bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2115: (2R,6R)-Hydroxynorketamine Prevents Myocardial Ischemia Reperfusion Injury By Regulating Immune And Inflammatory Response
Authors: Chen Baihe , Luo Tao , Kitakaze Masafumi
Published: October 2023

Background:
The ketamine metabolite 2R,6R-hydroxynorketamine (HNK) has recently attracted increasing attention by its potential as a novel fast-acting antidepressant candidate to treat depression. HNK has anti-inflammatory properties which contribute to its protective effects against ischemia and reperfusion (I/R) injury. However, whether HNK produces beneficial effects on myocardial I/R injury has not been elucidated yet. Here we tested the hypothesis that HNK might protects the heart from myocardial I/R injury by limiting myocardial inflammation.

Methods and Results:
Twelve male C57BL/6J mice (8-10 weeks old, 20-25 g) were used to produce acute myocardial I/R (30 min/24 h) by micro-surgery. Mice were divided into four groups: Sham, HNK+Sham, I/R, HNK+I/R. A single-dose of HNK (30 mg/kg) was intraperitoneally injected one hour before the I/R. Echocardiographic imaging and TTC staining showed that HKN administration before myocardial I/R increased fraction shortening by 26.5% and limited infarct size by 40% compared to I/R alone. Proteomics analysis showed HNK caused 81 proteins down-regulated and 18 proteins up-regulated in the left ventricle after I/R (HNK+I/R
vs
. I/R). The gene name of the top 5 down-regulated proteins are Ighg2b, Igkv17-121, Igkc, Igtp and Cd68. The go enrichment analysis showed that HNK in the I/R group could modulate immune response via downregulating signal transducer and activation of transcription 1 and 2 (STAT1 and STAT2) and type 1 interferon pathway. The KEGG enrichment analysis showed that HNK affected antigen processing and presentation via down-regulating beta-2-microglobulin, TAPBP and TAP1/2. Cytokine analysis showed that HNK attenuated the increased tissue levels of TNF-alpha, IL-1 beta, CTnI and BNP induced by I/R. HNK also significantly decreased I/R induced-neutrophil infiltration, apoptosis and oxidative stress in the myocardium as detected by Ly6G staining, tunel staining and DHE probe staining.

Conclusion:
We demonstrated for the first time the administration of HNK regulates immune response through STAT1 and STAT2 and type 1 interferon pathway in the heart, leading to the cardioprotection against I/R injury. The use of HNK may become an immunomodulatory therapy for the ischemic heart disease.
Abstract P2128: Insight Into Glycogen Synthase Kinase 3 Beta Localization Within The Heart: Role On Ischemia Reperfusion Injury
Authors: Federico Marilen , Hurst Stephen , Tsai Hui-Ying , Barraque Facundo , Sheu Shey-Shing
Published: October 2023

Glycogen synthase kinase 3 beta (GSK3β) is a cytosolic serine-threonine kinase first described for inhibiting glycogen synthase. GSK3β structure shows a small sequence that can work as a mitochondrial target sequence, however mitochondrial GSK3β (mtGSK3β) in the heart, and its role have not been investigated. It is known the enzyme can be activated under ischemia-reperfusion (I/R) injury and modulating mitochondrial permeability transition pore (MPTP) opening. In the nervous system, the truncation of GSK3β activates the enzyme, but in the heart, the truncation function remains unknown.
We hypothesized that a small subset of GSK3β is truncated and translocated to the mitochondrial matrix, which might modulate MPTP opening
. To test our hypothesis, we studied GSK3β expression by Western blot in mitochondrial fractions isolated from differential centrifugation. We found a percentage of GSK3β present in purified mitochondria (Pmito) truncated at the C-terminal side. To evaluate if mtGSK3β is involved in I/R injury, we study its expression in the fractions isolated from hearts subjected to I/R protocol. We found GSK3β is being truncated and its expression was increased in Pmito from I/R-hearts in comparison to normoxia-hearts (controls). To study the role of mtGSK3β in MPTP regulation we measure the sensitivity of the pore under a GSK3β inhibitor, SB216763 (SB21) by fluorescente of Fura-2-FF in a spectrofluorometer. MPTP Ca
2+
-sensitivity was reduced in crude mitochondria fraction (Cm, mitochondria with other organelles) and increased in Pmito under SB21 treatment. This data suggests mtGSK3β is inactive in Pmito and SB21 is inducing MPTP opening by another mechanism. Since GSK3β can be activated and inhibited by phosphorylation at T390 site (pT390) and S9 site (pS9), respectively, we measure it on mtGSK3β. We found pT390 is not present in Pmito likely missing during truncation, while pS9 is in a low amount. Moreover, pS9 increases on Pmito from hearts subjected to I/R protocol. All the data together showed that a small percentage of GSK3β is truncated and translocated within the mitochondrial matrix. However, this mtGSK3β is not involved in the modulation of MPTP opening, since the lack of a C-terminal side causes its inactivation.
Abstract P2147: Inhibition Of Cdk7 Attenuates Doxorubicin Cardiotoxicity And Enhances Anticancer Efficacy
Authors: Chen Jingrui , Wei Jing , Xia Peng , Liu Yuening , Cheng Zhaokang
Published: October 2023

Background:
The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle-reentry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy.

Methods and Results:
DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of
Cdk7
attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumor growth when used together with DOX in an immunocompetent mouse model of breast cancer.

