bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3086: Aldose Reductase Deletion Protects Ischemic Murine Hearts By Modulating Immune Cell Dynamics
Authors: Hasan Syed Nurul , Yepuri Gautham , Quadri Nosirudeen , Bhallamudi Sangeeta , Bernadin Rollanda , Mangar Kaamashri , Schmidt Ann , Ramasamy Ravichandran
Published: October 2023

Cardiac injury remains a major cause of death worldwide. Previous studies have demonstrated that chronic unchecked inflammation, during post ischemic reperfusion, adversely affects adaptive repair and augments tissue damage. Nature of the infiltrating immune cells determines the extent of repair vs exacerbation of tissue damage during post myocardial ischemia. Understanding mechanisms by which ischemia-mediated inflammatory responses exacerbate tissue damage and mitigate repair pathways are vital to discover therapeutics for myocardial infarction. Our earlier studies demonstrated that aldose reductase (AR) is a key mediator of myocardial ischemia-reperfusion (I/R) injury. Specifically, it established that cardio myocyte AR drives metabolic and signalling mechanisms leads to injury and impaired function in hearts. In this study we investigated the role of AR in influencing time dependent changes in recruitment of immune cells, during in vivo myocardial I/R injury. AR knockout (ARKO) and littermate controls (WT) mice were subjected to LAD sugery followed by reperfusion ( In vivo I/R) and the temporal changes in recruitment of immune cells were determined at earlier (day 3) and later (day 7) time points. We found ablation of AR leads to significant (p<0.0001) cardiac protection during post I/R in mice. Cardiac function measurements revealed greater recovery of ejection fraction and fractional shortening after I/R in ARKO vs WT hearts (p<0.05). Deletion of AR leads to a significant (p =0.0053) reduction in T cells infiltration post I/R in the heart. We observed 2-fold reduction in CD8+ cells, and 2-fold increase in CD4+ cells in ARKO vs WT mice hearts post I/R injury. Specifically, 2-fold reduction in pro-inflammatory Th1, Th2 and Th17 cells and 3-fold increases in anti-inflammatory Treg cells (p = 0.0091) were observed in ARKO vs WT hearts at post I/R injury. In addition, we observed reduction in infiltration of pro-inflammatory macrophages, dendritic cells, and B cells in ARKO vs WT hearts post I/R. Taken together, our data suggests that deletion of AR protects I/R hearts by promoting increases in anti-inflammatory immune cells and paves the way for addressing immune cell-cardiomyocyte crosstalk mechanisms by which AR deletion protects from I/R injury.
Abstract P3099: Pathogenic Innate Immune Responses Blunt The Activation Of Pro-reparative Fibroblasts After Myocardial Infarction
Authors: Ninh Van K , Yu Justin , Sehgal Rahul , King Kevin
Published: October 2023

Acute myocardial infarction (MI) leads to the development of a borderzone (BZ) that separates the ischemic zone from the surviving myocardium. The BZ is the site in which injured cardiomyocytes interact with diverse stromal and infiltrating immune cells that determine the course of tissue repair of the infarcted heart. We have recently identified spatially distinct transcriptional niches of cellular subsets using capture-based spatial transcriptomics in mice subjected to permanent ligation of the left anterior descending artery. Interestingly, we observed focal patterns of innate immune responses localized to the BZ microenvironment and a concomitant lack of stromal responses. We thus hypothesize that pathogenic BZ innate immune responses antagonize protective stromal cell responses important in the evolution and remodeling of the BZ.We tested the effects of the pro-inflammatory type I interferon response
in vitro
using cultured L929 fibroblasts treated with 10 ng/μL of IFNβ1, 10 ng/μL TGFβ1, or both. Fibroblast transcriptional activation was determined by gene expression of
Postn, Sparc, Bgn, Acta2,
and
Col1
α
1
by qPCR analysis. The potent IFNβ1 cytokine significantly inhibited TGFβ-mediated fibroblast activation compared to TGFβ1-treated cells (0.42±0.05 vs 1.0±0.15, p < 0.005).Fibroblast wound contracture and matricellular proteins have been shown to be critically important and protective in acute MI. This process was functionally measured by seeding fibroblasts into a 3D collagen gel and treated with IFNβ1, TGFβ1, or both and quantifying the difference in gel area. Using this functional assessment of fibroblast function, we show that IFNβ inhibits TGFβ-stimulated contraction of a 3D collagen hydrogel by again attenuating TGFβ-mediated fibroblast function (0.25±0.1 vs.60±0.11, p < 0.005). This IFNβ1-mediated contractile inhibition was blocked by treatment with a neutralizing antibody against the IFNβ1 receptor. Our data enables us to understand how a given cell state changes based on the cells’ neighborhood and how this relates to cardiac remodeling post-MI. Understanding the exact cellular and molecular mechanisms of wound healing processes during the acute inflammatory event will be key to developing novel therapeutics.
Abstract P3100: Mice Lacking S-nitrosoglutathione Reductase (gsnor
-/-
), A Mouse Model Of Preeclampsia, Exhibit Increases In Oxidative And Nitrosative Stress And Deterioration Of Cardiovascular Structure And Function In The Mother Postpartum.
Authors: Dulce Raul A , Balkan Wayne , Hare Joshua M , Kulandavelu Shathiyah
Published: October 2023

Introduction:
Preeclampsia (PE), a leading cause of maternal mortality, is linked to persistent pathological changes in the heart, predisposing the mother to increased risk of chronic cardiovascular (CV) disease later in life. PE is characterized by increased S-nitrosylated (SNO) proteins and nitrosative stress. We recently showed that mice lacking S-nitrosoglutathione reductase (GSNOR

-/
-

mice), a denitrosylase that regulates protein S-nitrosylation, exhibit the clinical features of PE including maternal hypertension, blunted increase in cardiac output, abnormal remodeling of left ventricular (LV) cardiac structure, dysregulation in nitrosylation and increased nitrosative stress. We hypothesize that the aberrant nitrosylation and nitrosative stress seen during pregnancy persist postpartum (PP), leading to increased maternal susceptibility to CV injury.

