bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2121: Relative Importance Of Reactive Oxygen Species From Reverse Electron Transfer At Mitochondrial Complex I In Reperfusion Injury
Authors: Tabata Fukushima Caio , Dancil Ian-Shika , Shah Nidhi , Brookes Paul S
Published: October 2023

The metabolite succinate accumulates during tissue ischemia. In early reperfusion, succinate is rapidly metabolized, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. It is widely believed that the source of succinate-driven ROS in early reperfusion is reverse electron transfer (RET) at respiratory complex I (Cx-I). However, evidence for Cx-I RET ROS is largely based on experimental evidence from isolated mitochondria with succinate as the sole substrate. We hypothesized that numerous other conditions present during early reperfusion may impact Cx-I RET ROS. Thus, experimental perturbations were applied to mitochondria to model early reperfusion, with measurement of ROS via the fluorescent probe PHPA. These conditions included: (i) A high concentration of NADH; (ii) A high concentration of lactate; (iii) A mildly acidic pH; (iv) A non-zero concentration of ADP buffered by a creatine / creatine phosphate / creatine kinase system; (v) Presence of purine nucleotide breakdown products; (vi) Presence of calcium. These studies revealed that under early reperfusion-like conditions mitochondrial ROS generation still occurred via Cx-I RET, but at a magnitude only half that previously reported. Furthermore, residual ROS that was insensitive to a specific Cx-I RET inhibitor (S1QEL) was slightly elevated under early-reperfusion like conditions, and this residual ROS was mostly inhibited by S3QEL, a blocker of ROS from respiratory complex III (Cx-III). Together, these data suggest that the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and that of Cx-III ROS underestimated.
Abstract P2135: A Novel
Pkp2
Mouse Model Of Genetic Arrhythmogenic Right Ventricular Cardiomyopathy And Its Rescue By Gene Therapy
Authors: Xu Emma , Van Pell Melissa , Zeng Aliya , Greer-Short Amara , Li Cindy , Cheng Ze , Wu Iris , Song Xiaomei , Jones Samantha , Ivey Kathy , Yang Jin , Lombardi Laura , Hoey Tim , Yang Jane
Published: October 2023

Background:
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder in which cardiomyocytes lack structural integrity and signaling is compromised resulting in the disruption of normal rhythms and formation of fibrosis. Patients suffer from arrhythmia and are at increased risk of sudden cardiac death and heart failure. Previous work has demonstrated that heterozygous mutations in the desmosomal gene,
plakophilin-2
(
PKP2
), are the most common cause of ARVC. Our objective was to investigate the mechanisms of ARVC development and test a potential ARVC gene therapy strategy using a novel mouse model of Pkp2.

Methods:
A knock-in (KI) mouse model with a point mutation corresponding to human
PKP2
(
PKP2
c.2146-1G>C or
Pkp2
KI) was generated via CRISPR/Cas9 technology. High fat diet (HFD) was used to induce the ARVC phenotype in the
Pkp2
KI heterozygous (het) mice. The
Pkp2
KI het mice were crossed with a cardiac-specific
Pkp2
cKO (αMHC-Cre
ERT
) mouse to generate a tamoxifen inducible model (KIxKO). Cardiac function was assessed by echocardiography and arrhythmia burden by EKG. The KIxKO animals received AAV:PKP2 via retro-orbital injection.

Results:
Pkp2
KI het animals with normal chow had slow and mild ARVC development, and 50% decrease in PKP2 protein expression, with right ventricle (RV) dilation not occurring until animals were 9 months old. Induction with HFD from 8 weeks hastened and worsened the RV dilation. In contrast, the KIxKO mice exhibited >90% decrease in PKP2 protein expression and a correspondingly severe ARVC phenotype starting from 3 weeks after Tamoxifen induction. This included a high arrhythmia burden and both RV area dilation and decreased ejection fraction, indicating the mice eventually progressed to biventricular heart failure. This resulted in 100% mortality in KIxKO animals by 9 weeks post-tamoxifen induction. A single dose of AAV:PKP2 gene therapy (6E13 vg/kg) maintained ejection fraction, prevented ARVC development and improved survival in the KIxKO mice after 9 weeks of treatment.