Conclusions:
Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity, but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.
Abstract P2151: A Novel, Micellized Naringenin Nanoparticle Rescues Hemodynamic Function Post-Myocardial Infarction Via MARK4 Inhibition
Authors: Weingarten Noah , Iyengar Amit , Meldrum Danika , Guevara-Plunkett Sara , Dominic Jessica , Amirshaghaghi Ahmad , Nie Daphne , Belec Andrew , Patel Mrinal , Ho Joyce , Herbst David A , Lee Benjamin , Eaton Deborah , Cheng Zhiliang , Tsourkas Andrew , Atluri Pavan
Published: October 2023

Impaired contractility and associated shock are significant contributors to morbidity after myocardial infarction (MI). Elevated MARK4 activity after MI impairs contractility by increasing α-tubulin detyrosination. As such, we developed a nanoparticle-based therapeutic strategy to enhance myocyte function with a small molecule MARK4 inhibitor (naringenin) and tested its ability to improve myocyte function in a rat model of MI. Naringenin was encapsulated in PEO-PCL to augment bioavailability. Adult Wistar rats underwent thoracotomy and were randomized to receive either: MI + myocardial injection of micellized naringenin (0.3 mg/kg) [MI-Nar], MI + empty micelle [MI-Mic], MI alone [MI-Untreated], or no MI [Sham]. MI was achieved with left anterior descending artery ligation. On pressure-volume hemodynamic assessment 24 hours post-thoracotomy, MI-Nar rats had higher ejection fractions than MI-Mic or MI-Untreated rats (63±3% v. 48±5% vs. 39±4%, p<0.05) and similar levels to Sham rats (61±1%, p=0.97). MI-Nar rats had greater stroke work, and lower end-diastolic pressure and tau than MI-Untreated rats (all p<0.05). Isolated myocytes from infarct border zones had increased contraction and relaxation velocity in MI-Nar versus MI-Untreated rats (both p<0.0001; Fig. 1A, B). Improved function was associated with decreased α-tubulin detyrosination in MI-Nar versus MI-Untreated rats on Western blot (p<0.05; Fig. 1C, D). These data support small molecule inhibition of MARK4 via micellized naringenin as a novel strategy to rescue hemodynamics post-MI by mitigating myocardial α-tubulin detyrosination and enhancing myocyte function.
Abstract P2173: Naltrindole Offers Potent Cardioprotection From Ischemia Reperfusion Injury; Opioid Antagonism Or Calcium Handling?
Authors: Boakye Tanoh Desmond , Singh Sunit G , Frimpong Smith , Nair Arjun , Dean Tameka C , Chen Qian , Barsotti Robert , Young Lindon H
Published: October 2023

Coronary Artery Disease accounts for approximately 25% of deaths annually, and remains theleading cause of mortality inthe United States. Myocardial ischemic (MI) damage is the initialinjury, characterized by irreversible hyper-contracture of cardiac muscle. Currently, there is nopharmacologic treatment tomitigate MI damage that may occur inthe 250,000CABGs,215,000percutaneous coronary interventions (PCI), and heart transplantation. We havepreviously reported that naltrindole (NTI) dose-dependently reduces infarct size upto82% ininvivomyocardial ischemia-reperfusion(MI/R) studies whengivenprior toregional ischemia. Werecently have shownthat NTI elicits this robust infarct size reducing effect by anovelmechanism since NTI alsoreduces polymorphonuclear leukocyte (PMNs) superoxide (SO)release at concentrations corresponding tothe blood concentrationinthe invivoMI/R study(8mg/kg = ~200uM; 4mg/kg = ~100uM). PMNs donot express opioid receptors, and NTI isknowntobe adeltaopioid receptor antagonist.Inour ex vivoLangendorff rat MI/R model, male SD rats (~300g) were anesthetized, heartswere removed, perfused with 37°C Krebs’ buffer and apressure transducer was placed intheleft ventricle (LV). Baseline recordings were made during the first 10minand the hearts werepretreated with NTI (5uM, n=8), naloxone (NX; panopioid receptor antagonist; 10uM, n=6)and binaltorphimine (BNI; kappaopioid receptor antagonist, 5uM, n=7) or controls (n=12) for 5mins prior toglobal I(30min)/R(45min).Ischemic PeakPressure (IPP) incontrolI/R heartswas39±3mmHg comparedtopre-ischemicLVEDP of9±1mmHg atbaseline(n=12,p<0.01),resultinginsubstantialinfarctatthe endof45minR (32±4%).IPP wasreduced byNTI (18±3mmHg/s)comparedtoallgroups(p<0.05).FinaldP/dt maxandinfarctsize were mostimprovedwith NTI (1830±90mmHg/s, 7±2%)comparedtocontrol(777±142mmHg/s, 32±4%,p<0.01).Cardiacfunctionandinfarctsize did notimprovewith BNI orNX. The resultssuggested that NTI exerted robust cardioprotectionwith significant reductionininfarct sizeindependent of deltaor kappaopioid receptor which led toinvestigating its possible underlyingmechanism of action.
Abstract P2175: Induced Pluripotent Stem Cell-derived Cardiac Myocytes To Assess Anthracycline Cardiotoxicity
Authors: Jacobs Johanna E , Duelen Robin , Gillijns Hilde , Johar Angad , Heggermont Ward , Sampaolesi Maurilio , Segers Heidi , Herrmann Joerg , Van Aelst Lucas , Janssens Stefan P
Published: October 2023

Background:
Anthracyclines are a class of highly effective chemotherapeutics, yet their use is regularly complicated by anthracycline-induced cardiotoxicity (AIC). The advent of induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) offers the potential for disease modeling and prediction of genetic cardiomyopathies.