Methods:
Pregnant control (WT [C57Bl/6J]) and GSNOR

-/-

mice (N=5) were examined at baseline, late-stage pregnancy (17.5 dpc) and 6 weeks PP. LV structure, cardiac output and stroke volume were determined using echocardiography (Vevo 2100). Cardiomyocytes (CM) were isolated at 6 weeks PP and ROS and peroxynitrite levels determined using fluorescent dyes.

Results:
In WT mice, all normal maternal CV adaptions to pregnancy, reverted to pre-pregnant levels 6 weeks PP. In contrast, GSNOR

-/-

mice exhibited increased cardiac output (+21% vs. -19% in WT), stroke volume (+21% vs. -41% in WT), and LV chamber dimension (+13% vs. -4% in WT) at 6 weeks PP, indicating high cardiac output LV cardiomyopathy. CM isolated from GSNOR

-/-

hearts showed ~5-fold more ROS generation (9.9x10
-4
±9x10
-5
vs. 2.1x10
-4
±1.5 x10
-4
ΔF/F
max
.min
-1
) and elevated peroxynitrite levels (0.318±0.011 vs. 0.243±0.012 ΔF/F
0
) compared to WT at 6 weeks PP, suggesting the presence of oxidative and nitrosative stress. All changes P<0.05.

Conclusion:
Postpartum GSNOR

-/-

(preeclamptic) mothers exhibited cardiac dysfunction characterized by high cardiac output LV cardiomyopathy, which was accompanied by elevated levels of oxidative and nitrosative stress. These changes may predispose the mother to higher risk for future CV injury.
Abstract P3110: Sex Differences In Ventricular Tachycardia Ablation In-hospital Outcomes: An Analysis From The National Inpatient Sample Database
Authors: Taha Yasmeen , Mahmoud Ahmad , Elzeneini Mohammed , Ruzieh Mohammed , Pepine Carl J , Agarwal Zubin
Published: October 2023

Background:
There are limited data available comparing outcomes of ventricular tachycardia (VT) ablation in men versus women.

Objectives:
For patients undergoing ablation for VT, we sought to determine if sex-based differences in outcomes and procedural complications exist.

Methods:
We queried the National Inpatient Sample (NIS) database between 2016 and 2018 to identify patients aged ≥18 years hospitalized with a primary diagnosis of VT who underwent ablation. Patients with other cardiac arrhythmias or who had an implantable defibrillator/pacemaker placement were excluded. Multivariable logistic regression analysis was performed to assess independent predictors of any procedural complication.

Results:
Out of 7,200 identified admissions for VT involving VT ablation, 1,280 (18%) were women and 5920 (82%) were men. Men were older (mean age 64.8 vs 57.4 years in women, P<0.01). Comorbidities as prior myocardial infarction (MI), prior CABG, Chronic kidney disease (CKD), COPD, prior cardiac pacemaker, chronic heart failure (CHF) and peripheral vascular disease (PVD) were all significantly more prevalent in men.The primary outcome of in-hospital mortality occurred in 3% of both men and women. Interestingly, the length of stay was longer in men (6.53±4.2 vs 5.26±2.5 days, p<0.01). The incidence of acute MI, acute ischemic stroke, bleeding, need of blood transfusion, tamponade, complete heart block, pulmonary complications, vascular complications similar between men and women, however acute kidney injury occurred more in men (21% vs. 10%, p< 0.001). The overall complication rate was significantly higher in men (28.8% vs 20.7 %, P= 0.0084). The presence of CKD, as well as CHF were independent predictors of any procedural complication (OR 1.27, 95% CI 1.19-1.36, P <0.01) and (OR 1.08, 95% CI 1.03-1.14, P=0.001).

Conclusion:
Men had higher rates of in hospital complications following VT ablation which might be related to higher rates of comorbidities. There was no difference in in-hospital mortality between men and women. Women represented only 17.8% of VT ablation admissions. Further studies are needed to distinguish the sex-based differences in VT ablation referrals and to compare long term outcomes post ablation.
Abstract P3118: Gender Differences In The Efficacy Of A Mobile-based Self-management Program For Patients With Heart Failure
Authors: Ahn Jeong-Ah
Published: October 2023

Background:
This study was to develop a mobile application (App) program, presenting educational materials and giving interactive feed-backs on self-management for heart failure patients and to evaluate the gender differences in the efficacy of the program.

Methods:
We developed a mobile App program for heart failure patients’ self-management, which included comprehensive educational materials and daily record logs for the patients. Also, it took into account the mobile use characteristics and convenience of aged heart failure patients, as well as active and instant interactions and feedback via group chat among the patients and healthcare providers. Regarding the experimental study, we employed a randomized controlled trial to evaluate the effects of the program in 66 heart failure patients. The intervention group was guided to use the mobile App program in their daily life at home for 3 months.

Results:
Male patients in the experimental group exhibited significantly decreased waist circumference and improved NYHA functional class than the control group after 3 months. As for female patients in the experimental group, they showed significantly improved NYHA functional class, depressive symptoms, and quality of life than the control group after 3 months.

