bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P384: Distinct Roles Of Cellular And Extracellular TRNA-derived Small RNAs In Myocardial Fibrosis
Authors: Li Guoping , Aidan Manning , Bagi Alex , Gokulnath Priyanka , Spanos Michail , Li Haobo , Muehlschlegel Jochen D , Kitchen Robert , Lowe Todd , Das Saumya
Published: November 2021

Heart failure (HF) remains the leading cause of morbidity and mortality in the US, affecting approximately 6.2 million Americans with an anticipated 46% increase in overall prevalence by 2030. Myocardial fibrosis, defined as increased cardiac fibroblast (CF) activation and excessive extracellular matrix (ECM) deposition, is a leading contributor to the progression of adverse heart remodeling that leads to HF. Despite this clinical importance, no FDA-approved antifibrotic drug is available to prevent the development of HF. There is therefore an urgent need to understand pathways important in myocardial fibrosis that can be targeted with novel therapies. Non-coding RNAs, particularly miRNAs and lincRNAs, have emerged as promising novel biomarkers or therapeutic targets for myocardial fibrosis. As an emerging category of small regulatory non-coding RNAs that was discovered only a decade ago, tRNA-derived small RNAs (tDRs), have been shown to play important roles in cancer and neurological disorders. However, the knowledge of tDRs in myocardial remodeling remains scarce. Here, by using a recently developed tDR sequencing technique, called ARM-seq, we have discovered the unique extracellular tDR fragmentation profile and created an atlas of stress-specific cellular and extracellular tDR signatures in both CFs and cardiomyocytes (CMs). Notably, we identified a nutritional deprivation-induced cellular tRNA-Asp-GTC-3’tDR (cl-tRNA-Asp-GTC-3’tDR) that demonstrates potent antifibrotic effects.
In vitro
mechanistic studies and transcriptome analysis suggest that the lysosome-localized cl-tRNA-Asp-GTC-3’tDR inhibits CF activation through targeting ECM deposition program and cellular response to TGFβ stimulus and the G-quadruplex motifs are required for its functionality. Meanwhile, a related extracellular tDR derived from the same canonical tRNA, ex-tRNA-Asp-GTC-3’tDR, which has two additional nucleotides at both 5’ and 3’ end of cl-tRNA-Asp-GTC-3’tDR, is specifically enriched in the extracellular vesicles derived from CFs and CMs. Our pilot data suggest that the secretion of ex-tRNA-Asp-GTC-3’tDR may be required for maintaining the homeostasis of CFs and CMs. Importantly, we found that cardiac ischemia significantly decreases the circulating ex-tRNA-Asp-GTC-3’tDR level in both cell culture models and human patients during cardiopulmonary bypass surgery or following acute coronary syndrome. Together, our findings identify a cellular tDR that represents an attractive therapeutic target against myocardial fibrosis and an extracellular tDR with the same origin that has a distinct function and may be translated into novel biomarkers for cardiac ischemia.
Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.
Authors: Vite Alexia , Matsuura Timothy , Lai Ling , Bedi Kenneth , Kelly Daniel P , Margulies Kenneth B
Published: November 2021

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior
in vivo
studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS
Glucose
: 0.1315±0.012; FS
FFA
: 0.1428±0.0127; FS
3OHB
: 0.1343±0.014; FS
MIX
: 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS
Glucose
: 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS
FFA
: 0.109±0.0127; FS
3OHB
: 0.107±0.012; FS
MIX
: 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.
Abstract P389: Free Fatty Acid Receptor 4 Is Necessary For An Adaptive Response Following Heart Failure Induced By Metabolic Syndrome In Mice
Authors: Zhang Naixin , Murphy Katherine A , Gyberg Dylan J , O'Connell Timothy D
Published: November 2021

Non-resolving inflammation is central to the pathogenesis of heart failure (HF). Heart failure preserved ejection fraction (HFpEF) is a type of HF that is particularly associated with inflammation provoked by metabolic syndrome (MetS). The G-protein coupled receptor, free fatty acid receptor 4 (Ffar4), is a receptor for medium and long chain fatty acids (FA) that regulates metabolism and attenuates inflammation. Ffar4 is expressed in the human heart, and downregulated in heart failure. Furthermore, polymorphisms in Ffar4 have been associated with eccentric remodeling in a patient cohort. Previously, Ffar4 was shown to protect the heart from pathologic stress by attenuating oxidative stress in a mouse model of pressure overload. Here, we tested the hypothesis that Ffar4 would attenuate the development of heart failure using a mouse model of MetS-induced HFpEF. Metabolic syndrome was induced in mice by feeding a high-fat, high-sucrose diet (42% fat, 30% sucrose) to produce obesity and delivering the nitric oxide synthase inhibitor, L-NAME, in the drinking water to induce hypertension. The combined intervention (referred to as HFpEF diet) resulted in mice developing excessive adiposity, glucose intolerance (in males only), and mild hypertension. After 20 weeks on the HFpEF diet, both male and female WT mice, developed diastolic dysfunction (increased E/A and E/e’) and preserved ejection fraction (EF), consistent with clinical HFpEF. In Ffar4KO male mice HFpEF diet induced a greater degree of diastolic dysfunction compared to WT mice, despite equivalent metabolic parameters. Female Ffar4KO mice fed the HFpEF diet had a greater increase in weight gain and adiposity compared to WT female mice. Surprisingly, diastolic function was equivalent between WT and FFAR4KO female mice, suggesting a sex-based difference in FFAR4 cardioprotection. Our data show that Ffar4 attenuates HFpEF secondary to MetS.
Abstract P396: Noncanonical Wnt5a Increases QT Interval And Susceptibility To Ventricular Arrhythmias
Authors: Wang Jerry , Lu Aizhu , Xia Ying , Wang Hongwei , Cimenci Cagla , Suuronen Erik J , Toeg Hadi , Ruel Marc , Davis Darryl R , Liu Peter , Beanlands Rob S , Liang Wenbin
Published: November 2021