Purpose:
We aim to use iPSC-CMs to assess susceptibility to AIC.

Methods:
Five AIC heart transplant patients and five anthracycline treated patients without AIC were included in our study. We reprogrammed lymphocytes from both groups toward iPSCs using Sendai Virus, followed by differentiation into CM-like cells. Cytotoxicity following chemotherapy exposure was assessed using flow cytometry (FC), electron microscopy (EM) and reactive oxygen species generation (ROS). Furthermore, whole genome sequencing (WGS) was performed and variants were filtered based on allele frequency, pathogenicity using HumVar trained variant effect predictors and presence in patients versus controls.

Results:
The cytotoxic effect of increasing concentrations of doxorubicin was quantifiable using FC on SIRPA positive iPSC-CMs (
A
). There was a higher rate of cell death at any concentration of doxorubicin in patients with AIC as opposed to controls (
B
). On the ultrastructural level, doxorubicin treated iPSC-CMs manifested darker and more condensed mitochondria as seen with EM (
C
). Increased ROS production was observed following doxorubicin exposure (
D
). WGS analysis showed 2 pathogenic variants in the PRAMEF6 gene present in cases and not in controls (
E
).

Conclusion:
iPSC-CMs represent a robust disease model to study mechanisms of and susceptibility to AIC.
Abstract P2183: Validation Of A Methodology For Left Ventricular Global Longitudinal Strain Measured With Deformation Encoding Mri To Detect Cardiotoxicity
Authors: Kar Julia , Cohen Michael V , Revere Cherie , Judice Angela , McQuiston Samuel , Malozzi Christopher
Published: October 2023

This study sought to validate left-ventricular (LV) global longitudinal strain (GLS) derived from an MRI deformation encoding technique for detecting cancer therapy-related cardiac dysfunction (CTRCD) in breast cancer patients. Displacement Encoding with Stimulated Echoes (DENSE) cardiac MRI and 2D echocardiographic images for validation were acquired on 32 breast cancer patients at baseline and 3- and 6-month follow-ups after chemotherapy with antineoplastic agents. The DENSE images were processed with automated Artificial Intelligence segmentation techniques for chamber quantification and 3D strain analysis. The corresponding echocardiographic data were processed with 2D speckle-tracking echocardiography (2D-STE). To establish the reliability of the DENSE-AI tool, intraclass correlation coefficients (ICC) were computed between DENSE and 2DE-derived GLS and other parameters at each time point with Cronbach’s Alpha (C-α) indices. CTRCD risk analyses were conducted on the DENSE-AI and 2D-STE measured parameters with Cox proportional hazard (PH) regression models. The ICC reliability results between DENSE and 2DE-derived GLS were C-α = 0.89 at baseline, C-α = 0.93 at 3 months, and C-α = 0.94 at 6 months. Both DENSE and 2D-STE GLS worsened from baseline to the 3- and 6-month follow-ups (-19.1 ± 2.1% vs. -16.0 ± 3.1% vs. -16.1 ± 2.9%, P<0.001) (Fig. 1). Cox regression showed the 3-month DENSE (HR-per-SD: 2.1; 95% CI: 1.4-3.1, P<0.001) GLS measurements as an independent prognosticator of CTRCD occurrence. In conclusion, LV GLS measurement with the DENSE-AI tool was validated to detect CTRCD in breast cancer patients treated with antineoplastic chemotherapy.
Abstract P2185: Hydrogen Sulfide Donor Inhibits Cisplatin-induced Foam Cell Death In An In Vitro Model Of Atherosclerosis
Authors: Carter Nigeste M , Mauro Adolfo G , Palladino Elisa , Roden Allison L , Mezzaroma Eleonora , Toldo Stefano A , Salloum Fadi N
Published: October 2023

Introduction:
Cisplatin (CPDD) is used to treat various cancers, including testicular, lung, head and neck, and ovarian. CPDD use is associated with long-term cardiovascular morbidity and mortality risk, including acute coronary syndrome (ACS). Pro-inflammatory activation and death of macrophage foam cells (MFC) within the atheroma contribute to plaque destabilization and coronary artery thrombosis. The mechanisms underlying CPDD-induced ACS remain largely obscure. Hydrogen sulfide (H
2
S) has been shown to regulate cell survival and reduce the residual inflammatory risk in the setting of myocardial infarction.

Hypothesis:
We hypothesize that H
2
S protects MFC following CPDD administration.

Methods:
Human monocyte THP-1 cells were differentiated into MFC with 150 nM of phorbol 12-myristate 13 acetate (PMA) for 48 hours, followed by 100 μg/mL of OxLDL in serum-free media for 24 hours. Lipid content was assessed by Oil Red O staining (Fig A). A Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay was used to assess the cytotoxic effects on MFC subjected to 24-hour treatment with 25 μM CPDD following 30-minute pre-treatment with 40 μM sodium sulfide (Na
2
S, a H
2
S donor) (Fig B).

Results:
Differentiated cells exposed to OxLDL showed a 25% increase in lipid accumulation compared to untreated cells (Fig A, **p<0.01 vs THP-1). Na
2
S alone showed no effect on cell death. Treatment with CPDD induced ~20% increase in cell death while pre-treatment with Na
2
S reduced MFC death by ~50% (Fig B, **p<0.01 vs OxLDL, *p<0.05 vs OxLDL+CPDD).