Conclusion:
This mobile App program showed effective improvements in heart failure patients' symptom classes for both men and women. Also, it showed that women had overall higher positive outcomes in psychosocial aspects, such as depressive symptoms and quality of life than men. Future study is needed to develop self-management programs considering gender differences and also to understand their effectiveness according to gender.
Abstract P3119: Identifying The Windows Of Cardiac Risk And Opportunity In Menopause
Authors: Barry Ciara , Rouhana Sarah , Saboune Jinan , Braun Jessica L , Geromella Mia S , Fajardo Val A , Pyle W. G
Published: October 2023

Post-acute myocardial infarction (AMI) mortality has significant sex differences with a bias towards higher rates in women. While treatment inequities and access to care profoundly impact outcomes, biological factors also contribute to survival discrepancies. One complicating factor that drives worse outcomes in women is menopause. Understanding the biological basis for AMI mortality risk and the exacerbating role of menopause has been hampered by the lack of an animal model that recapitulates the dynamic changes associated with the perimenopausal transition. We used a mouse model of menopause in which ovarian failure is gradually induced following treatment with 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg, 15d), allowing for examination of cardiac alterations established during perimenopause. Echocardiography revealed no contractile changes during perimenopause, but cardiac myofilament activation was significantly affected. Calcium re-uptake by SERCA was impaired by the end of perimenopause. Changes in calcium handling were associated with alterations in calcium handling protein expression and phosphorylation. Activation of RISK and SAFE signaling molecules that protect against ischemia-reperfusion (IR) injury was altered throughout perimenopause, and the RISK-SAFE response to IR differed at points during perimenopause. The myocardial response to cardioprotective estrogen receptor activation also varied throughout perimenopause. Together these data reveal significant, nonlinear alterations in myocardial physiology and endogenous protective signing pathways during perimenopause. Disruptions in endogenous protection and calcium handling are established prior to menopause, providing possible mechanisms for worse post-menopausal AMI outcomes. The altered response to estrogen receptor activation preceding menopause may explain the inconsistent effectiveness of post-menopausal estrogen replacement therapy.
Abstract P3129: Strain-induced Cardiac Arrhythmias Caused By A Noonan Syndrome Mutation In Raf1 Can Be Suppressed By Modulation Of The Ras-mapk Pathway
In Vitro
Authors: Haghighi Fereshteh , Boehmker Leonie , Liutkute Aiste , Kleemann Karolin , Busley Alexandra V. , Pietras Jan P. , Fakaude Funsho E. , Seibertz Fitzwilliam , Rubio Tony , Kutschka Ingo , Zenker Martin , Voigt Niels , Cyganek Lukas , Kensah George
Published: October 2023

The gain of function mutation RAF1
S257L/+
, accounting for more than 50% of Noonan syndrome (NS) cases, results in a hyperactive RAS-MAPK pathway. Individuals suffer from myocardial dysfunction including electrophysiological abnormalities. Resulting arrhythmias may be potentially life-threatening, if sinus rhythm is not restored expeditiously. Intensive research has provided insights into structural anomalies. However, electrophysiological abnormalities contributing to arrhythmia initiation and maintenance in affected NS patients have not yet been investigated in-depth.To delineate the role of RAF1
S257L/+
in cardiac rhythm disturbance
in vitro
, we used cardiomyocytes differentiated from NS patient-derived (RAF1
S257L/+
) and isogenic gene-corrected (RAF1
corr/+
) iPS cells to generate 3D bioartificial cardiac tissues (BCTs). In a custom-made bioreactor system, we applied mechanical preload to BCTs to resemble an
in vivo
physiological stress condition. Here, in RAF1
S257L/+
BCTs, we observed a high susceptibility to arrhythmias compared to RAF1
corr/+
myocardium. This abnormality came alongside with a severely stiffened myocardium, and an impaired myocardial relaxation, pointing towards a diastolic disfunction. Electrophysiological analysis revealed a potentially arrhythmogenic shortening in action potential duration in RAF1
S257L/+
BCTs. Furthermore, the presence of spontaneous ectopic foci resulted in unstable excitation patterns. Long-term modulation of the hyperactive RAS-MAPK pathway by treatment with a MEK inhibitor, significantly reduced these arrhythmogenic parameters accompanied with a normalization of RAF1
S257L/+
BCT compliance, eventually stabilizing beating rhythm significantly.For the first time, our study provides an in-depth characterization of the RAF1
S257L/+
-associated arrhythmogenic phenotype
in vitro
. Shortened action potential durations, instable pace maker regions and a stiffened myocardium may contribute to the initiation and maintenance of severe cardiac rhythm anomalies. Moreover, we successfully rescued this distinct phenotype by pharmacological intervention. Our findings may have implications for future preventive treatment strategies for NS patients.
Abstract P3130: Preventing Site-specific Calpain Cleavage Of Junctophilin-2 Protects Against Stress-induced E-c Coupling Dysfunction And Heart Failure Development In Mice
Authors: Wang Jinxi
Published: October 2023

