lymphocyte ratio suggestive of suboptimal inflammation. As a result of SF, resolution mediators (RvD2, RvD3, RvD5, LXA
4
, PD1, and MaR1) decreased and inflammatory mediators (PGD
2
, PGE
2
, PGF
2a
, 6k-PGF
1a
) were increased post- MI in OBD fed mice. At the site of infarction, the proinflammatory cytokines;
Ccl2, IL1β
, and
IL-6
were amplified in OBD+SF indicating a robust proinflammatory milieu. OBD+SF group showed signs of neutropenia in the spleen with pre-activation of CD169 macrophages. Also, brain circadian genes (
Bmal1, Clock)
were downregulated in SF subjected control mice but remained elevated in OBD mice post-MI.

Conclusion:
SF superimposed on obesity dysregulated physiological inflammation and disrupted resolving response thereby impaired cardiac repair and signs of pathological inflammation.
Abstract P3068: TNFR1 Regulates Cellular Proliferation And Not Pathogenicity Of Cd4
+
T-lymphocytes During Ischemic Heart Failure
Authors: Kumar Vinay , Rosenzweig Rachel , Asalla Suman , Nehra Sarita , Prabhu Sumanth D , Bansal Shyam S
Published: November 2022

Ischemic heart failure (HF) is associated with elevated inflammatory responses including pro-inflammatory cytokines, such as Tumor necrosis factor (TNF) α [and its cognate receptors, TNF receptor (TNFR) 1 and TNFR2] and pathological switching of CD4
+
T-cells. TNF-TNFR2 signaling is known to promote CD4
+
T-cell activation and proliferation. However, it is unknown if CD4
+
T-cells also express TNFR1, and if elevated TNFα-TNFR1 signaling during HF mediates pathological effects of CD4
+
T-cells. Using flow cytometry, we show that TNFR1
+
CD4
+
T-cells are amplified in the hearts at 3 days (d) and 8 weeks (wks) post-MI (HF) in mice. As compared to sham, while TNFα expression (and %) is blunted in CD4
+
T-cells at 3 d post-MI, it is increased during HF with significantly higher expression in TNFR1
+
T-cells as compared to TNFR1
-
T-cells. Splenic CD4
+
T-cells activated using anti-CD3/CD28 antibodies and treated with neutralizing anti-TNFR1 antibody showed increased T-cell proliferation, and Bcl-xL (pro-survival factor) and TNFα expression without affecting activation marker (CD69) suggesting a role of TNFR1 in mediating pro-apoptotic signaling without affecting activation. To confirm these results, we adoptively transferred (AT) HF-activated splenic WT and TNFR1
-/-
T-cells (CD45.2) into CD45.1 naïve mice, and measured changes in cardiac function of recipient mice by echocardiography (as an indicator of pathological activity), and donor T-cells (as a measure of cell survival) in the blood, heart, spleen and mediastinal lymph nodes (mLNs) at 14 wks post-AT. Compared to WT, we observed amplified levels of donor TNFR1
-/-
T-cells in the blood, spleen, and mLNs which were not different in the hearts. Consistently, recipient mice injected either with WT and TNFR1
-/-
T-cells showed similar degrees of cardiac dysfunction. Interestingly, at 14 wks post-AT the expression of ki67, a proliferation marker, was magnified in HF-activated TNFR1
-/-
T-cells (as compared to WT) infiltrated into the myocardium suggesting that TNF-TNFR1 mediates pro-survival and proliferative signaling in T-cells during HF, and not their pathogenicity. Our results also provide deeper insights into the paradoxical outcomes observed in the clinical trials antagonizing TNFα in human HF patients.
Abstract P3071: Ikkε Deficiency Reduces P38 Activity Of Macrophages To Increases Cardiac Injury
Authors: Kim Yong-sook , Cho Hyang H , Cho Dong-im , Cho Meeyoung , Ahn Young Keun
Published: November 2022

Background:
Inhibitor of NF-κB kinase ε (IKKε) is a non-classical modulator of nuclear factor-kappa B (NF-κB), and is reported to regulate inflammation in various diseases. In this study, the involvement of IKKε in pathophysiological responses was examined in a mouse myocardial infarction model.

Methods:
MI was induced in IKKε-/- mice by coronary artery ligation. Neutrophils and macrophages were isolated from the heart tissues or bone marrows for further comparison studies by Western blot, PCR, and flow cytometry. Cell phenotypes were identified by immunofluorescence staining.

Results:
The IKKε-/- group showed poor early survival rate, high circulating IL-6 level, large cardiac fibrosis, and low ejection fraction (30.33±5.25% vs. 34.02±5.53%, p<0.05) compared with the wild type group. Further study revealed that the numbers of neutrophils and macrophages in the heart were higher in the IKKε-/- group than in the PBS group. Macrophage phenotype was more inflammatory along with lower p38 activity in the IKKε-/- group. Importantly, we identified macrophage-myofibroblast transition (MMT) in the infarcted myocardium and found that the number of MMT F4/80(+)SMA(+) cells was 2-fold higher in the IKKε-/- group. The inactivation of p38 accelerated MMT in human macrophages, and the level of phosphorylated p38 was lower in infarct zone of human failing heart tissues.