Conclusion:
Our results indicate that CPDD may contribute to plaque destabilization by inducing MFC death, which is attenuated with H
2
S treatment.
Abstract P3008: A Computational Model Predicts Sex-specific Responses To Calcium Channel Blockers In Mesenteric Vascular Smooth Muscle
Authors: Hernandez Hernandez Gonzalo , O’Dwyer Samantha , Matsumoto Collin , Yang Pei-Chi , Fong Zhihui , Tieu Mindy , Santana Luis F , Clancy Colleen
Published: October 2023

The electrical activity of smooth muscle cells underlies the regulation of the diameter of small resistance arteries and arterioles to control blood flow and blood pressure. Historically, measurements of pressure in blood vessels and their responses to drug interventions have been based entirely on data from male animals. Recently, experiments have indicated sex-specific differences in smooth muscle. We hypothesize that the fundamental differences in ion channel expression measured in male and female cells may underlie variable responses to antihypertensive drugs between males and females. To determine if this hypothesis is plausible, we developed a computational model to investigate the effects of measured sex-specific differences in the electrophysiology and Ca
2+
signaling of vascular smooth muscle cells. Contrary to established models suggesting that K
V
1.5 channels are the most prominent K
V
channels regulating membrane potential, our model predicts that K
V
2.1 channels play an unexpectedly primary role in the control of membrane potential in female but not in male cells. In addition, we found that sex-specific differences in the amplitude and voltage dependencies of L-type Ca
V
1.2 currents between males and females cause higher Ca
2+
influx and higher [Ca
2+
]
i
, in female than in male mesenteric smooth muscle cells. To simulate sex differences in the regulation of resistance arteries and arterioles, we developed an idealized vessel model by connecting smooth muscle cells through simulated resistances to represent gap junctional coupling. The model predictions suggest that female arterial smooth muscle is more sensitive to clinically used Ca
2+
channel blockers than male smooth muscle. Our computational framework initiates the first steps toward the investigation of the potential sex-specific impact of antihypertensive drugs, which may lead to more effective treatment for hypertension.
Abstract P3011: Assessing Subclinical Atherosclerosis Using Carotid Intima-media Thickness In Inflammatory Bowel Disease Patients: A Metanalysis
Authors: Sharma Vagisha , BHASKAR ASHOK JAGDISH , Murtaza Syeda Fatima , Zaidi Syed Asjad Tauheed , Kamani Mayank Dineshbhai , Behera Ritika , Wadhwa Sohmika , Sutaria Muskaan , Konakanchi Vijaya Rupa , Joshi Advait , Roy Ritwik , Vasavada Advait
Published: October 2023

Objective:
Chronic inflammatory diseases are linked to atherosclerosis and cardiovascular events. However, there are conflicting results published in the literature regarding differences in carotid intima-media thickness, a marker of subclinical atherosclerosis, between inflammatory bowel disease patients and controls. We aimed to conduct a pooled analysis of the studies and determine if it is true.

Method:
We performed a systematic literature search of databases Google Scholar, Scopus and PubMed from Jan 01, 2012, until Feb 01, 2023. Unadjusted odds ratios (OR) were pooled using a random-effect model, and a p-value of <0.05 was considered statistically significant.

Findings:
A total of 4 studies with 354 participants were shortlisted for analysis. The results shown in the figure show an insignificant difference in the carotid intima-media thickness between inflammatory bowel disease patients and control groups of healthy individuals - the odds ratio came out -0.22 with a confidence interval of -0.89 to 0.45 and p-value of <0.05.

Conclusions:
Our pooled analysis revealed no impact of IBD on carotid-intima media thickness. Future studies should focus on scrutinizing the actual risk of atherosclerosis and its complications with the disease.
Abstract P3015: Mechanosensitive, Piezo2 Channels Mediate The Exaggerated Exercise Pressor Reflex In A Mouse Model Of Hind Limb Ischemia.
Authors: li qinglu , bjorge Johannes , Xie An , Das Satyabrata , Dudley Samuel C , Garry Mary G
Published: October 2023

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to afferent neuron activation by skeletal muscle contraction. Patients with peripheral artery disease (PAD) have an exaggerated EPR, contributing to exercise intolerance, morbidity and mortality. Recently, we developed a mouse model which we used to determine the effect of femoral artery ligation (FAL) on the EPR. We observed that the EPR is exaggerated 72 hrs following FAL in mice. Stimulation of metabolically active afferent neurons was not different between FAL and control. In contrast, the responses induced by static contraction (stimulation of both mechanically and metabolically sensitive afferent fibers) and passive stretch (preferential stimulation of mechanically sensitive afferent neurons) were exaggerated in FAL mice when compared to control. These exaggerated responses were not affected by SB-705498 (a TRPv1 receptor inhibitor) but were nearly abolished by Gadolinium (a mechanical sensitive group III afferent neuron inhibitor) and significantly reduced by GsMTx-4 (a selective inhibitor of the PIEZO2 receptor).. Furthermore, we observed that PIEZO2 expression was upregulated in the dorsal root ganglia (DRG) from FAL mice compared to controls. Importantly, FAL mice displayed a decreased exercise capacity when compared to control mice, verifying that this mouse model mimics human PAD and supporting the notion that an exaggerated EPR contributes to exercise intolerance. These results further indicate that the exaggerated EPR in FAL is mediated, in part, by the PIEZO2 receptor. To explore this, we generated mice with a conditional, DRG targeted, deletion of
Piezo2
. In these mice, we observed that the responses to static contraction and passive stretch were significantly reduced compared to control. Moreover, we performed whole-cell voltage-clamp on dissociated DRG observing increased negative pressure induced inward currents in the setting of FAL while these currents were decreased in the
Piezo2 -/-
DRG. These data indicate that PIEZO2 expression may serve as a therapeutic target in the treatment of PAD.
Abstract P3019: During Salt-sensitive Hypertension, CD8+ T Cells Increase Renal Sodium Transporters, NKCC2 And ENaC
Authors: Deck Katherine , Guo Yunping , Benson Lance , Liu Yunmeng , Mora Christoph , Mu Shengyu
Published: October 2023