Conclusion:
The current results demonstrated that AAV:PKP2 gene therapy may be a promising therapeutic approach to treat ARVC patients with
PKP2
mutations resulting in haploinsufficiency.
Abstract P2136: TpTe Interval Behavior And Mortality In Wistar Rats Submitted To Cardiac Ischemia And Reperfusion Treated With The Heparin Fragment Trisulfated Disaccharide
Authors: Vasques Enio R , Vasques Bruna K
Published: October 2023

Introduction:
The Tp-Te ekg interval reflects ventricular transmural dispersion, being used for prediction of ventricular arrhythmia and sudden death. The heparin fragment called Trisulfated Disaccharide (TD), which has no effect on blood coagulation and acts on the sodium-calcium exchanger accelerating the output of calcium from the cell, decreases cytoplasmic calcium concentrations in situations of calcium overload due to ischemia and reperfusion (I-R).

Objective:
To evaluate the behavior of the Tp-Te interval and mortality due to cardiac arest in Wistar rats submitted to surgicaly induced I-R under treatment with TD at reperfusion.

Materials and Method:
Wistar rats (n=8) with average weight of 300g were anesthetized and under assisted breathing were electrocardiographically monitored and submitted to surgical cardiac ischemia by ligation of the anterior descending artery for 10 min, with reperfusion thereafter. The Tp-Te interval was analyzed before ischemia, at 10 min after ischemia induction and at 10 min after reperfusion, The incidence of arrhythmias was determined throughout the recording period. In the study group intravenous (0.2 mg/kg) TD was injected concomitantly with the reperfusion. In the control group (n=8), the same procedure was performed, without the TD infusion.

Results:
During ischemia there was a similar alteration of the Tp-Te interval in both groups when compared to the pre-ischemic phase, with an increase of about 90% in magnitude, as well as the presence of ventricular arrhythmias, without animal mortality. During reperfusion Tp-Te interval returned to normal in the treated group and there was no mortality. In the control group, the magnitude of the Tp-Te increase was maintained. The presence of ventricular arrhythmias was similar, but total atrioventricular block and/or sustained arrhythmia occurred in 50% of the animals, followed by animals death.

Conclusion:
Treatment with TD at the time of reperfusion normalized the Tp-Te interval 10 min after TD infusion as well as reduced mortality in wistar rats submitted to I-R, and may be useful in the protection of organs submitted to the same I-R conditions (transplants, surgical and hemodynamic procedures), where calcium overload is determinant in morbidity and mortality.
Abstract P2138: Long-Term Clinical Value And Impact Of Mavacamten In Symptomatic Obstructive Hypertrophic Cardiomyopathy: A Systematic Review And Meta-Analysis
Authors: Sebastian Sneha Annie , Khan Ubaid , Padda Inderbir , Mahtani Arun , Nasir Muhammad M , Mubariz Muhammad , Iqbal Javed , Johal Gurpreet
Published: October 2023

Introduction:
Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disorder involving the cardiac sarcomere. Mavacamten is the first-in-class oral cardiac myosin inhibitor approved for treating HCM in adults targeting sarcomere hypercontractility. We aimed to evaluate the efficacy and safety of mavacamten in symptomatic obstructive HCM.

Methods:
We conducted a systematic literature search in Embase, PubMed, Scopus, and Web of Science databases from inception until March 05, 2023.

Results:
After screening 349 studies, our final analysis included 5 studies with 843 obstructive HCM patients and an average follow-up of 27.2 weeks. A fixed effects model was used to calculate risk ratios (RRs). Our meta-analysis revealed a significant improvement in NYHA functional class from baseline in patients on mavacamten compared to placebo [RR, 4.59 (95% CI: 2.94-7.16), p<0.00001]. Mavacamten use in obstructive HCM patients also resulted in significantly lower rates of septal reduction therapy (SRT) or patients who were guideline-eligible for SRT versus placebo [RR, 0.29 (95% CI: 0.22-0.39), p<0.00001]. There was no significant effect of mavacamten use on the Kansas City Cardiomyopathy Questionnaire (KCCQ) score (p = 0.22). Also, no significant difference was observed in the occurrence of ≥1 serious adverse event (p = 0.77), atrial fibrillation (p = 0.98), and non-sustained ventricular tachycardia (p = 0.44) between mavacamten and placebo group.