Background:
Wnt signaling plays a critical role in both embryonic cardiogenesis and cardiac remodeling in adult heart disease. We have previously demonstrated that the canonical Wnt/β-catenin pathway inhibits cardiac sodium current, but it remains unclear whether the noncanonical Wnt pathway affects cardiac electrophysiology.

Methods and Results:
Western blot analysis of ventricular tissues from patients with heart failure (n=6) demonstrated a 2.3x fold increase (p<0.01) in the protein level of Wnt5a, a noncanonical Wnt ligand, as compared to healthy ventricular tissues (n=5). To investigate if Wnt5a affects cardiac electrophysiology, adenovirus expressing Wnt5a and mCherry (Ad-Wnt5a) or control adenovirus expressing mCherry only (Ad-mCherry) was injected into the left ventricular free wall of adult rat hearts. At 4-5 days after virus injection, surface ECG revealed increased QT interval (p<0.01) in Ad-Wn5a-injected rats (90.1±2.3 ms n=7, vs 72.3±2.0 ms in control Ad-mCherry rats n=7). In addition, ventricular tachycardia was induced by programmed electrical stimulation in 92% (11/12) Ad-Wnt5a hearts, but only in 22% (2/9) control Ad-mCherry hearts (p<0.01). Patch-clamp recording of isolated single ventricular myocytes demonstrated that Ad-Wnt5a myocytes exhibited marked prolongation of action potential duration (APD
90
: 273±77ms, n=5) as compared to control cells (42±12 ms, n=7, p<0.05). In addition, the prolonged action potentials in Ad-Wnt5a myocytes were associated with frequent early afterdepolarizations and delayed afterdepolarizations, two mechanisms for triggered ventricular arrhythmias.

Conclusion:
Wnt5a is increased in the myocardium of patients with heart failure. Viral expression of Wnt5a in rat ventricular tissue increases QT interval and ventricular arrhythmia susceptibility, which is associated with prolongation of action potentials in cardiomyocytes. This may be an important target for future therapies.
Abstract P414: Systems Genetics Analysis Reveals Significant Eqtls Associated With Echocardiography Parameters In Genetic Reference Population Of Bxd Strains.
Authors: Orgil Buyan-Ochir , Xu Fuyi , Munkhsaikhan Undral , Alberson Neely R , Johnson Jason , Towbin Jeffrey A , Lu Lu , Purevjav Enkhsaikhan
Published: November 2021

Background:
Causal and modifier genes, genetic background and environment underlie clinical heterogeneity in cardiomyopathy (CM). The BXD recombinant inbred (RI) family represents a murine genetic reference population (GRP) that are descendants from crosses between C57BL/6J (B6) and DBA/2J (D2) mice. The parental D2 mouse is a natural model of hypertrophic CM (HCM). The study aimed to dissect genetic architecture of cardiac traits in BXD GRP using systems genetics analysis.

Methods:
Echocardiography was performed in 88 strains of male (M) and female (F) BXDs (N>5/sex) at 4-5 months of age. Cardiac traits were then associated with heart transcriptome, and expression quantitative trait loci
(
eQTL) mapping was performed.