Hypertension originates in part from impaired salt handling by the kidneys. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts) adoptively transferred from salt-sensitive hypertensive mice to normotensive mice stimulate Sodium Chloride Cotransporter (NCC) increasing sodium retention. However, whether other sodium transporters within the nephron are affected by CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize CD8Ts will also have an impact on other sodium channels. Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. In vitro, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously. In vivo, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. As for in vitro, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological or with additional corticosteroid levels. Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention and any effect of CD8Ts in increasing ENaC may be masked in physiologic conditions.
Abstract P3021: Circulating Exosomal MicroRNAs As Biomarkers For Pulmonary Embolism
Authors: Zhang Chongyu , Nikolovski Srdjan , Kantarcioglu Bulent , Siddiqui Fakiha , Fareed Jawed , Jones Keith
Published: October 2023

Pulmonary Embolism (PE) is the third leading cause of death worldwide. Current treatment for PE, including thrombolytics and anticoagulants, have serious side effects. Protein and RNA biomarkers presently used to diagnose PE do not have the necessary specificity or sensitivity. Therefore, discovery of predictive biomarkers for therapeutic effects and adverse outcomes is imperative. MicroRNAs (miRNAs) are known to regulate gene expression networks and their expression levels reflect the physiological state of their cell of origin. By investigating these circulating miRNAs, we can gain valuable insight into the state of the damaged cells or tissue. Here, we innovated a novel methodology to isolate exosomes from patient platelet-free plasma that provides better yield of both exosomes and their RNA content. We are the first to stratify the PE patient samples according to clinical severity (low-risk, sub-massive, massive PE) and analyze the correlations of the exosomal miRNA biomarkers with PE severity. We found that exosomes from PE patients are more heterogeneous, lower in concentration and larger in size compared to healthy controls. Further, exosomes from PE patient samples pack more RNA than healthy controls. With the results from ongoing miRNA sequencing, we will be able to identify miRNAs and other short non-coding RNAs as potential diagnostic biomarkers that best correlate with PE severity. Validation and predictive studies using qRT-PCR will push the frontiers on circulating small RNA biomarkers in PE and other cardiovascular diseases. Finally, network analysis on the potential RNA biomarkers will provide information for future studies on disease mechanisms and therapeutics development.
Abstract P3030: Reduced Exercise Capacity Occurs Before Intrinsic Changes In Skeletal Muscle In Experimental Rat Models Of Pulmonary Hypertension
Authors: Zhang Peng , da Silva Goncalves Bos Denielli , Vang Alexander , Feord Julia , McCullough Danielle , Zimmer Alexsandra , D'Silva Natalie , Clements Richard T , Choudhary Gaurav
Published: October 2023

Background:
Pulmonary Hypertension (PH) is defined with mean pulmonary arterial pressure greater than 20 mmHg at rest. Reduced exercise capacity in PH significantly impacts quality of life. While the cause of reduced exercise capacity in PH remains unclear, it is suggested that regional skeletal muscle adaptions may play a role. The objective of this study was to investigate whether intrinsic changes in skeletal muscle determine exercise capacity in PH using preclinical PH rat models with different PH severity.

Methods and Results:
PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 1-4 weeks of normoxia exposure. Rats injected with vehicle and housed in normoxia were used as controls. Echocardiography and hemodynamics were performed to assess PH and cardiac function. Exercise capacity was assessed by VO
2
max. Skeletal muscle structural changes (atrophy, fiber type switch, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed in soleus muscles. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7 weeks timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4 weeks timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged.

Conclusions:
Our study demonstrated that reduced exercise capacity in PH occurs before intrinsic changes in skeletal muscle, suggesting that alterations in skeletal muscle are not causative to exercise intolerance in PH, while they may worsen exercise capacity collaboratively with primary factors in the late stage of PH. In addition, our study supports the notion that improving central cardiopulmonary impairments at the early stage of PH may be critical in preventing or delaying the occurrence of exercise limitation in settings of PH.
Abstract P3049: Exploring The Therapeutic Potential Of Aav-mediated Restoration Of Col6a2 In A Mouse Model Of Ullrich Muscular Dystrophy
Authors: Havens Julian R , Prasad Vikram , Blair Scott , Molkentin Jeff D
Published: October 2023