Conclusions:
A substantial improvement in NYHA functional class without any serious adverse events, and a significant reduction in the number of patients requiring SRT, makes mavacamten a promising drug for obstructive HCM.
Abstract P2142: Evaluation Of Novel A3 Adenosine Receptor Agonist In Hypertension
Authors: Zapata-Sudo Gisele , Rocha Bruna , Da Silva Jaqueline S , Montagnoli Tadeu , Pedreira Julia G , Barreiro Eliezer J
Published: October 2023

The search for alternative strategies for treatment of cardiovascular diseases, lead to the design and synthesis of
N
-acylhydrazone derivative, named LASSBio-2062, which is a new ligand of adenosine receptor and that is a possible therapeutic target to reduce blood pressure because of its vasodilatory action and antioxidant properties. This work investigated the effects of a new A
3
adenosine receptor agonist (LASSBio-2062) in spontaneously hypertensive rats (SHR). Animal Care and Use Committee at Universidade Federal do Rio de Janeiro approved the protocols (017/19). Vascular reactivity was evaluated using isometric tension recording of pre-contracted thoracic aorta from male Wistar rats after exposure to increasing concentrations of LASSBio-2062 (0.1 - 100 μM) in the absence or presence of antagonist of adenosine A
2A
receptor, ZM-241385 (0.1 μM), an antagonist of adenosine A
3
receptor, MRE 3008F20 (0.1 μM), a blocker of K
ATP
channel, glibenclamide (10 μM), an inhibitor of Kv channels, 4-AP, inhibitor of BK
Ca
, TEA (3 mM).

Mechanical removal of endothelium increased the EC
50
for vasodilation from 15.5 ± 6.5 to 49.4 ± 10.0 μM, indicating partial dependence on the functional integrity of vascular endothelium. Vascular relaxation was not altered by ZM-241385 but the concentration-response curve shifted to the right with MRE 3008F20 and reduced maximum relaxation from 72.1 ± 4.0 to 23.6 ± 7.7%, indicating that vasodilation occurred by activation of the adenosine receptor type A
3
, but not A
2A
. Reduced maximum vascular response by glibenclamide, TEA and 4-AP suggests that activation of A
3
adenosine receptor promotes hyperpolarization as a result reduces intracellular calcium. Antihypertensive effect was investigated in spontaneously hypertensive rats (SHR, 12-15 wks old), through the measurement of mean blood pressure (MBP) after intravenous injection of 10 and 30 μmol/kg of LASSBio-2062. After intravenous administration of LASSBio-2062 (10 umol/kg) in SHR, systolic and diastolic pressures reduced from 155.1 ± 10.1 to 110.6 ± 8.0 and from 111.0 ± 9.1 to 63.9 ± 14.7 mmHg, respectively. HR was also changed from 248.1 ± 12.1 to 146.5 ± 21.0 bpm. In conclusion, the
N
-acylhydrazone, LASSBio-2062 is a potential therapeutic agent to treat arterial hypertension.
Abstract P2143: New Rock Inhibitor Improves Pulmonary Vascular Resistance And Right Ventricle Function In Animal Model Of Pulmonary Arterial Hypertension
Authors: Zapata-Sudo Gisele , Montagnoli Tadeu , Da Silva Jaqueline S , Rocha Bruna , Santos-Carlos Bianca , Fraga Carlos
Published: October 2023

Pulmonary arterial hypertension (PAH) is characterised by increased pulmonary arterial pressure and vascular thickening, which causes RV dysfunction and failure. Current therapies lack efficacy for preventing disease progression and lead to poor survival rate in patients. Rho-activated protein kinases (ROCK) play a key role in smooth muscle contraction and proliferation and cardiomyocyte hypertrophy and survival, which indicate the potential of ROCK inhibitors to treat PAH. This work aims to assess the efficacy of a novel ROCK inhibitor, named LASSBio-2065, on cardiac and vascular function in monocrotaline (MCT)-induced PAH. Intraperitoneal injection of 60 mg/kg MCT induced PAH in Wistar rats, which protocol was approved by the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. After 2 wks, echocardiography confirmed the PAH and rats were randomly divided in groups and treated with either vehicle or LASSBio-2065 (60 μmol/kg/day, i.p.) for 14 days. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and RV catheterization. Tissues were collected for histologic and morphometric analysis. Data expressed as mean ± SEM was analysed using One-way ANOVA followed by Dunnett post-test. LASSBio-2065 improves pulmonary vascular resistance and RV afterload because the ratio of pulmonary acceleration time-to-total ejection time reduced from 26.7 ± 2.0 to 21.7 ± 1.6 (p< 0.05) and RV output from 64.2 ± 6.1 to 39.1 ± 8.0 (p<0.05). Vascular and cardiac remodelling were prevented by LASSBio-2065, since medial wall thickness of distal pulmonary arterioles reduced and short axis RV-to-LV internal area increased from 85.8 ± 11.4 to 115.2 ± 17.8 (p<0.05). The new ROCK inhibitor also improved the RV diastolic function, restoring both RV end-diastolic pressures increasing from 5.3 ± 0.8 to 11.1 ± 1.9 mmHg (p<0.05) and Tau from 16.9 ± 2.2 to 27.1 ± 2.8 (p<0.05). In conclusion, LASSBio-2065 significantly improved RV function in MCT-induced PAH. In conclusion, the new ROCK inhibitor represents a potential alternative for management of PAH-associated RV dysfunction and remodelling.
Abstract P2159: Naltrindole Attenuates Phorbol 12-myristate 13-acetate Induced Superoxide Release In Polymorphonuclear Leukocytes Independently Of Delta Opioid Receptor Antagonism
Authors: Johnson Devani , DiLisi Taylor , Melnik Juliet , Pinto Denise , Barrera Alexandra , Nair Arjun , Talukder Zinya , Dean Tameka , CHEN Qian , Barsotti Robert , Young Lindon
Published: October 2023