In heart failure (HF), excitation-contraction (E-C) coupling processes become disrupted, resulting in abnormal Ca
2+
homeostasis that triggers cardiac dysfunction. Junctophilin-2 (JP2) is an essential E-C coupling constituent for maintaining cardiomyocyte integrity and cardiac function both at baseline and in response to stress. We previously showed that calpain-mediated proteolytic cleavage of JP2 at arginine 565 / threonine 566 (R
565
/T
566
) is key to its downregulation in the diseased heart. However, it remains unclear whether preventing site-specific proteolysis of JP2 can prevent cardiac remodeling and HF progression following stress. To investigate this, calpain-resistant mutant JP2 knockin mice (JP2
CR
) were generated by deleting residues 563-568 spanning the primary calpain cleavage site. Studies in cultured cardiomyocytes treated with isoproterenol (ISO) showed that JP2
CR
is resistant to stress-induced loss of JP2 integrity and T-tubule disorganization compared to wild-type (WT) cardiomyocytes. Further, in vivo studies demonstrate JP2
CR
mice are more resistant to pressure overload stress having significantly less abnormal Ca
2+
homeostasis, cardiac dysfunction, hypertrophy, lung edema and fibrosis relative to WT mice. Western blot analysis revealed JP2
CR
hearts have preserved expression levels of several key E-C coupling proteins, including RyR2, SERCA2a and Ca
V
1.2. RNA-Sequencing analysis further revealed attenuated pressure overload induced hypertrophic and heart failure-related transcriptional reprogramming in JP2
CR
hearts. These findings indicate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain-cleavage of JP2 offers cardiac protection against stress-induced hypertrophy and heart failure. Our data indicate that preventing JP2 cleavage by gene therapy approaches may hold great therapeutic potential for treating heart failure.
Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation
Authors: Liutkute Aiste , Pavez-Giani Mario , Sobitov Izzatullo , van Wijk Stan , Sossalla Samuel , Cyganek Lukas , Jjm Brundel Bianca J , Voigt Niels
Published: October 2023

Background:
Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear.

Objective:
We hypothesize that impaired intracellular Ca
2+
-homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis.

Methods:
LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca
2+
current and intracellular Ca
2+
concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively.

Results:
R331Q iPSC-CMs demonstrated increased L-type Ca
2+
current density followed by increased amplitude of systolic Ca
2+
transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca
2+
content was evaluated from caffeine-induced CaT amplitude and integrated Na
+
-Ca
2+
exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca
2+
spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca
2+
alternans at lower threshold frequency (Fig. D).

Conclusions:
LMNA mutation R331Q is associated with disturbed intracellular Ca
2+
cycling, promoting occurrence of diastolic Ca
2+
sparks and arrhythmogenic Ca
2+
alternans. Collectively, our findings suggest that impaired Ca
2+
homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.
Abstract P3136: Alternative Polyadenylation Of Scn5a Generates A Novel N-terminal Nav1.5 Microprotein
Authors: Witmer Nathan H , McLendon Jared M , Zhang Xiaoming , Stein Colleen , Yoon Jin-Young , London Barry , Boudreau Ryan L
Published: October 2023

SCN5A
encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates the cardiac action potential. Its expression and activity are highly regulated, and variants can cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently discovered a well-conserved alternative polyadenylation (APA) signal downstream of the first coding exon in
SCN5A.
The APA signal yields a short
SCN5A
transcript, resulting in the presence of mRNA-seq reads mapping to this intronic region in heart tissues from several species (e.g. human, pig, and cat; rodents lack the upstream APA). Analysis of cardiac APA data revealed a decrease in upstream APA signal usage in humans with dilated cardiomyopathy (~30%, p=0.08). Reanalysis of human cardiac RNA-seq data indicated the ratio of short/full-length
SCN5A
mRNA is reduced ~25% in failing hearts (p<0.05, versus non-failing hearts). Notably, the short transcript isoform has the potential to produce a novel microprotein (referred to as SCN5A-NT), which is composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from “intronic” sequence. Western blot of human heart tissue using an N-terminal NaV1.5 antibody shows a band migrating identically to transgene-derived SCN5A-NT, supporting its expression in human hearts. SCN5A-NT is predicted to contain a mitochondrial targeting sequence, and immunostaining of transgene-derived SCN5A-NT in neonatal rat cardiomyocytes colocalizes with mitochondria. These findings were corroborated in mice treated with AAV9 encoding SCN5A-NT, which showed myocardial SCN5A-NT expression by western blot and immunostaining that colocalized to cardiomyocyte mitochondria. We are currently devising cell culture and mouse experiments to test if SCN5A-NT alters mitochondrial functions and/or NaV1.5 activity, perhaps by modulating mitochondria ROS or NAD+/NADH. We also generated knock-in mice harboring the human APA signal to investigate how upstream APA influences NaV1.5 expression and whether upstream APA usage is dynamically regulated during cardiac stress. Overall, this work describes a new regulatory mechanism of NaV1.5 expression that yields an undescribed microprotein.
Abstract P3139: Unsupervised Anomaly Detection For Identifying Arrhythmogenic Rhythms In Atrioventricular Block Hearts Using Deep Convolutional Autoencoders
Authors: Park Hyeong Kyun , Son Young Hoon , Kim Nam K , Jang Jeongin , Sheng Christina , Choi Dahim , Park Junbeom , Cho Hee Cheol , Park Sung-Jin
Published: October 2023