Conclusion:
Collectively, IKKε-p38 axis may lead macrophages to aggravated inflammation and accelerated MMT, resulting in poor outcome after myocardial infarction. Here, we suggest that restoring IKKε or p38 in macrophages could be the target to attenuate cardiac inflammation.
Abstract P3087: Single-cell Transcriptomics Reveals Cell-type Specific Transcriptional Responses In Murine Embryonic Hearts Exposed To Maternal Hyperglycemia
Authors: Manivannan Sathiyanarayanan , Mansfield Corrin R , Zhang Xinmin , Kodigepalli Madhav Karthik , Majumdar Uddalak , Garg Vidu , Basu Madhumita
Published: November 2022

Congenital heart disease (CHD) is a significant pediatric health burden in the United States and a leading non-infectious cause of mortality in the infants. According to CDC, ~1 in 4 babies born with cardiac defects develop critical CHD and require medical interventions during first year of their lives. Despite the strong heritable basis of CHD, genetic variations explain nearly 30% of cases. The phenotypic heterogeneity and incomplete penetrance of the disease implies a vital role of environmental factors as non-genetic contributors of CHD. Women with pregestational diabetes mellitus (PGDM) are about four times more likely to have a pregnancy affected by CHD compared to women without diabetes. Previous studies have established that maternal hyperglycemia (matHG) before pregnancy and in first trimester profoundly increases the risk of CHD among the offspring, with a preponderance of conotruncal and septal defects. However, the cell-type-specific sensitivity to matHG responsible for such cardiac malformations remains unclear. Using a chemically induced murine PGDM model, we first demonstrated increased incidence of CHD in matHG-exposed embryos compared to control (CNTRL) embryos. To evaluate the underlying cellular basis of CHD risk in diabetic pregnancies, we performed in vivo single-cell RNA-sequencing in CNTRL and matHG-exposed embryonic hearts. The transcriptomic data revealed cardiac cell-type-specific molecular changes associated matHG. Bioinformatics analysis showed significant gene expression differences in Isl1+ multipotent progenitor cells, Tnnt2+ cardiomyocytes, Tbx18+ epicardial cells, Sox9+ fibromesenchymal cells, Cdh5+ endocardial/endothelial cells, and in Dlx5+ neural crest cell populations. Pseudotime trajectory analysis suggested HG-induced impairments in genes critical for cardiomyocyte differentiation. Furthermore, in vivo Isl1-derived cell fate mapping studies suggested matHG-mediated perturbations in cardiomyocyte proliferation and differentiation at critical stages of cardiac development. In combination with our scRNA-seq and cell fate mapping data, future mechanistic studies will delineate cell-type-specific gene regulatory network perturbed in matHG environment. Early diagnosis and treatment of diabetes in pregnancy is of great clinical significance and more in-depth studies will enable the development of preventative therapeutic measures to ameliorate PGDM associated CHD.
Abstract P3089: Etv2 Functions As A Pioneer Transcription Factor To Regulate Endothelial Lineage Development And Reprogramming
Authors: Das Satyabrata , Sierra-Pagan Javier , Gong Wuming , Dsouza Nikita , Larson Thijs , Garry Mary G , Luzete-Monteiro Edgar , Zaret Kenneth S , Garry Daniel J
Published: November 2022

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic heart disease. The ETS-related transcription factor 2 (ETV2) is a transcription factor that is both necessary and sufficient for the development of endothelial and blood lineages. However, the molecular mechanism by which ETV2 promotes endothelial and blood development is not known and warrants further investigation. Here, we report that ETV2 functions as a novel pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. Pioneer factors are a subset of transcription factors that bind closed chromatin and promote chromatin relaxation in order to drive the development and reprogramming of cell lineages. By comparing embryonic stem cell differentiation and fibroblast reprogramming models with single cell RNA-seq, ATAC-seq, ChIP-seq and in-vitro nucleosomal binding techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We also determined that ETV2 executed a pioneering role by recruiting and directly interacting with the ATP-dependent chromatin remodeling enzyme BRG1 to remodel chromatin around endothelial genes to help maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results that define a novel pioneer factor and epigenetic regulatory pathways that govern the specification and differentiation of endothelial progenitor cells, will serve as a platform for targeted therapies to promote cardiovascular regeneration.
Abstract P3105: Improving Cardiovascular Toxicity Of Chronic Myeloid Leukemia Therapy.
Authors: Hnatiuk Hnatiuk Anna , Bruyneel Arne , Taylor Dhanir , Pandrala Mallesh , Serrano Ricardo , Feyen Dries , Nakauchi Yusuke , Vu Michelle , Amatya Prashila , Majeti Ravindra , Malhotra Sanjay , Mercola Mark
Published: November 2022

Introduction:
Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects.