Naltrindole (NTI) is a selective delta opioid receptor antagonist that recently has been shown to be cardioprotective in both
in vivo
and
ex vivo
studies.
In vivo,
NTI showed cardioprotective effects that were dose-dependent significantly reducing infarct size up to 82% compared to controls subjected to ischemia-reperfusion (I/R) injury (p<0.05). In
ex vivo
isolated rat I/R hearts, NTI significantly reduced infarct size compared to controls (p<0.01). NTI restored cardiac left ventricular end diastolic pressure to near baseline values compared to controls or other general opioid receptor antagonists in
ex vivo
hearts, suggesting a tissue salvaging mechanism independent of opioid delta receptor antagonism. Polymorphonuclear leukocytes (PMNs) do not express opioid receptors. In this study, we investigated NTI attenuation of PMN superoxide (SO) release
in vitro
. We hypothesized that NTI would attenuate PMN SO release via a calcium handling mechanism, a similar mechanism that explained cardioprotective effects of NTI pretreatment mitigating ischemic hypercontracture due to an increase in intracellular calcium. Rat PMNs (5x10
6
) were incubated for 15 min at 37
o
C in the presence or absence (dH
2
O vehicle controls) of NTI (10μM, 50μM, or 200μM). PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after phorbol 12-myristate-13 acetate (PMA) stimulation (100nM). Cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of the assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test. NTI significantly decreased PMN SO release from 30 sec to 420 sec. Changes in final absorbance of NTI 200μM (0.244±0.07, n=8, p<0.05) and 50μM (0.293±0.06, n=4, p<0.05), but not NTI 10μM (0.412±0.07, n=5), were significant compared to controls (0.487±0.12, n=9). Cell viability was not significantly different compared to PMA across all NTI concentrations. These results suggest that NTI reduces I/R injury and PMN SO release, by a means independent of delta opioid receptor antagonism. Future studies will assess NTI SO attenuation in human umbilical vein endothelial cells. We will use different concentrations to investigate optimal concentration response.
Abstract P2161: Stem Cell-derived Exosome Nebulization Therapy (SCENT) Improves Cardiac Function And Tissue Repair In Mice And Pigs
Authors: Li Junlang , ZHU DASHUAI , Huang Ke , Li Yuan , Cheng Ke
Published: October 2023

Stem cell-derived exosomes show promise as therapeutics for cardiac repair after injury. However, due to their short half-life in vivo, effective treatment typically requires repeated dosing. Previous methods for delivering exosomes to the heart involve invasive injection routes. Here, we report a noninvasive and repeatable approach to exosome delivery via inhalation after myocardial infarction (MI), called Stem Cell-derived Exosome Nebulization Therapy (SCENT). In a mouse model of MI, exosomes were recruited to the ischemic heart after repeated dosing via nebulization. SCENT ameliorated cardiac repair by significantly improving left ventricular function, reducing infarct size, and promoting cardiomyocyte proliferation. Further investigation via single-cell RNA sequencing of the mouse heart after SCENT revealed a significant downregulation of CD36 in endothelial cells (ECs). In an EC-
Cd36
fl/-
mouse model, we found that inhibiting CD36, a fatty acid transporter in ECs, led to a compensatory increase in glucose utilization in the heart and higher ATP generation, resulting in enhanced cardiac contractility. In a pig model of acute MI, we evaluated functional outcomes using cardiac magnetic resonance imaging (MRI) and found that SCENT led to reduced left ventricular (LV) end-systolic volume (ESV) at day 28 (19.07 ± 3.22 ml) compared to controls (23.60 ± 3.06 ml). Additionally, pigs treated with SCENT exhibited increased LV ejection fraction (EF) (59.36 ±2.84%) and fractional shortening (FS) (29.56 ± 1.25%), further confirming cardioprotection in pigs. Our results pave the way for development of a non-invasive exosome delivery method for rescuing heart injury.
Abstract P2163: The Genetic And Pharmacological Inhibition Of Classic Transient Receptor Potential Channels 3 And 6 (trpc3/c6) Improve Pulmonary Arterial Hypertension.
Authors: Moriuchi Kenji , Nakagawa Yasuaki , Kinoshita Hideyuki , Inazumi Hideaki , Yanagisawa Hiromu , Kanamori Takahiko , Nishikimi Toshio , Oya Miku , Nishida Motohiro , Mori Yasuo , Nagata Ryu , Nakamura Kazufumi , Kuwahara Koichiro , Nakao Kazuwa , Kimura Takeshi
Published: October 2023