Atrioventricular block (AVB) is a condition that can cause delayed or absent impulse conduction and increase the risk of arrhythmic complications such as atrial fibrillation (AF), premature ventricular contraction (PVC), and ventricular tachycardia (VT). Therefore, long-term monitoring of the heart's AVB rhythms and the ability to identify and predict abnormal rhythms is crucial for clinical decision-making in AVB patients. However, current tools to analyze irregular AVB rhythms and identify anomalies are limited. While supervised deep learning algorithms have been effective for anomaly detection during regular ordered sinus rhythm, they lack practicality for analyzing irregular AVB ECG rhythms. To address this challenge, we developed an unsupervised anomaly detection approach using deep convolutional autoencoder (CAE) models. We created three different CAE models consisting of either residual blocks (ResBlocks) or standard and transpose convolutional layers (TransConv1Ds) to identify anomalies in AVB ECG data. We acquired 30 days of continuous ECG data from seven porcine AVB hearts with complete AV node blockage and optimized the models accordingly. Both ResBlocks- and TransConv1Ds-based CAE models demonstrated high sensitivity and specificity to identify AF, PVC, and VT compared to a PCA-based traditional autoencoder model, with a true positive rate of 92-99% and a true negative rate of 85-92%. The ResBlocks-based CAE models showed high robustness across different ECG data acquired at different days and various porcine models compared to a TransConv1D-based model. As a case application of our anomaly detection approach, we applied the CAE to evaluate the efficacy and toxicity of TBX18-based biological pacemakers for treating AVB hearts. Our CAE analysis revealed that injecting these pacemakers at the ventricular high septum increased the occurrence of AF and PVC compared to the control group, suggesting that the rhythms from the biological pacemakers competed with the ventricular rhythms of the AVB heart. Moreover, the CAE demonstrated high sensitivity, specificity, and robustness in identifying anomalies from human AVB ECG rhythms, indicating the potential for unsupervised algorithms to improve AVB management and treatment.
Abstract P3153: Human Bone Mesenchymal Stem Cell Lentiviral Transduction For The Production Of Extracellular Vesicles Targeted To The Heart Limits Cell Line Expansion
Authors: Xu Cynthia M , Broadwin Mark , Brinck Teixeira Rayane , Sellke Frank W , Abid Ruhul
Published: October 2023

Objectives:
Human bone mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) have been found to have regenerative and anti-inflammatory effects in heart disease, but one of the main obstacles of this treatment modality is the lack of practical delivery to the heart. To make intravenous delivery possible, this study sought to create a human bone mesenchymal stem cell (HBMSC) that secretes HBMSC-EV which will avoid macrophage uptake and home to the heart through lentiviral genetic modification.

Methods:
The following HBMSC extracellular genetic modifications were selected: CD-47, an anti-phagocytic marker; cardiac targeting peptide (CTP, sequence APWHLSSQYSRT) and ischemic myocardium targeting peptide (IMTP, sequence CSTSMLKAC), peptides both found to have selective cardiac and ischemic myocardium uptake, respectively, through previous biopanning research. Through a 3
rd
generation lentiviral system and subsequent antibiotic selection process, wild-type HBMSC were altered to carry these modifications.

Results:
Three HMBSC lines were created: 1) CD-47 only, 2) CD-47 and CTP anchored to the outer cell membrane by Lamp-2b, and 3) CD-47 and IMTP anchored by Lamp-2b. Preliminary findings show hindered cell proliferation and adherence - cell doubling time was significantly slowed by passage 6 (versus passage 10 in wild-type HBMSC). Also, the genetically modified HBMSC required a 1:2 subculture ratio (versus 1:3-4 in wild-type HBMSC).

Conclusions:
These genetically-modified HBMSC, engineered with a goal to be better suited for HBMSC-EV production in the treatment of cardiac disease, have significant limitations in cell proliferation and expansion, which will be a significant obstacle to the production of a meaningful quantity of HBMSC-EV. Ongoing studies are being carried out to characterize changes in HBMSC differentiation and in the cell-matrix.
Abstract P3159: Exosomes Derived From Podoplanin Positive Cells Induce Endo-Mt And Myofibroblast Transformation In Healthy Mouse Heart And After Ischemic Injury
Authors: Cimini Maria , Mallaredy Vandana , Estaras Conchi , Truongcao May , Wang Chunlin , Benedict Cindy , Kishore Raj
Published: October 2023

Specific understanding of the differences in cell communications between physiologicaland pathological conditions harbor the understanding of mechanisms to recover theequilibrium in the injured tissue and restore the organ function. Neurohumoral factorsreleased after myocardial infarction (MI) induce the expression of platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN) in mesenchymalstromal cells, which pivot their structural support function and actively participate in theremodeling of the ischemic tissue releasing an abundance of extracellular vesicles calledexosomes. Exosomes (exo) derived from activated PDPN+ cells conditioned media wereused in vivo for the treatment of healthy mouse hearts, and in vitro for the treatment ofmouse cardiac endothelial cells (mCECs), NHI-3T3 and adult cardiac fibroblast. Ourongoing experiments showed that in vivo, PDPN-exo could independently alter healthyheart physiology and structure; mice hearts treated with PDPN-exo developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increaseof the end diastolic and systolic volumes. The fibrotic area was characterized by ECMdeposition, increased lymphatic vessels and infiltrating CD45+ cells. In vitro, PDPN-exoreprogramed mCECs, NHI-3T3 and adult cardiac fibroblast, upregulating the expressionof fibrotic markers suggesting an Endothelial-Mesenchymal Transition (EndoMT) andmyofibroblast transformation. Proteomic analysis of PDPN-exo suggests these transitionsmay depend upon the delivery of NOTCH1 via exosomes and the cleavage of it throughγ-Secretase. Experiments of gain and loss of NOTCH1 function showed thatoverexpression of NOTCH1 intracellular domain (NICD1) in vitro was sufficient to derivea lineage switching in mCECs and fibroblasts on the contrary in vivo delivery and in vitrotreatment with exosomes derived from PDPN+ cells silenced for NOTCH1 or treatmentwith γ-Secretase inhibitor did not alter the heart structure and function or the endothelialcells and fibroblast lineage. Thus, NOTCH1 delivery via PDPN-exo after MI play a majorrole in scar remodeling and its biology can be pharmacologically modulated.
Abstract P3160: Myocarditis Treatments Within Our Sights: Targeting Inflammatory Conversations In The Heart Using Extracellular Vesicles
Authors: Beetler Danielle , Bruno Katelyn , Wolfram Joy , Fairweather DeLisa
Published: October 2023