Results:
More than 2-fold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular (LV) volumes at end-systole and end-diastole (Vol;s and Vol;d), internal dimensions (ID;s and ID;d), posterior wall (PW), and interventricular septum (IVS) thickness was found among BXDs. Traits seen in dilated CM (DCM) patients such as reduced EF%, FS%, and LVPW and increased Vol;s and ID;s are found in BXD78 (M, F), BXD32, 111, and 68 (F) strains. Strains D2, BXD90 and 155 (M, F), BXD44 and 65 (M), and BXD113, 16, 77 (F) had significantly greater LV mass, LVPW and IVS thickness compared to sex-matched controls, suggestive for traits seen in HCM patients. A 6.4 Mb QTL (peak LRS=18.50) was identified on chromosome (Chr) 8 to be significantly associated with ID;s, ID;d, Vol;s and Vol;d among male BXDs. eQTL mapping for each of 131 genes on Chr8 QTL identified 6 genes (
Coq9
,
Ndrg4
,
Crnde, Irx3, Rpgrip1l, and Rbl2
) being
cis
-regulated and
Ndrg, Slc6a2 and Ces1d
being significantly (p < 0.05) correlated with LV volumes. In female BXDs, a significant QTL on Chr7 (40.2 Mb) with 9 genes that significantly correlated with LVPW;d was identified. A suggestive 92.6 Mb QTL on Chr3 with
Snapin
,
Tpm3
, and
Wars2
correlated with EF% and FS% (p < 0.05).

Conclusions:
Our study found cardiomyopathy-associated traits are segregated among BXD family and these traits vary among BXD lines. Multiple associated QTLs demonstrate that the BXD family is suitable to map gene variants and identify genetic factors and modifiers that influence cardiomyopathy phenotypes.
Abstract P415: Etv2 Interacts With Vezf1 And Co-regulate Endothelial Gene Expression
Authors: Das Satyabrata , Gupta Vinayak , Gong Wuming , Sierra-pagan Javier , Skie Erik , Garry Mary G , Garry Daniel J
Published: November 2021

Background:
Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood.

Results:
Using a yeast two-hybrid assay, we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatics analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as candidate genes and have previously been shown to function as regulators of endothelial development. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histones. Additionally, we have undertaken ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) and Vezf1 ChIP-Seq studies to profile the epigenetic modifications and genome-wide occupancy of Vezf1 in the presence or absence of Etv2 in endothelial progenitor cells.

Conclusion:
In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and we further demonstrate their combined role in the regulation of downstream target genes, Amotl2 and Hdac7, during endothelial development.
Abstract P423: Nox2 Mediates Increased Oxidative Stress And Ros Production Leading To Atrial Ion Channel And Structural Remodeling In Obesity-induced Af.
Authors: Sridhar Arvind , Hong Liang , Ly Olivia T , Chen Hanna , Perike Srikanth , Arif Pavel Mahmud , Rehman Jalees , McCauley Mark , Darbar Dawood
Published: November 2021

Background:
Inducible atrial fibrillation (AF) in diet-induced obese (DIO) mice is mediated in part by a combined effect of ion channel remodeling and atrial fibrosis. NADPH oxidase 2 (NOX2), a major source of reactive oxygen species (ROS) production in human atria, is increased in DIO mice. Although a mitochondrial antioxidant (MitoTEMPO) reduced AF burden, and reversed ion channel and structural remodeling, the role of NOX2 in increased ROS production and atrial remodeling in obesity-induced AF remains unclear.

Objective:
To test the hypothesis that increased NOX2 modulates atrial remodeling in obesity-induced AF, we treated DIO mice with apocynin, NOX2 inhibitor, and fed a 60% high fat diet (HFD) to
Nox2
-KO mice.

Methods:
Weight, BP, plasma glucose, trans-esophageal rapid (TE) pacing and F
2
-isoprostanes were measured in DIO mice and compared to controls. Echocardiography, electrophysiology (EP), immunohistochemistry, Western blotting, cellular patch clamping and optical mapping studies were performed.

Results:
The average weight of DIO mice treated with apocynin (DIO-A) and vehicle control mice was 38.4 ± 3.8 g versus 44.0 ± 7.5 g versus 31.7 ± 1.19 g respectively (P≤0.0001). Both groups of DIO mice displayed progressive increase in weight over 10 weeks of HFD + drug treatment compared to controls (P≤0.0001). After TE pacing, DIO mice treated with apocynin showed significantly reduced pacing-induced AF burden when compared to DIO mice treated with vehicle. DIO mice treated with apocynin displayed 20.2 ± 26.1 sec versus 162.3 ± 133.7 sec and 18.3 ± 18.1 sec in DIO mice treated with vehicle and control mice respectively (P≤0.0001). Western blotting experiments showed that potassium channels, Kv7.1 and Kv1.5 protein expression is restored in
Nox2
-KO HFD mice compared to DIO mice thus indicating restoration of ion channel remodeling upon Nox2 inhibition.