Hypothesis:
Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity.

Methods:
Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an
in vivo
xenograft mouse model of CML.

Results:
The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth
in vitro
and
in vivo
. Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs
in vitro
. The therapeutic window was increased
in vivo
, leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity.

Conclusions:
This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.
Abstract P3114: Intermittent Fasting Potentiates Anthracycline Cardiotoxicity
Authors: Ozcan Mualla , Guo Zhen , Valenzuela Ripoll Carla , Asnani Aarti , Margulies Kenneth B , Bergom Carmen , Diwan Abhinav , Javaheri Ali
Published: November 2022

Background:
Anthracyclines such as doxorubicin (Dox) cause cardiotoxicity and associated autophagic impairment, though controversy exists increasing autophagy ameliorates or exacerbates Dox cardiotoxicity. Because intermittent fasting (IF), a potent stimulus of autophagy, is presently under clinical investigation in multiple human clinical trials of cancer patients receiving chemotherapy, we tested whether IF would ameliorate or exacerbate Dox cardiotoxicity in multiple murine models.

Methods:
Chow-fed C57BL/6 mice (n=150) were randomized to
ad libitum
feeding (
adlib
) or IF (every other day) and treated with vehicle or Dox (5 mg/kg IP x 4 doses). Alternatively, to stimulate autophagy and lysosomal biogenesis transcriptionally, we employed adeno-associated virus 9 (AAV9)-driven overexpression of transcription factor EB (TFEB) vs null, followed by vehicle or Dox (n=40). We further analyzed TFEB nuclear content in myocardial tissue from patients with anthracycline cardiomyopathy vs non-failing donors and patients with other non-ischemic cardiomyopathies (n=17).

Results:
In Dox-treated mice (but not in vehicle controls), IF significantly reduced survival (18% absolute reduction or AR) (
figure A
), left ventricular ejection fraction (LVEF) (8% AR) (
figure B
) and heart weight index (12% relative reduction) (
figure C
) compared to adlib group. Mechanistically, in Dox-treated mice, IF significantly decreased phosphorylation of mammalian target of rapamycin, increased active, nuclear TFEB content, and increased levels of the atrophy factor muscle RING-finger protein-1. In myocardial tissue from patients with anthracycline cardiomyopathy, we observed a 3.5-fold increase (p<0.01) in nuclear TFEB content compared to both tissues from donors without heart failure and tissues from patients with other non-ischemic cardiomyopathies. Overexpression of TFEB exacerbated Dox-induced cardiotoxicity, including significant decreases in LVEF, left ventricular mass index, and survival.

Conclusions:
IF exacerbates Dox-related mortality and cardiomyopathy, likely by stimulating the autophagy-lysosomal transcriptional regulator TFEB. Our data raise concerns about the safety of IF and augmentation of TFEB activity in the context of anthracycline chemotherapy.
Abstract P3120: Cardio And Neurotoxicity Of Repurposed Anti-COVID-19 Drugs
Authors: Nicholson Martin , Huang Ching-Ying , Wang Jyun-Yuan , Chien-Yu Ting , Cheng Yu-Che , Chan Darien , Lee Yi-Chan , Hsu Ching-Chuan , Chang Cindy M , Hsieh Marvin L. , Cheng Yuan-Yuan , Lin Yi-Ling , Chen Chien-Hsiun , Wu Ying-Ta , Hacker Timothy A , Wu Joseph C , Kamp Timothy , Hsieh Patrick C
Published: November 2022

In December 2019, the novel coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spread around the globe resulting in ~435 million confirmed cases and ~6 million related deaths as of March 2022, according to the World Health Organization. To combat COVID-19 quickly, there have been many attempts to repurpose current FDA-approved drugs or to revive old drugs with anti-viral properties. However, aside from the biological stress imposed by the virus, many of the current treatment options have been known to cause adverse drug reactions. We established a population-based human induced pluripotent stem cell drug screening platform to assess the toxicity of the first line of anti-COVID-19 drugs and to understand viral infection of cardiomyocytes and neurons. We found that iPSC-derived cardiomyocytes express the ACE2 receptor which correlated with a higher infection of the SARS-CoV-2 virus (r=0.86). However, ACE2 expression was undetectable in neurons which correlated with low infection of neurons. We then assessed the toxicity of anti-COVID-19 drugs and identified two cardiotoxic compounds (remdesivir and arbidol) and 4 neurotoxic compounds (arbidol, remdesivir, hydroxychloroquine, and chloroquine) which were validated by dose-response curves. These data show that this platform can quickly and easily be employed to further our understanding of cell-specific infection and identify drug toxicity of potential treatment options helping clinicians better decide on treatment options.