Viral myocarditis is an inflammatory cardiomyopathy with no pharmacologic agent currently able to quell the immune mechanisms that damage the heart in their inflammatory crossfire to quell infections, without risk of immunocompromise. Sex differences are well known in inflammatory and/or infectious diseases. Females display more efficient, anti-inflammatory responses to external threats like viruses, while males launch a pro-inflammatory defensive response. Myocarditis is a classic case of an infectious, inflammatory disease being more frequent and more severe in men, while pre-menopausal women are most protected. Extracellular vesicles are biocompatible, highly abundant, stable messaging carriers with no transformative risk, and hold great promise as therapeutics due to their functional cargo. As EVs are soluble mediators of the conversations between cells, we seek to redirect protective anti-inflammatory messaging from pre-menopausal women to male mice, using a highly translational model of murine myocarditis. We derive EVs from omental fat, repurposing this source to derive a purified, therapeutic component. Our results show robust immune system control, with EV treatments effectively attenuating pro-inflammatory inflammasome and complement markers, while leaving regulatory components like M2b macrophages and complement component CR1 unchanged. Treated mice are healthier during acute disease and demonstrate better heart function than control mice, despite being a strain susceptible to myocarditis and subsequent dilated cardiomyopathy. Tests using EVs from similarly aged men do not demonstrate this anti-inflammatory capacity. Proteomic and RNA-sequencing content comparisons of these EVs from these two donor groups reveal important proteins and regulatory micro RNAs. Our data suggest that EVs from fat may be a novel anti-inflammatory treatment for viral myocarditis, however individual donors show varying therapeutic capacity, with EV content heavily influenced by sex and age. This study not only addresses a major therapeutic gap in the myocarditis field, but also provides functional and -omic based comparisons of EVs from donors differing only by sex, to test if female cardioprotection from myocarditis is transferrable.
Abstract P3164: Podocalyxin, A Renal Podocyte Specific Protein Expression In Urine Exosome Acts As A Marker For Podocyte Injury During Sars-cov-2 Infection
Authors: Nandula Seshagiri Rao R , Sen Sabyasachi
Published: October 2023

COVID-19 attributed to SARS-CoV2 infection has been associated with cardiovascular disease and diabetes. COVID-19 has been associated with endothelial cell dysfunction (ECD), which is manifested by entities such as microvascular thrombosis. ECD is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. We explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether SARS-CoV2 infection puts an individual at a higher risk of developing cardio-metabolic complications such as kidney disease (DKD), with podocyte damage. We evaluated albuminuria and podocyte protein in urine derived exosomes in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post SARS-CoV2 infection.

Methods:
Blood and Urine samples from SARS-CoV2 patients post-acute phase of infection were procured. Peripheral blood mononuclear cells (PBMNCs) and urine exosomes were isolated and podocyte protein marker Podocalyxin (PODXL) was identified by western blot analysis.

Results:
Podocyte specific proteins were examined in urine exosomal fraction such as Podocalyxin and Nephrin (normalized to CD9 exosomal protein band intensities) levels increased significantly compared to urine samples from type-2 diabetes subjects with documented evidence of nephropathy. Podocalyxin levels were significantly high at 12month time point (p=0.001; n=9) and Nephrin levels were noted to be high at 10week (p=0.01; n=8), 6months (p=0.04; n=14 ) and 12Months (p=0.001; n=6) time points. Podocalyxin and Nephrin obtained from urine exosome samples collected from non diabetic subjects who never had COVID-19, showed little or barely detectable corresponding western blot bands. Interestingly, there was no significant differences noted on urine albumin: creatinine (UAR) ratios between the two groups, indicating that urine exosomal protein estimation may be an ideal biomarker to monitor podocyte damage in early phases of CKD.

Conclusion:
A persistent high levels of podocyte specific proteins was noted in urinary exosomes even at 12 months post acute Covid which may be secondary to long-standing podocyte inflammation leading to long term renal damage.
Abstract P3172: Elamipretide Reduces Mitochondrial Hyperacetylation In Left Ventricular Myocardium Of Dogs With Chronic Heart Failure
Authors: Gupta Ramesh C , Gupta Vinita S , Castle Kristina , Sabbah Hani N
Published: October 2023

Background:
The failing heart manifests abnormalities of mitochondrial (MITO) function characterized by reduced respiration and membrane potential and increased acetylation of MITO proteins. The cause(s) of MITO protein hyperacetylation is unclear, but can be due to reduced sirtuin 3 (Sirt3) possessing deacetylase activity and increased CD38 possessing NADase activity and increased NADH/NAD ratio. Long-term therapy with elamipretide (ELAM), a MITO-targeting tetra-peptide, was shown to improve MITO function in dogs with microembolization-induced heart failure (HF).

Hypothesis:
We tested the hypothesis that MITO hyperacetylation caused by dysregulation of Sirt3 and CD38 and increased NADH/NAD ratio in MITO of left ventricular (LV) myocardium of HF dogs can be normalized after chronic therapy with ELAM.

Methods:
MITO fractions were isolated from LV tissue of 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELAM (HF+ELAM, 0.5 mg/kg once daily, n=7) or no therapy at all (control, HF-CON, n=7) and from LV tissue of 6 normal (NL) dogs for comparison. An NAD/NADH quantitation kit was used to determined levels of NADH and NAD in MITO fractions. Protein levels of CD38, Sirt3, and porin, an internal MITO loading control, were determined in Mito-SDS-extracts using Western blotting and band intensity expressed in densitometric units (du). Protein levels of CD38 and Sirt3 were normalized to porin.