Conclusions:
We showed that genetic and pharmacological inhibition of NOX2 abrogates ion channel remodeling and reverses obesity-induced AF burden. Our findings may have important implications for the management of obesity-mediated AF in patients.
Abstract P431:
Trypanosoma Cruzi
Induces Alterations In The Piwi-interacting Rna Expression Profile Of Human Cardiomyocytes During The Early Phase Of Infection
Authors: Rayford Kayla J , Cooley Ayorinde , Lima Maria F , Nde Pius N
Published: November 2021

Trypanosoma cruzi
, the causative agent of Chagas Heart Disease, is an intracellular parasite that was originally endemic to Central and South America. However, due to globalization, the disease is now present in most industrialized countries. About 40% of
T. cruzi
infected individuals will develop a form of cardiomyopathy including aneurysms, arrythmias, fibrosis, hypertrophy. Host gene expression profile has been shown to be significantly dysregulated to facilitate parasite infection and pathogenesis. The role of gene expression regulatory molecules such as small noncoding RNAs during infection has yet to be fully elucidated. In this study, we aim to evaluate if the parasite can regulate the expression profile of an emerging class of small non-coding RNAs, piwi-interacting RNAs (piRNAs), during the early phase of
T. cruzi
infection in primary human cardiacmyocytes (PHCM). We challenged PHCM with invasive
T. cruzi
trypomastigotes and purified RNA for RNA sequencing and analysis. We found that an average of 21,595,866 clean reads (88.4%) mapped to the human reference genome. We observed that the parasite induced significant differential expression of 217 host piRNAs using NOISeq. Furthermore, we employed miRanda and RNA22 to predict mRNA targets of the differentially expressed piRNA. We utilized WEBGESTALT to map piRNAs of interest to their biological pathways.
In silico
analysis showed that both dysregulated known and novel piRNAs could target several genes of interest including NFATC2, which has been reported to play an important role in the onset of cardiomyopathies reported in Chagasic patients. Our novel findings are the first to show that
T. cruzi
can induces differential expression of piRNAs during the early phase PHCM infection.
Abstract P437: Deep Learning-based Models For Complete Atrioventricular Block Heart Rhythm Analysis
Authors: Choi Dahim , Kim Nam Kyun , Son Young H , Gao Yuming , Sheng Christina , Jang Jeongin , Lee Kihong , Cho Hee C , Park Sung Jin
Published: November 2021

Atrioventricular block (AVB), caused by impairment in the heart conduction system, presents extreme diversity and is associated with other complications. Only half of AVB patients require a permanent pacemaker, and the process determining the pacemaker implantation is associated with an increase in cost and patient morbidity and mortality. Thus, there is a need for models capable of accurately identifying transient or reversible causes for conduction disturbances and predicting the patient risks and the necessity of a pacemaker. Deep learning (DL) is brought to the forefront due to its prediction accuracy, and the DL-based electrocardiogram (ECG) analysis can be a breakthrough to analyze a massive amount of data. However, the current DL models are unsuitable for AVB-ECG, where the P waves are decoupled from the QRS/T waves, and a black-box nature of the DL-based model lowers the credibility of prediction models to physicians. Here, we present a real-time-capable DL-based algorithm that can identify AVB-ECG waves and automate AVB phenotyping for arrhythmogenic risk assessment. Our algorithm can analyze unformatted ECG records with abnormal patterns by integrating the two representative DL algorithms: convolutional neural networks (CNN) and recurrent neural networks (RNN). This hybrid CNN/RNN network can memorize local patterns, spatial hierarchies, and long-range temporal dependencies of ECG signals. Furthermore, by integrating parameters derived from dimension reduction analysis and heart rate variability into the hybrid layers, the algorithm can capture the P/QRS/T-specific morphological and temporal features in ECG waveforms. We evaluated the algorithm using the six AVB porcine models, where TBX18, a pacemaker transcription factor, was transduced into the ventricular myocardium to form a biological pacemaker, and an additional electronic pacemaker was transplanted as a backup pacemaker. We achieved high sensitivity (95% true positive rate) and quantified the potential risks of various pathological ECG patterns. This study may be a starting point in conducting both retrospective and prospective patient studies and will help physicians understand its decision-making workflow and find the incorrect recommendations for AVB patients.
Abstract P447: Layer-by-layer Fabrication Of Thick Cardiac Muscle Patches With High Viability And Maturity
Authors: Wang Lu , Zhang Jianyi
Published: November 2021