Ullrich muscular dystrophy is a genetic disease characterized by joint contractures and muscle weakness which leads to loss of ambulation and respiratory insufficiency early in life. This disease is caused by mutations in any of the three major genes of collagen VI - Col6a1, Col6a2, or Col6a3. Together, these three genes are required to generate a complex quaternary protein assembly that is secreted to help form the basement membrane. Currently there is no cure or greater treatment for Ullrich muscular dystrophy although 2 of the genes (Col6a1, Col6a2) are within the size limit of adeno-associated virus (AAV) and could be addressed by gene therapy. However, it is unclear what cell type in muscle generates the bulk of Collagen VI during development, or if it could be replaced at a later time by gene therapy from expression in myofibers or from fibroblasts. Given that mutations in the Col6a2 gene are the most common cause of Ullrich muscular dystrophy, here we generated Col6a2 gene deleted (Col6a2
-/-
) mice which showed the anticipated histopathologic evidence of dystrophic disease as young adults. Adult mice were then subject to retro-orbital injection of MyoAAV-Col6a2, which resulted in robust expression of Col6a2 within the myofibers of all skeletal muscles analyzed (and heart), although this was ineffective in restoring the collagen VI complex within the basement membrane. Further characterization into the effects of Col6a2 re-expression from the myofibers versus the fibroblasts in these gene targeted mice is underway with the goal of defining a combinatorial (with Col6a1 and Col6a3) or cell type restricted infection protocol to successfully achieve full restoration of Collagen VI in muscle.
Abstract P3052: RNA Binding Protein Csdc2 Is Required For Metabolic And Cardiac Stress Responses
Authors: McLendon Jared M , Zhang Xiaoming , Boudreau Ryan L
Published: October 2023

Post transcriptional gene regulation (PTGR) mediated by networks of microRNAs and RNA binding proteins (RBP's) controls mRNA processing, localization, and expression. Hundreds of microRNAs and RBPs are expressed in heart, and much remains unknown regarding PTGR’s influence on cardiovascular health and disease. Through bioinformatic analyses, we identified Csdc2 as a cardiac-enriched RBP that is consistently downregulated in failing hearts. Csdc2’s paralog, Carshp1, controls metabolic gene expression in liver through PPARα. We hypothesize that Csdc2 plays key roles in cardio-metabolic stress. To address this, we first assessed mRNA-seq changes in neonatal rat cardiomyocytes after overexpression and knockdown of Csdc2. Pathway analyses revealed reciprocal dysregulation of fatty acid metabolism, ion channels, and cytoskeletal and sarcomere genes. We generated constitutive Csdc2-KO mice, which are viable with no overt baseline phenotypes. Cardiac phenotyping revealed that Csdc2-KO mice have slightly decreased PR interval (~10%, p=1.3e-4, n=16-22) but maintain normal cardiac structure/function. To assess Csdc2’s potential role in metabolic stress, mice were fed a high-fat-diet for 10 weeks and monitored for lean/fat mass and insulin/glucose tolerances. Csdc2-KO mice gained 20% more weight than wildtype littermates (p=0.04, n=13-18) and developed insulin resistance (p<0.01). To test if Csdc2 loss influences cardiac stress, normal chow cohorts were subjected to microsurgery-induced myocardial infarction (MI); Csdc2-KO mice showed ~2-fold increase in mortality within two weeks (70% versus 40% for wildtype). Echocardiographic measures support that Csdc2-KO hearts have larger infarcts, dilation without compensatory remodeling and worse systolic function. Together, these data highlight Csdc2 as a disease-relevant RBP that controls cardio-metabolic stress responses.
Abstract P3066: TSC2-mTOR Axis Exerts Biased Control Over Macrophage Infiltration Following Myocardial Infarction
Authors: Keykhaei Mohammad , Koleini Navid , Meddeb Mariam , Ranek Mark J , Farshidfar Farnaz , Yoo Edwin , Mulligan Abigail , Kass David A
Published: October 2023

Introduction:
The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved serine/threonine kinase, which regulates pivotal physiological processes. mTORC1 activity is tightly regulated upstream by the tuberous sclerosis complex (TSC) which consists of TSC1 and TSC2. In macrophages as throughout the immune system, mTORC1 is a potent regulator of cell activation and function. Myocardial infarction (MI) is associated with stimulation of mTORC1; however, studies vary as to whether this activation is beneficial or detrimental, and its influence on macrophage differentiation, and infiltration after MI is unknown.

Hypothesis:
We tested the hypothesis that cardiac infiltration of macrophages following MI is regulated by their expression of TSC2.

Methods/Results:
Macrophage-specific TSC2 knock-out mice (TSC2-LyzM) were made by crossing mice with floxed-TSC2 and LysM-Cre that display a significant reduction of macrophage TSC2 protein. TSC2-LyzM bone marrow-derived macrophages (BMDMs) have constitutive mTORC1 activation reflected by phosphorylation of downstream targets S6K1 and 4E-BP1 and were correspondingly larger. To assess macrophage polarization, cells were stimulated with LPS to promote an M1-like phenotype or IL-4 to induce an M2-like phenotype. LPS- treated TSC2-LyzM BMDMs secreted more of the proinflammatory cytokine TNF-a, while IL-4 stimulation resulted in less of an M2 program, with significantly less Arg1, Mrc1, and Rentla expression. These findings show TSC2 is required for normal BMDM polarization. We next tested if TSC2 impacts macrophage myocardial infiltration after MI. Macrophages were identified as CD45+CD11b+CD64+ and further parsed by CCR2 and MHC-II. In concordance with prior studies, we found substantial infiltration of CCR2+ (BMDMs) macrophages in both genotype groups after MI. However, more TSC2-LyzM had increased CCR2+ MHC-II+ phenotype (infiltrating macrophages) versus wild-type.