Results:
Protein levels of porin were similar among NL, HF-CON and HF+ELAM dogs. In HF-CON dogs, NADH/NAD ratio increased (0.70 ± 0.14 versus 0.32 ± 0.05, p<0.05 versus HF) compared to NL dogs and decreased significantly in HF+ELAM (0.36 ± 0.02, p<0.05 versus HF-CON). In HF-CON dogs, Sirt3 (0.50 ± 0.0.05 versus 1.24 ± 0.13, p<0.05 versus NL) was significantly reduced and CD38 (1.69 ± 0.12 versus 0.70 ± 0.03, p<0.05 versus NL) was significantly increased compared to NL dogs. Treatment with ELAM normalized Sirt3 (1.0 ± 0.11, p<0.05 vs. HF-CON) and CD38 (0.92 ± 0.051, p<0.05 vs. HF-CON).

Conclusions:
ELAM normalized levels of Sirt3 and CD38 and reduced NADH/NAD ratio in MITO of LV of HF dogs suggesting normalization of MITO acetylome. The later may be partly responsible for improved MITO function and possibly LV function in HF dogs treated with ELAM.
Abstract P3175: Mitochondrial Fission And Extracellular Mitochondria Mediate Cardiac Dysfunction In Obesity Induced Cardiomyopathy
Authors: Li Sin-Jin , Chen Ching-Yi , Ostberg Nicolai , Tetri Laura , Lopez Ivan , Cornell Timothy , Mochly Rosen Daria , Haileselassie Bereketeab
Published: October 2023

Background:
Recent studies have demonstrated that dysregulation of mitochondrial dynamics, including excessive mitochondrial fission, lead to extracellular release of damaged mitochondria (exMito) as an alternate mechanism for mitochondrial quality control. However, the exact role of this mechanism on obesity cardiomyopathy (OCM) has not been explored. This project aims to determine if Drp1/Fis1 dependent excessive mitochondrial fission mediates the release of damaged mitochondria and propagate cardiomyocyte injury in OCM.

Methods:
H9c2 cardiomyoblast were treated with 200 μM palmitate (PA) to induce lipotoxicity. C57BL6/J mice were subjected to a high-fat diet (HFD) for 12 weeks to induce OCM. Changes in cardiac Drp1 activity, mitochondrial morphology, bioenergetics and contractile function was evaluated. Simultaneously, exMito were collected and characterized from supernatant and plasma. Using peptide P110 co-treatment , we determined the impact of pharmacologic inhibition of Drp1/Fis1 dependent mitochondrial fission on cardiac mitochondrial function as well as the quality and quantity of exMito.

Results:
H9c2 cells treated with PA demonstrated excessive mitochondrial fission, increased oxidative stress and decreased ATP level. The excessive mitochondrial fission seen following PA treatment was associated with the release of damaged exMito. The exMito isolated from PA treated cells were noted to mediate cytotoxicity to healthy H9c2s independent of PA. P110 treatment was protective against PA mediated mitochondrial damage, represented by improved bioenergetic profile, increased ATP and lower oxidative stress. P110 treatment also lowered the release of damaged mitochondria from PA treated H9c2s. Similarly, treatment of HFD mice with P110, improved mitochondrial function and glucose utilization, exMito release and subsequently improved cardiomyocyte contractile function.

Conclusion:
Lipotoxicity induced mitochondrial fission, disrupted mitochondrial function leading to release of exMito. P110 limited lipotoxicity mediated alterations in mitochondrial dynamics, mitochondrial function and exMito, and then improved cardiac function. Therefore, pathologic fission and exMito may play a critical role in OCM.
Abstract P3181: A Cyclophilin D-mitochondria-redox Differentiation Pathway Regulates Neonatal Heart Bioenergetics And Function
Authors: Kile Hope , Agyarko Joshua , Beutner Gisela , Porter George A
Published: October 2023

Background:
During birth, demands on the heart increase when exposed to high oxidative stress. Cardiac myocyte differentiation is dependent on changes in mitochondrial function that regulate cell stress pathways to enhance differentiation. We have shown these changes are dependent on closure of the mitochondrial permeability transition pore (mPTP) controlled by the protein cyclophilin D (CypD). These studies aim to determine the effects of independently manipulating CypD, mitochondrial membrane potential, and reactive oxygen species (ROS) production on mitochondrial function, myocyte differentiation, and cardiac function in the neonate.

Methods:
Neonatal C57Bl6 mice were intraperitoneally injected on postnatal day (P) 1-6 of with mitoparaquat (0.025-0.05 mg/kg), Tempol (0.7 mg/kg), mitoTempo (0.7 mg/kg), 2,4-dinitrophenol (2 mg/kg), or vehicle. Hearts were harvested at P7 and mitochondrial respiration, enzymatic activity of the electron transport chain (ETC) complex (Cx) I and 2, and assembly of mitochondrial supercomplexes were measured by native gel electrophoresis.

Results:
Treatment with mitoparaquat to increase mitochondrial ROS caused a dose-dependent decrease of Cx I activity measured by oxygen consumption and enzyme assays. This corresponded to decrease in assembly of Cx-I containing supercomplexes. Decreasing mitochondrial ROS using mitoTempo had no effect on Cx I activity but increased assembly of Cx-III containing supercomplexes. Decreasing ROS in the cell with Tempol decreased Cx-I activity but had no effect on supercomplex assembly. Uncoupling at the mitochondrial membrane using 2,4-dinitrophenol increased electron flux from Cx I to Cx III but may decrease supercomplex assembly. Treatment of isolated mitochondria with cyclosporin A to inhibit CypD had no effects on these measures.