Background:
Pulmonary artery hypertension (PAH) is a progressive and fatal disease. Currently, there are only three types of PAH drugs, targeting the endothelin pathway, NO pathway, and prostacyclin pathway, and all these drugs are pulmonary vasodilators. However, the pathogenesis of PAH develops through various mechanisms, including vascular endothelial dysfunction, pulmonary artery smooth muscle cell (PASMC) proliferation, fibrosis and inflammation as well as vasoconstriction. Recently, calcium signaling has been reported to be involved in these multiple mechanisms in PAH. Classic transient receptor potential channel 3 and 6 (TRPC3/C6), known as receptor-operated calcium channels, have been reported to be involved in the pathogenesis of PAH.

Methods and Results:
Initially, we evaluated TRPC3 knockout mice (TRPC3KO), TRPC6 knockout mice (TRPC6KO), and TRPC3/C6 double-knockout mice (dKO) in the PAH models, to evaluate the effects of genetic inhibition of TRPC3/C6. All of TRPC3KO, TRPC6KO, and dKO have weakened PAH phenotypes such as the systolic pulmonary artery pressure, the right ventricular hypertrophy, and the tunica media thickening of the pulmonary artery in monocrotaline pyrrole-induced PAH models. On the other hand, in the hypoxia-induced PAH model, TRPC6KO and dKO ameliorated these PAH phenotypes, but TRPC3KO didn’t. Next, we evaluated the effect of pharmacological inhibition of TRPC3/C6 in PAH model rats. We have developed a novel selective TRPC3/C6 inhibitor, L862. This compound showed excellent pharmacokinetic and safety properties. L862 ameliorated monocrotaline-induced and hypoxia-induced PAH in rat models on prevention protocol. Moreover, L862 significantly suppressed PDGF-BB-induced proliferation of human PASMC derived from idiopathic PAH patients in a dose-dependent manner. L862 also improved PAH in Sugen/hypoxia rat model on the therapeutic protocol.

Conclusions:
Both genetic and pharmacological inhibition of TRPC3/C6 ameliorated PAH in several PAH models. These results suggested that TRPC3/C6 had essential roles in the pathogenesis of PAH and L862 had potential clinical application.
Abstract P2166: Cardioprotective Effect Of Angiotensin Converting Enzyme 2 Activation In Hesc-derived Cardiomyocytes Under Diabetic Condition
Authors: Cho Sung Woo , Sung Ji Hee , Han Jin , Kim Hyoung Kyu
Published: October 2023

Diabetes mellitus (DM) is on the rise and has quickly become one of the most common and expensive chronic diseases in the world. DM and cardiovascular disease (CVD), the leading cause of morbidity and mortality in diabetic patients, have a close relationship. Diabetic cardiomyopathy (DCM) is a phenomenon comprising the deterioration in cardiac function and structure independent of coronary artery disease and other causes. However, there is still no specific and effective treatment of DCM. We investigated the cardioprotective effect of pharmacological activation of angiotensin converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE) in DCM in vitro model by using human embryonic stem cell (hESC)-derived cardiomyocytes under diabetic condition. In hESC-derived cardiomyocytes under diabetic condition, DIZE decerased the cardiomyocyte size and stabilized beating phenotypes. Furthermore, DIZE reduced the HF, cardiac hypertrophy, fibrosis, oxidative stress related gene expression, and ROS level and increased the mitochondrial function related gene expression and mitochondrial membrane potential. In transcriptome analysis, SERPINE1/PAI-1 and CCN2/CTGF are highly upregulated after DIZE treatment and might be candidate target genes of ACE2 activation. Collectively, ACE2 activation have a direct cardioprotective effect in hESC-derived cardiomyocytes under diabetic condition and can be a potential therapeutic target of DCM.
Abstract P2171: Lentivirus TAZ Gene Therapy Restores Metabolic Functions In Taz Ko Mice Model Of Barth Syndrome
Authors: Raghav Rahul , Awata Junya , Martin Gregory , Strathdee Douglas , Blanton Robert M , Chin Michael T
Published: October 2023