Conventionally manufactured human cardiac muscle patches (hCMPs) typically rely on diffusion for oxygen and nutrient delivery to the cells and, consequently, the thickness of the hCMP is limited to 100-200 μm. Here, we tested our hypothesis that our layer-by-layer (lbl) manufacturing protocol could increase hCMP thickness without compromising the viability of individual cell populations. Human induced-pluripotent stem cells were differentiated into cardiomyocytes (CMs) or endothelial cells (ECs); then, layers composed of CMs or ECs were fabricated by mixing a cell-containing fibrinogen solution with thrombin and pouring the mixture into a mold, and the layers were stacked into a CM-EC-CM “sandwich”. hCMP viability was assessed via TUNEL staining, vascularization via expression of the EC marker CD144, and maturation via immunofluorescence and qPCR assessments of the expression of contractile (cTnT, cTnI), connective (Cx43, N-cad), and calcium-handling (RyR2, SERCA2, BIN1, Kir2.1) proteins. Sarcomere length was evaluated by staining for α-sarcomeric actinin, and hCMP ultrastructure was examined via transmission electron microscopy. The lbl-manufactured hCMP was 10 mm x 10 mm and 1.8 mm thick after fabrication. Minimal cells in the lbl-hCMP were apoptotic or necrotic. Compared to assessments in a single-layered patch containing the same number and proportion of cells, the lbl-hCMP was vascularized with higher levels of contractile, connective, and calcium-handling protein expression. Contractile proteins were also better aligned in the lbl-hCMP (deviation from global alignment 12.3 ± 3° vs. 24.3° ± 1.9°, p<0.05). The lbl-hCMP contained longer sarcomeres (2.05 ± 0.015 vs. 1.74 ± 0.026 um, p<0.05) with well-ordered I-bands, A-bands, M-lines, Z-lines, desmosomes, and intercalated discs. Thus, our lbl-manufacturing technique produced thicker and more mature patches that can be potentially clinically applicable for hearts with postinfarction LV remodeling.
Abstract P450: Single-cell Transcriptomic Profiling Of The Type-2 Diabetic Mouse Heart
Authors: Cohen Charles D , De Blasio Miles J , Farrugia Gabriella E , Dona Malathi S , Hsu Ian , Prakoso Darnel , Krstevski Crisdion , Nash David M , Li Mandy , Gaynor Taylah L , Deo Minh , Drummond Grant R , Ritchie Rebecca H , Pinto Alexander R
Published: November 2021

Background:
Diabetes is associated with a significantly elevated risk of heart failure. However, the precise cellular and molecular protagonists underpinning the development of heart failure in diabetes remains unclear. Moreover, very little is known, of how disparate non-myocyte populations of the heart contribute to diabetic cardiomyopathy.

Methodology:
To address this gap in knowledge, we conducted single-cell transcriptomic analysis of non-myocytes from heart ventricles of spontaneous type-2 diabetic (
db/db
) male mice. Findings were corroborated by flow cytometry, histology and computational analysis of publically available bulk RNA sequencing datasets from alternative diabetes models.

Results:
Single-cell transcriptomic analysis of
db/db
mouse hearts revealed a concerted diabetes-induced cellular response driving cardiac pathological remodelling. We identified diabetes-induced up-regulation of pathways contributing to known features of diabetic cardiomyopathy such as dysregulation of vascular homeostasis and lipid metabolism, as well as augmented inflammation, in cell specific contexts. We also identified unexpected characteristics in the diabetic heart, including impaired protein folding and cellular trafficking within lymphatic vessels. Using flow cytometry and histology, increase in inflammatory cells, such as Ly6C
hi
monocytes, shifts in macrophage phenotype, and increased abundances of fibroblasts and endocardial cells were confirmed. Finally, integration of single-cell transcriptomic data with publically available bulk-RNA sequencing datasets from alternative diabetes models revealed shared hallmarks of diabetic heart and disease context-dependent features.

Conclusions:
Together, this work offers a new perspective for understanding the cellular and molecular mediators of diabetes-induced cardiac pathology. Targeting these mediators may offer new therapeutic avenues to address the cardiac complications associated with diabetes.
Abstract P455: Acetylated Cyclophilin D Regulates Mitochondrial Electron Transport Chain Activity In The Developing Heart
Authors: Beutner Gisela , Burris Jonathan , Porter George A
Published: November 2021

Background:
Mitochondria generate energy in form of ATP in mature eukaryotic cells. The components of the electron transport chain (ETC) are expressed at embryonic (E) day 9.5 in mouse hearts, but ETC activity and oxidative phosphorylation (OXPHOS) are uncoupled, because the permeability transition pore (PTP) is open. In mouse hearts the mPTP closes around E11.5. Opening and closing of the PTP is regulated by cyclophilin D (CypD), but the regulatory mechanism are not understood.

Hypothesis:
Maturation of ETC activity is regulated by acetylated CypD.

Methods:
Cardiac tissue homogenates or isolated heart mitochondria from mice, ranging in age from E 9.5 to adult, were used to measure oxygen consumption and the calcium retention capacity. The expression of proteins of the ETC and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status were assessed by western blotting and densitometry. The enzymatic activity of the ETC complexes and CypD was measured using spectrophotometry.

Results:
In the heart of mouse embryos at E 9.5 mitochondrial ETC activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 μM cyclosporin A, an inhibitor of CypD and the PTP acutely increases the RCR to 3.69 ± 0.59 (n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. The total expression of CypD increases as the mouse heart matures, but the enzymatic activity of CypD decreases. In addition, the ratio of acetylated CypD to the total expressed CypD decreased from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart.