Conclusions:
Changes in ROS and mitochondrial membrane potential have selective effects on ETC assembly, activity in the neonatal period, and are not inhibited by closure of the mPTP. This data suggests a pathway where CypD regulates the mPTP to regulate the ETC, membrane potential, and ROS production to regulate myocyte differentiation; such a pathway could be targeted to increase cardiac maturation in infants with poor cardiac function.
Abstract P3183: Cardiac Troponin I Directly Binds Mitochondrial Proteins And Inhibits Mitochondrial Functions
Authors: Elezaby Aly , Lin Amanda J , Vijayan Vijith , Pokhrel Suman , Bechara Luiz , Ostberg Nicolai , Queliconi Bruno B , Campos Juliane , Ferreira Julio , Haileselassie Bereketeab , Mochly-Rosen Daria
Published: October 2023

The heart relies on mitochondria for its extensive energetic demands, and some cytoskeletal elements can regulate mitochondrial function. Cardiac troponin I (cTnI) is a structural protein involved in sarcomeric contraction. Here we demonstrate that some cTnI localizes to mitochondria, inhibits mitochondrial functions, and increases opening of the mitochondrial permeability transition pore under oxidative stress. Purified cTnI directly binds immune-captured ATP synthase in saturable fashion and doubles "reverse" F
1
F
0
-ATPase activity
in vitro
, whereas skeletal troponin I (ssTnI) does not. Relevant to human disease, several pathogenic variants of cTnI associated with hypertrophic cardiomyopathy do not increase F
1
F
0
-ATPase activity. Using a rational search, we identified the d subunit of ATP synthase as a potential cTnI-binding partner, and a ten-amino acid peptide inhibitor of cTnI’s interaction with ATP synthase, peptide P888, inhibited cTnI binding to ATP synthase and abolished the cTnI-induced increase in ATPase activity. P888 treatment greatly reduced cardiac LDH release and improved fractional shortening
in vivo
after transient coronary artery occlusion in rats, indicating that mitochondrial cTnI exacerbates cardiac ischemia-reperfusion injury. We explore the mechanisms governing cTnI’s mitochondrial localization and additional mitochondrial binding partners for cTnI. Together, our studies suggest that mitochondria-associated cTnI inhibits ATP synthase under basal conditions. Preventing cTnI from binding to ATP synthase during cardiac ischemia may increase the reservoir of functional mitochondria to mitigate post-ischemic cardiac injury. We also suggest that cTnI has additional roles in regulating mitochondrial functions. Finally, pathogenic cTnI mutations associated with hypertrophic cardiomyopathy do not affect ATP synthase activity, suggesting a potential mechanism that contributes to the diverse pathologies associated with these mutations.
Abstract P3184: Catalase Knockdown Reverses The Activation Of Akt And The Increase In Mitochondrial Complex I Induced By MnSOD Overexpression In Mouse Heart Endothelial Cells
Authors: Brinck Teixeira Rayane , Xu Cynthia M , Chang Christopher , Sellke Frank W , Abid Ruhul
Published: October 2023

Introduction:
We have recently showed that endothelial cell (EC)-specific overexpression of mitochondrial antioxidant MnSOD (MnSOD-OE) induced Akt and ERK activation, mitochondrial complex I (C-I) biogenesis, supercomplex assembly, and a shift from glycolysis to mitochondrial respiration in mouse heart ECs (MHEC).
In vivo,
MnSOD-OE induced coronary angiogenesis and improved cardiac function in a transgenic animal model of myocardial infarction.

Hypothesis:
We hypothesize that antioxidant catalase mediated neutralization of MnSOD-induced conversion of superoxide to H
2
O
2
within mitochondria (mito) is crucial for C-I biogenesis, ATP synthesis, and Akt and ERK activation in MnSOD-OE MHEC.

Methods:
We used an EC-specific, tetracycline (Tet)-controlled, transgenic MnSOD-OE model. Isolated MHECs (Tet-ON=Control, Tet-OFF=MnSOD-OE) were reverse transfected with either negative control (NC)- or catalase-siRNA (Tet-ON NC or CAT siRNA, Tet-OFF NC or CAT siRNA) and evaluated by western blot and ATP luminescence. Data were analyzed by Two-Way ANOVA and Tukey’s post-hoc. A
p
-value ≤0.05 was considered statistically significant.

Results:
Catalase-siRNA resulted in 54% decrease in catalase expression (2.01±0.05 in Tet-OFF NC vs. 0.93±0.05 in Tet-OFF CAT siRNA) in MHEC, resulting in 69% increase in mito-ROS (17.33±1.71 in Tet-OFF NC vs. 29.33±2.42 in Tet-OFF CAT siRNA). Catalase knockdown and increased mito-ROS resulted in 30% decrease in p-44/42 ERK (1.92±0.08 in Tet-OFF NC vs 1.35±0.17 in Tet-OFF CAT siRNA) and 17% decrease in p-Akt
ser473
/t-Akt ratio in MnSOD-OE MHECs (1.57±0.07 in Tet-OFF NC vs 1.30±0.08 in Tet-OFF CAT siRNA). MnSOD-OE MHECs also showed decreased expression of mitochondrial C-l (by 31%; 1.43±0.06 in Tet-OFF NC vs 0.99±0.03 in Tet-OFF CAT siRNA) and C-III (by 29%; 0.90±0.06 in Tet-OFF NC vs 0.64±0.03 in Tet-OFF CAT siRNA), and decreased ATP luminescence when compared with the control group (by 27%; 12836±568.6 in Tet-ON CAT siRNA vs 9319±650.8 in Tet-OFF CAT siRNA).

Conclusions:
Catalase appears to play crucial role in reducing mito-ROS and inducing C-I biogenesis, ATP synthesis, and activation of Akt and ERK in MnSOD-OE MHECs.