Introduction:
Barth Syndrome is an X-linked disorder caused by mutation in thegene encoding tafazzin (or
TAZ
), characterized by dilated cardiomyopathy, muscularhypotonia and cyclic neutropenia.
TAZ
is a mitochondrial acyltransferase that generatesmature cardiolipin (CL) from monolysocardiolipin (MLCL) and is essential formitochondrial morphology and function. Patients with BTHS exhibit a characteristicreduction in the levels of CL and an accumulation of MLCL. Currently, there is no targetedtreatment exists for these patients. We characterized a global
TAZ
knockout mousemodel of BTHS and tested the efficacy of Lentivirus-mediated
TAZ
gene therapy.

Methods:
We generated third generation lentiviral constructs without an insert, withGreen Fluorescent Protein, with recombinant tafazzin and with recombinant tafazzinmodified to contain a cellular penetrating peptide (hTAZ-CPP) that promotes uptake intotafazzin-deficient cells. We tested the efficacy of these recombinant lentiviruses, first inmouse embryo fibroblasts (MEF) isolated from tafazzin knockout mice and then intafazzin knockout mice after systemic administration.

Results:
We have found that treatment with hTAZ-CPPs reduces the MLCL/CL ratio tonear wild type levels, thereby rescuing the defect in cardiolipin remodeling in TAZ KOMEFs. Serial echocardiograms revealed significant increase in septal wall thickness andsignificant decrease in ejection fraction, fractional shortening and global longitudinal strainin TAZ KO mice. The TAZ KO mice were treated systemic lentivirus at 5 weeks of ageand then echocardiograms were performed at every 4 weeks until 16 weeks. After thetreatment septal wall thickness continued to increase and ejection fraction, fractionalshortening and global longitudinal strain continued to decrease in the empty virus or GFPtreated mice, whereas they are remained stable in those treated with hTAZ-CPPs.

Conclusion:
These findings suggest that Lentivirus-mediated
TAZ
gene replacementtherapy enhanced with cell penetrating peptides has potential as a future treatment forBTHS.
Abstract P2192: Growth Hormone-releasing Hormone Receptor Antagonist Attenuates Cardiopulmonary Injury Induced By A Bsl2 Rvsv-sars-cov-2-s Virus Mimic Of Sars-cov-2 Infectivity
Authors: Condor Capcha Jose Manuel , Kamiar Ali , Robleto Emely , Cui Tengjiao , Saad Ali , Wong Amanda , Villano Jason , Pekosz Andrew , Ranek Mark J , Webster Keith A , Schally Andrew , Jackson Robert M , Shehadeh Lina A
Published: October 2023

Background:
COVID-19 causes severe pulmonary injury that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to modulate cellular immune responses to bleomycin lung injury and decrease inflammation in models of sarcoid granuloma. Using the BSL2-friendly rVSV-SARS-CoV-2-S of K18 hACE2tg mice to mimic native SARS-CoV-2 infection, we tested our hypothesis that MIA-602 attenuates cardiopulmonary injury in this COVID-19 model.

Methods:
Male and female K18 hACE2tg mice were inoculated with SARS-CoV-2 Washington (WA-1) native strain, recombinant VSV-SARS-CoV-2-Spike virus (rVSV-SARS-CoV-2-S), or PBS and lung viral load, weight loss and histopathology compared between groups (N=5-8). K18 h
ACE2
tg mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed; and blood and tissues collected for histopathological analyses, H&E staining and ICAM-1 immunohistochemistry. T-test or One-way ANOVA-test was used for statistical analysis.

Results:
Native SARS-CoV-2 and rVSV-SARS-CoV-2-S presented with similar patterns of weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung perivascular inflammation and pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued respiratory rate, increased expiratory parameters (Te, PEF, EEP) and mitigated dysregulated measures of airway obstruction (Penh and Rpef) compared to vehicle.