Conclusion:
The activity of acetylated CypD regulates maturation of mitochondrial ETC activity in the developing mouse heart.
Abstract P457: Systems Analysis To Understand The Impact Of The CDK Module On Cardiomyocyte Proliferation
Authors: Harris Bryana N , Woo Laura , Saucerman Jeffrey J
Published: November 2021

Rationale:
Heart failure is caused by the inability of adult mammalian hearts to overcome the loss of cardiomyocytes (CMs). This is due partly to the limited proliferative capacity of CMs, which exit the cell cycle and do not undergo cell division. Current knowledge in cardiac regeneration lacks an understanding of the molecular regulatory networks that determine whether CMs will progress through the cell cycle to proliferate. Our goal is to use computational modeling to understand the expression and activation levels of the core cell cycle network, specifically cyclins and cyclin-cyclin-dependent kinase (CDK) complexes.

Methods:
A model of core cell cycle dynamics was curated using previously published studies of CM proliferation regulators. This model incorporates those regulators known to stimulate G1/S and G2/M transitions through the core CDKs. The activity of each of the 22 network nodes (22 reactions) was predicted using a logic-based differential equation approach. The CDK model was then coupled with a minimal ODE model of cell cycle phase distributions and validated based on descriptions and experimental data from the literature. To prioritize key nodes for experimental validation, we performed a sensitivity analysis by stimulating individual knockdown for every node in the network, measuring the fractional activity of all nodes.

Results:
Our model confirmed that the knockdown of p21 and Rb protein and the overexpression of E2F transcription factor and cyclinD-cdk4 showed an increase in cells going through DNA synthesis and entering mitosis. A combined knockdown of p21 and p27 showed an increase of cells entering mitosis. Cyclin D-cdk4 and p21 overexpression showed a decrease and increase of Rb expression, respectively. Of the 14 model predictions, 12 agreed with experimental data in the literature. A comprehensive knockdown of the model nodes suggests that E2F (a key transcription factor driving DNA synthesis) is positively regulated by cyclin D while negatively regulated by GSK3B, SMAD3, and pRB.

Conclusion:
This model enables us to predict how cardiomyocytes respond to stimuli in the CDK network and identify potential therapeutic regulators that induce cardiomyocyte proliferation.
Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy
Authors: Halder Saiti S , Sewanan Lorenzo R , Rynkiewicz Michael J , Moore Jeffrey R , Lehman William J , Campbell Stuart G
Published: November 2021

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.
Abstract P460: Role Of Hopx In Antiretroviral Drugs Induced Cardiac Hypertrophy And Epigenetic Modification
Authors: Kashyap Shiridhar , Kondrachuk Olena , Gupta Manish K
Published: November 2021

Background:
Heart failure is the one of the leading causes of death in HIV patients. Application ofantiretroviral therapy (ART) raise the life expectancy of HIV patients, but survival population show higherrisk of cardiovascular disorder. The aim of this study is to understand the underlying molecular mechanismof antiretroviral drugs (ARVs) induced cardiac dysfunction in HIV patients.

Method and Results:
To determine the mechanism of ARVs induced cardiac dysfunction, we performeda global transcriptomic profiling in primary cardiomyocytes treated with ARVs. Differentially expressedgenes were identified by DESeq2. Functional enrichment analysis of differentially expressed genes wereperformed using clusterProfiler R and ingenuity pathway analysis. Our data show that ARVs treatmentcauses upregulation of several biological function associated with cardiotoxicity and heart failure.Interestingly, we found that ARV drugs treatment significantly upregulates the expression of a set of genesinvolved cardiac enlargement and hypertrophy in the heart. Global gene expression data were validated inthe cardiac tissue isolated from the HIV patients having history of ART treatment. Interestingly, we foundthat the homeodomain-containing only protein homeobox (HOPX) expression was significantly increasedin transcriptional and translational level in cardiomyocytes treated with ARV drugs as well as in heart tissueof ART treated HIV patients. Further, we performed adenovirus mediated gain in and siRNA mediatedknockdown approach to determine the role of HOPX in ARVs mediated cardiac hypertrophy and epigeneticmodifications. Mechanistically, we found that HOPX expression level plays a key role in ARV drugsmediated increased cardiomyocytes cell size and reduced acetylation level of histone 3 at lysine 9 and lysine27. Furthermore, we found that knockdown of HOPX gene expression blunted the hypertrophy effect ofARV drugs in cardiomyocytes. It is known that HOPX reduces cellular acetylation level through interactionwith HDAC2. In our study, we found that histone deacetylase inhibitor Trichostatin A can restore cellularacetylation level in presence of ARVs.