Conclusion:
Overall, our findings highlight a key role for the TSC2-mTORC1 pathway in regulating macrophage fate specification and cardiac infiltration following a MI. This supports novel signaling control over BMDMs by TSC2-phosphorylation mediated mTORC1 activation or inhibition.
Abstract P3071: Targeting Neutrophil Sting And Type I Interferon Activation In Myocardial Ischemia-Reperfusion Injury
Authors: Brockman Maegan L , Wang Lanfang , Calvert John W , Levit Rebecca D
Published: October 2023

Introduction:
Inflammation following myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and numerous cells in the post-MI/R heart at 24 hours post reperfusion. Preliminary single-cell RNA sequencing data shows type I interferon (IFN) responding neutrophils are present in the hearts of mice 24 hours post-MI/R. The following study aims to address how the type I IFN responding neutrophil phenotype identified in single-cell RNA sequencing data affects cardiac inflammation in MI/R. We hypothesize that STING and type I IFN activation in neutrophils in the early stages of inflammation can exacerbate inflammation, causing further damage to the heart in MI/R.

Methods:
Male C57BL/6J mice (10-11 weeks old) underwent 60 minutes of ischemia followed by reperfusion, and were dosed with either vehicle, a STING inhibitor (H151), or an IFNAR1 antibody (ab) at reperfusion and at 24 hours following reperfusion. Cardiac function was assessed two weeks post-reperfusion via echocardiography and tissue samples were collected for histological analysis.

Results:
Mice treated with IFNAR1 ab showed significant improvement in ejection fraction (44.9% ± 6.8) compared to mice treated with vehicle (31.9% ± 6.2; One-way ANOVA, N=5-6, p<0.05). Additionally, mice treated with IFNAR1 ab showed significant improvements in left ventricular end systolic volume (LVESD) (3.16mm ± 0.52) compared to vehicle treated mice (3.89mm ± 0.40; One-way ANOVA, N=5-6, p<0.05). Mice treated with H151 showed significant improvement in ejection fraction (43.8% ± 7.1) compared to mice treated with vehicle (31.9% ± 6.2; One-way ANOVA, N=5-6, p<0.05). Treatment with H151 or IFNAR1 ab reduced scar size (7.22% ± 3.6; 7.88% ± 3.1%) compared to mice treated with vehicle (9.74% ± 3.7; N=5-6).

Conclusion:
These data show that targeting the early IFN response in MI/R can improve cardiac function, and provide a basis for studies identifying the effects of the neutrophil specific STING and type I IFN activation following MI/R. If correct, this pathway could be a powerful and clinically feasible point of intervention to improve cardiac recovery after MI/R.
Abstract P3072: Effects Of Antiviral Innate Immune Activation On Cytokine Secretion And Function Of Human Ipsc-derived Cardiomyocytes
Authors: Ashok Deepthi , Liu Ting , Prakash Meghana , Kim Jason , Ho Bryan , Papanicolaou Kyriakos , Kim Deok-Ho , O'Rourke Brian
Published: October 2023

Introduction:
Acute and long-term cardiovascular complications of COVID-19 have been reported, ranging from arrhythmias, ischemic heart disease, and heart failure to cerebrovascular and cardiometabolic disorders, yet the underlying molecular mechanisms are not well understood. Here, we examine the effects of stimulating the double-stranded RNA (dsRNA) pattern recognition receptor pathway, which is strongly induced to activate interferon-stimulated gene (ISG) expression in the heart during COVID-19, on excitation-contraction coupling and mitochondrial function. While providing antiviral protection, we hypothesize that, even in the absence of cardiac viral replication, this pathway might contribute to COVID-19-related cardiac dysfunction.

Results:
We used the viral dsRNA mimetic polyinosinic: polycytidylic (PIC) to induce a type I interferon response in iPSC-CM or A549 (lung epithelial) cells. In iPSC-CM, PIC induced robust protein expression of STAT1/pSTAT1, OAS2, OAS3, IRF7, pIRF7, IRF9 and MX1. A marked increase in cytokine protein secretion was also observed, with significant increases in CXCL-10 (~160 fold), IL6, CCL5 (~20-fold) IL8 (15-fold), and CXCL1 (~10-fold), and a 2-5-fold increase in ~30 other cytokines. Negative effects on excitation-contraction coupling were observed after 72 hours of PIC treatment, manifested as a 25% reduction in iPSC-CM Ca2+ transient amplitude. Microelectrode array analysis of iPSC-CM monolayers revealed electrophysiological dysfunction: PIC treatment increased cardiac field potential duration and spike amplitude, but reduced beat periods. A549 cells also showed increases in interferon pathway genes (STAT1, pSTAT1, IRF3, pIRF3, OAS1,2,3 and IFNb1), along with increased rates of RNA degradation after PIC treatment, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. PIC-treated A549 cells also showed a large decrease in oxidative phosphorylation (maximal respiration and spare respiratory capacity), which was prevented by the mitochondrial antioxidant, mitoTEMPO.