Conclusions:
rVSV-SARS-CoV-2-S is an accurate and safe alternative to native SARS-CoV-2 for preclinical studies. Daily treatment with the synthetic peptide GHRH-R antagonist MIA-602 attenuates pulmonary dysfunction and heart inflammation in this new preclinical mouse model of COVID-19 pneumonia. We hypothesize that the molecular mechanism involves anti-inflammatory actions of MIA-602.
Abstract P3007: Rosuvastatin-eluting Gold-nanoparticle-loaded Perivascular Wrap For Enhanced Arteriovenous Fistula Maturation In A Murine Model
Authors: Martin Benjamin M , Klusman Carleigh L , Perez Joy Vanessa D , Barcena Allan John R , Bernardino Marvin R , San Valentin Erin Marie D , Damasco Jossana A , Del Mundoa Huckie C , Court Karem A , Godin Biana , Fowlkes Natalie , Bouchard Richard , Cheng Jizhong , Huang Steven Y , Melancon Marites P
Published: October 2023

Background:
Arteriovenous fistulas (AVFs) are a vital form of arteriovenous access for patients requiring hemodialysis, but they are linked to overall morbidity and mortality when they fail to mature. The most common cause of AVF non-maturation is neointimal hyperplasia (NIH). To minimize the effects of NIH, a perivascular wrap composed of polycaprolactone (PCL), rosuvastatin (ROSU), and gold nanoparticles (AuNPs) was created.

Objectives:
This study assessed the impact of ROSU-eluting, radiopaque resorbable perivascular wraps on pathologic NIH in a chronic kidney disease (CKD) rodent model of AVF.

Methods:
Electrospun PCL wraps containing AuNPs and/or ROSU were monitored for in vitro AuNP release, ROSU elution, and cell viability. The wraps were implanted around an AVF in a CKD rodent model for in vivo ultrasound (US) and micro-computed tomography (mCT) imaging. AVF specimens were collected for histological analyses.

Results:
Cell viability was preserved in the presence of both AuNP- and ROSU-containing wraps. In vitro release of ROSU and AuNPs correlated with in vivo findings of decreasing radiopacity on mCT over time. AuNP-loaded wraps had increased radiopacity compared with PCL-alone wraps and control AVFs without wraps. The addition of ROSU decreased US and histologic measurements of NIH.

Conclusions:
The reduced NIH seen with ROSU-loaded perivascular wraps suggests a synergistic effect between mechanical support and anti-hyperplasia medication. Furthermore, AuNP loading increased wrap radiopacity. Together, our results show that AuNP- and ROSU-loaded PCL wraps induce AVF maturation and suppress NIH while facilitating optimal implanted device visualization.
Abstract P3010: The C10X Polymorphism In The
CARD8
Gene Is Associated With The Levels Of CCL20 And IL-6 Proteins In Healthy Young Men
Authors: Fransén Karin , Hiyoshi Ayako , Hurtig-Wennlof Anita
Published: October 2023

Introduction:
The Caspase Activation and Recruitment Domain 8 (CARD8) protein is a component of innate immunity which has been associated with regulation of proteins involved in inflammation. We have previously identified CARD8 to act as a regulator of cytokines and chemokines in atherosclerotic lesions, and via knockdown of
CARD8
in human umbilical vein endothelial cells (HUVECs).

Aim:
The present study examines the role of the genetic variation rs2043211 in the
CARD8
gene (encoding C10X variant) in relation to a selection of markers of inflammation (Olink inflammation panel; https://olink.com). The
CARD8
polymorphism is assumed to mimic the knockdown of
CARD8.

Methods:
DNA was extracted from blood samples from 233 Swedish young healthy men (aged 18.0-25.9 years), collected in the Lifestyle, Biomarkers and Atherosclerosis (LBA) cohort, Örebro University, Sweden. Genotyping of the
CARD8
polymorphism was performed with TaqMan real time PCR on DNA from blood samples from young healthy men (
n
=233). Protein levels across genotypes were analyzed by additive model (TT vs TA vs AA) by analysis of variance (ANOVA). The recessive (TT + TA vs AA) and dominant (TT vs TA + AA) models were analyzed by t-test.

Results:
The minor (A) allele of the rs2043211 polymorphism in the
CARD8
gene was associated with lower levels of CCL20 and IL-6. The dominant model resulted in p= 0.023 and p= 0.037 for CCL20 and IL-6, respectively. The association remained significant also after adjustment for age and potential intermediate variables (Body Mass Index, low density lipoprotein cholesterol, triglycerides, insulin, and systolic blood pressure). The results agree with previous observations in other cell types.