Conclusion:
ART treatment causes cardiotoxicity through regulation of fatal gene expression incardiomyocytes and in adult heart. Additionally, we found that HOPX expression is critical in ARVsmediated cardiomyocytes remodeling and epigenetic modification.
Abstract P462: Bscl2/seipin Deficiency In Heart Causes Energy Deficit And Heart Failure Via Inducing Excessive Lipid Catabolism
Authors: Zhou Hongyi , Li Jie , Su Huabo , Li Ji , Lydic Todd A , Young Martin E , Chen Weiqin
Published: November 2021

Heart failure (HF) is one of the leading causes of death world-wide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the
BSCL2
gene. Global lipodystrophic

Bscl2
–/–

mice exhibit hypertrophic cardiomyopathy. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown. Here we show that mice with cardiac-specific deletion of
Bscl2
(

Bscl2
cKO

) developed dilated HF. Myocardial BSCL2 deletion led to elevated ATGL expression and FA oxidation (FAO) along with reduced cardiac lipid contents. Cardiac dysfunction in

Bscl2
cKO

mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, heart failure in

Bscl2
cKO

mice could be partially reversed by pharmacological inhibition of FAO, or prevented by high fat diet (HFD) feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in

Bscl2
cKO

hearts, which were partially normalized by FAO inhibition or HFD. Our study reveals a new form of HF with excessive lipid catabolism, and identifies a crucial cardiomyocyte-specific role of BSCL2 in controlling cardiac lipid catabolism, energy state and contractile function. It also provides novel insights into metabolically treating energy-starved HF using FAO inhibitor or HFD.
Abstract P464: Cardiac Dysfunction In A Mouse Model For Autosomal Dominant Polycystic Kidney Disease
Authors: Perez William , Hendrickson Troy , Schiattarella Gabriele G , Hill Joseph A , Altamirano Francisco J
Published: November 2021

Mutations in polycystin-1 (PC1) cause autosomal dominant polycystic kidney disease (ADPKD), a disorder that manifests with cardiac hypertrophy and dysfunction. We recently showed that cardiomyocyte-specific PC1 KO mice exhibit both systolic and diastolic dysfunction without signs of cardiac hypertrophy. The purpose of this study was to determine the effects of ADPKD-causing PC1 mutations on cardiac function using a mouse model for ADPKD harboring a mutation in PC1 R3277C (RC/RC). We used echocardiography to determine cardiac function and Western blot analysis to explore signaling pathways in WT and RC/RC mice (2-4 months of age).We observed a slight but significant decrease in ejection fraction (77.2±0.7 vs 86.2±2.4 %, N=5, 4, P=0.015) without signs of left ventricular hypertrophy (LV mass 78.4±5.5 vs 93.5±4.2 mg, N=5, 4, P>0.05) in RC/RC compared to WT mice. Western blot analysis from total heart lysates (WT and RC/RC; N=5) revealed no changes in protein levels of hypertrophic markers: beta myosin heavy chain (β-MHC) and regulator of calcineurin 1 (RCAN1). In addition, we studied multiple signaling pathways involved in cardiac hypertrophy by analyzing their phosphorylation status by Western blot (phosphorylated/total protein). We observed no changes in mTOR, S6K1 and S6 phosphorylation. However, a decrease in p-4EBP1 and p-eIF4B was observed in RC/RC compared to WT. Moreover, we observed a significant increase in p-ERK and p-CaMKII. Our data suggest that alterations in PC1 signaling promote cardiac dysfunction but do not promote hypertrophy in young mice (2-4 months of age). Published evidence (PMID: 32730856) suggest that RC/RC hearts become hypertrophic at 6 months of age. However, our data suggest there may be dysfunction prior to cardiac hypertrophy. This warrants further investigation into the more primary role of ADPKD-associated co-morbidities. More studies, with a larger animal cohort, are necessary to unveil the effects of mutant PC1 on cardiac function.
Abstract P466: Sqstm1/p62 Regulates Hypoxia Inducible Factor 1 Alpha Stabilization And Protects The Heart From Hypoxic Injury
Authors: Ghosh Rajeshwary , Fatahian Nima , Vishaka Vinod , Symons J D , Boudina Sihem
Published: November 2021