Conclusions:
Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, might contribute to mitochondrial and cardiac dysfunction in COVID-19.
Abstract P3074: Alkylating Chemotherapy Reshapes The Immune Landscape Of The Heart
Authors: He Ruijun , Lavine Kory J
Published: October 2023

Cancer survivors typically have concomitant cardiac comorbidities and were exposed to various chemotherapeutic agents. Despite extensive investigation, we still have a poor understanding of how chemotherapy agents impact cardiac physiology and disease pathogenesis. Alkylating agents, for instance, carboplatin, are the backbone of many chemotherapy regimens and have been applied in cancer treatment for decades. Cardiac macrophages are the primary component of the cardiac immune landscape and play essential roles in homeostasis and disease. My data implied that alkylating drugs induced cell death of the reparative cardiac macrophages (CCR2-) through activating apoptosis and necroptosis. However, the pro-inflammatory subset (CCR2+) is not decreased. Intriguingly, the reparative macrophages recovered four weeks after the carboplatin treatment through proliferation but showed different gene expression profiles. To investigate the functional influence of these reshaped macrophages in heart diseases, I established a hypertensive heart injury model and an ischemic heart injury model using carboplatin-exposed mice. Interestingly, these mice showed less fibrosis and smaller infarct size. Further, I used CD169-DTR mice that allow selective ablation of CCR2- macrophages. The attenuated remodeling phenotype is abolished without the reshaped CCR2- macrophages. Moreover, the type I interferon signaling in CCR2- macrophage is significantly upregulated in carboplatin-exposed mice upon hypertensive injury. Using neutralizing antibodies to block the IFN-I signaling, I found heart remodeling in carboplatin-treated mice became more advanced. Together, my study revealed that alkylating chemotherapy reshaped the immune landscape of the heart and mitigated adverse heart remodeling by activating the IFN-I signaling pathway in cardiac resident macrophages. These findings highlighted the role of CCR2- macrophages and the IFN-I signaling in the long-term cardiac effects of alkylating chemotherapy.
Abstract P3075: Neutrophil Secreted Chitinase 3-like-1 Antagonizes Macrophage Reparative Polarization And Contributes To Ventricular Dysfunction After Myocardial Infarction
Authors: Stephan Jonah , Zoubul Taylor , Nong Yibing , Brainard Robert , Wysoczynski Marcin
Published: October 2023

Clinical studies show that chitinase 3-like-1 (CHI3L1) is a biomarker and predictor of all-cause mortality in heart failure. Studies from our lab demonstrated that CHI3L1 is elevated in hearts post-MI, and CHI3L1 deficiency in mice results in preserved ventricular structure and function post-MI. Furthermore, we found that CHI3L1 is secreted from activated neutrophils but has no effect on neutrophil recruitment in hearts after MI. The goal of the current study was to elucidate mechanisms whereby CHI3L1 contributes to exacerbated cardiac dysfunction after MI. Post-MI neutrophil recruitment is followed by the sequential programming of macrophages to pro-inflammatory or pro-reparative phenotypes. We sought to determine the impact of neutrophil-secreted CHI3L1 on macrophage polarization. Activated neutrophils isolated from

Chil1
+/+

and

Chil1
-/-

mice were co-cultured with bone marrow-derived macrophages. We found that macrophages directly co-cultured with

Chil1
-/-

but not

Chil1
+/+

neutrophils showed upregulation of the pro-reparative markers,
Chil4
and
Arg1
(n=5/group, p<0.05). Additionally, direct co-culture of

Chil1
-/-

neutrophils and macrophages showed trends towards downregulation of the pro-inflammatory polarization markers,
Il1b
and
Tnfa
relative to macrophages co-cultured with

Chil
+/+

neutrophils. These data suggest that neutrophil-derived CHI3L1 may antagonize signaling to polarize macrophages towards a pro-reparative phenotype that may explain delayed scar formation and exacerbated ventricular dysfunction after MI. Future efforts will elucidate the signaling role of CHI3L1 associated with neutrophil extracellular traps (NETs) on macrophage function.
Abstract P3106: Sex-specific Limitation Of Cardiac Capacity: Return To Fundamental Structure And Function.
Authors: Guo Meihan , Diaz-Canestro Candela , Montero David
Published: October 2023

Objective:
The physiology underlying the sex-specific limitation of cardiac pumping capacity remains unresolved. This study experimentally manipulated the pressure gradient (PG) driving venous return to evidence whether cardiac structure and/or function explain sex differences in cardiac capacity in young and older individuals.

Methods:
Healthy women and men matched by age and physical activity were included within young (
n
=40, age=25±4 yr) and older (
n
=55, age=60±8 yr) age groups. Cardiac volumes and output (Q) were assessed via transthoracic echocardiography from moderate to peak exercise under two hemodynamic conditions (‘low’ and ‘high’ PG between lower and upper body) generated via opposite PG identically delivered in a lower body pressure chamber. Main outcomes included sex differences in delta (‘high’ ‘low’ PG) left ventricular (LV) end-diastolic volume (ΔLVEDV), stroke volume (ΔSV) and Q (ΔQ).

Results:
In young individuals, ‘high’ PG increased exercise LVEDV and SV in men (
P
≤0.002), but not in women (
P
≥0.562), relative to ‘low’ PG. Accordingly, ΔLVEDV, ΔSV and ΔQ were enhanced in young men compared with young women, specifically at the highest exercise intensities (
P
≤0.019). In contrast, right and left atrial volumes were similarly increased by ‘high’ PG in both sexes (
P
≤0.007). ‘High’ PG exclusively prolonged LV filling time during exercise in young men (
P
≤ 0.036). In older women and men, ‘high’ PG did not modify exercise cardiac volumes (
P
≥0.124) and impaired established markers of LV diastolic function (
P
≤0.049).

Conclusions:
The female young heart is not constrained by venous return or structural factors external to the myocardium. An impaired ability to lengthen LV filling time with augmented venous return may limit female-specific cardiac capacity. With older age, cardiac chambers are not distended and LV diastolic function is impaired with increased PG in both sexes.