Ischemic heart disease (IHD) is characterized by cardiac tissue hypoxia, dysregulatedmetabolism, and cell death. Hypoxia inducible factor 1a (HIF1α) is a major component of thehypoxia pathway that regulates metabolic and angiogenic genes. Under normoxia, HIF1α getshydroxylated by the prolyl hydroxylases (PHDs), followed by its ubiquitination, and degradationby the Ubiquitin Proteasome System (UPS). Given the short half-life of HIF1α (>5mins), PHDinhibitors are employed to stabilize HIF1α and improve cardiac function in animal models ofischemia/infarction. Because PHD inhibitors exert off-target effects, alternative strategies tostabilize HIF1α are needed. In cancer cells, p62 stabilizes HIF1α by binding PHD3. Wehypothesized that p62 would stabilize HIF1α and provides cardioprotection from hypoxia. Wegenerated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (cip62KO mice)and exposed them to 7% O2 for 6h. Compared to wild-type (WT) littermates, cip62KO micedisplayed increased oxidative stress and enhanced cell death. Hypoxia caused contractiledysfunction in cip62KO vs. WT mice. To gain mechanistic insights as to how lack of p62exacerbated hypoxic injury, cultured H9c2 cardiac myoblasts were exposed to 21% (normoxia)or 1% (hypoxia) O2 for 24h. Hypoxia increased HIF1α and p62 protein expression in H9c2 cells(p<0.05). To determine whether hypoxia-induced p62 accumulation is required for HIF1αstabilization, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, andexposed to hypoxia. Compared to ctrl siRNA cells, hypoxia-induced HIF1α protein accumulationwas reduced (p<0.05) after p62 knockdown. Additionally, hypoxia increased expression ofHIF1α downstream targets:
Egln1
,
Vegfa
,
Bnip3
and
Hmox1
mRNA, in ctrl siRNA cells, but theresponse was blunted (p<0.05) after p62 knockdown. These data indicate that p62 contributes tohypoxia-induced HIF1α stabilization and transcriptional activation. Defining how p62contributes to HIF1α stabilization and hypoxia tolerance is relevant clinically and could identifyp62 as a therapeutic target for treating IHD.
Abstract P476: Determinants Of Atrial Fibrillation Mechanisms Using Metabolomic Profiling
Authors: Sirish Padmini , overton James , Yang Jun , Nader Carol , Kiaii Bob , Srivatsa Uma , Hammock Bruce D , Chiamvimonvat Nipavan
Published: November 2021

Introduction:
Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. Oxylipins are derived from arachidonic acid, which is metabolized through three pathways including the cytochrome P450 (CYP450) pathway. The CYP450 products, epoxyeicosatrienoic acids (EETs), are major anti-inflammatory metabolites with several cardioprotective effects. The central hypothesis to be tested is that patients with AF will demonstrate an increase in oxylipin profiles towards inflammatory features compared to patients with normal sinus rhythm (NSR). We further hypothesize that lipid mediators may represent a new therapeutic target for AF.

Methods:
In the cross sectional study, human atrial appendage specimens and blood from informed and consented patients undergoing coronary artery bypass graft surgery were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling and inflammatory cytokines were assessed from the plasma of patients using LC-MS/MS. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology.

Results:
Metabolomic profiling shows a significant decrease in the EETs/DHETs ratios and EpOMEs/DiHOMEs ratios in the cohort with AF compared to patients in NSR. There was a significant increase in inflammatory cytokine, chemokine levels and oxidative stress in patients with AF compared to patients in NSR. There was a significant increase in nuclear translocation of NF-κB (nNF-κB) from patients with AF compared to NSR using western blot analysis. Assessment of total IκB and phosphorylated-IκB (pIκB) levels showed a decrease in IκB level and an increase in the pIκB levels associated with the activation of NF-κB in AF compared to the NSR cohort.

Conclusions:
The study provides important insights into metabolomic profiles in patients with AF and reveals the prospect of oxylipins being a novel therapeutic target in the treatment of AF.
Abstract P478: Farnesyl Transferase Inhibitor Tipifarnib Reduces Circulating Exosomes And Protects Against Pressure Overload-mediated Cardiac Hypertrophy
Authors: Mallaredy Vandana
Published: November 2021

Clinically, Hypertrophic cardiomyopathy (HCM) in response to pathophysiological stress is one of the major initiating factors for the onset of cardiac remodeling leading to heart failure. Studies have revealed that HCM characterized by left ventricular hypertrophy, hypercontractility, and impaired relaxation is mainly driven by an intricate crosstalk among the multiple cellular and molecular mechanisms, which leads to heart failure. In agreement with this observation, we investigated if the Tipifarnib-mediated reduction/alteration of circulating exosomes mediates cardiac cell communication during HCM. Several studies have shown Tipifarnib as a potential Farnesyl transferase inhibitor. However, in recent past Tipifarnib has been shown to target exosomes biogenesis by several mechanisms such as inhibiting Ras pathway, ESCRT complex etc. Tipifarnib treatment in mice significantly reduced the number of circulating plasma exosomes. We examined the response of Tipifarnib treatment (10 mg/kg body weight) in C57BL6J male mice subjected to transverse aortic constriction (TAC) surgery. Untreated TAC mice had worsening of systolic Left Ventricular function at 4 weeks that further deteriorated at 8 weeks, while the treatment with Tipifarnib substantially improved cardiac functions by reducing cardiac hypertrophy and fibrosis. Exosomes isolated from the serum of sham and TAC mice with or without tipifarnib were used for
in vitro
cell based analyses. We observed that the exosomes isolated from Tipifarnib treated TAC mice reduced isoproterenol (ISO)-induced cardiomyoblast hypertrophy and fibrosis-associated genes in adult cardiac fibroblasts. Taken together, our studies suggest Tipifarnib protects against pressure overload induced cardiac remodeling and dysfunction by altering hypertrophic and fibrotic gene expression, by potentially reducing circulating exosomes or by altering exosome contents. Ongoing studies will clarify the molecular mechanisms of these observations.