Conclusions:
We have shown an association between the polymorphism rs2043211 encoding a truncated variant of the
CARD8
gene and lower levels of CCL20 and IL-6 levels in men. Our data indicate that CARD8 may be involved in the regulation of these proteins. Additional
in vitro
studies are needed to reveal the functional mechanistic effects of the polymorphism. Future longitudinal studies of the polymorphism are warranted to elucidate the clinical effect of these proteins.
Abstract P3017: TNF-α-mediated Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Expression In Endothelial Cells
Authors: Kleefeldt Florian , Götz Lisa , Rueckschloss Uwe , Ergün Süleyman
Published: October 2023

The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a central regulator of vascular physiology. Previously we have shown that progressive age leads to a mutual upregulation of vascular CEACAM1 and TNF-α expression that maintains a chronic pro-inflammatory milieu in the vasculature. This age-dependent CEACAM1 upregulation is crucial to vascular aging processes, e.g. oxidative stress and arterial collagen deposition. In the current study, we analyzed the signaling pathways that are involved in the TNF-α-mediated CEACAM1 expression. Using endothelial cell cultures, we conducted pharmacological as well as biochemical experiments. We found that the upregulation of endothelial CEACAM1 expression in response to TNF-α stimulation shows a biphasic pattern. Inhibitor studies point to an early response that is mediated by activation of NFκB, whereas at later time-points the persistent upregulation might depend on Catenin-driven expression. Our data support a novel mechanism of CEACAM1 expression regulation. Especially in inflammatory settings with enhanced TNF-α signaling, i.e. atherosclerosis or cancer that regulation might significantly be involved in the pathology.
Abstract P3029: The Role Of Fatty Acid Synthase In The Modulation Of Vascular Smooth Muscle Cells Into A Foam Cell Phenotype
Authors: Bogan Bethany J , Williams Holly C , Holden Claire , Rezvan Amir , San Martin Alejandra
Published: October 2023

Background:
The contractile phenotype of differentiated VSMCs is essential for vascular function. When exposed to Cholesterol (CHO), VSMCs dedifferentiate and acquire characteristics of foam cells, lipid-laden macrophage-like cells that comprise the Atherosclerotic plaques. CHO uptake by VSMCs is either removed by ABCA1-mediated efflux or is stored in lipid droplets as cholesterol esters (CEs). CEs are formed by the reaction of CHO with fatty acids catalyzed by SOAT1. Thus, we hypothesize that fatty acid biosynthesis controls the formation of VSMC-derived foam cells.

Results:
In VSMCs, CHO treatment upregulates the lipogenic enzymes FASN (3.6±1 vs. 9.0±0.9 AU p<0.01) and acetyl-CoA carboxylase (ACC) (2.8±0.5 vs. 10.2±2.3 AU p=0.02) and induces the phosphorylation of ATP Citrate Lyase (ACLY) (1.4±0 vs. 6.2±1 AU). Consistent with the literature, these changes are accompanied by increased expression of the macrophage-related markers CD68 and KLF4. Significantly, downregulation of FASN inhibits the CHO-induced CD68 (9.1±0.8 vs. 4.6±1,9 p=0.002) and KLF4 (p=0.02) expression upregulation. FASN-deficient VSMCs failed to accumulate lipids and transition to a foam cell phenotype after CHO treatment measured by Oil Red O staining. Interestingly, FASN-deficient VSMCs have decreased expression of SOAT1 (p=0.04) and increased expression of the CHO efflux transporter ABCA1 (p=0.04).

Conclusion:
Our study shows lipogenic enzymes are upregulated during the VSMC to foam cell transition triggered by CHO treatment. FASN expression is required for VSMC-derived foam cell formation. Our data suggest that FASN participates in the esterification of CHO and foam cell formation and may contribute to atherosclerotic plaque accumulation. Thus, FASN may represent a novel target for therapeutic intervention.
Abstract P3031: The Cross Talk Between Increased Abdominal Adiposity And Calcification Of Aortic Valve
Authors: Bouchareb Rihab
Published: October 2023

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations affecting nearly 2.5% of individuals over 65. Currently, there is no viable pharmacological treatment to stop the disease's progression or activate mineral regression. The only effective therapy to treat CAVD is surgical valve replacement or trans-catheterization. Obesity is the major common nutritional disorder worldwide and is one of the important risk factors for CAVD. It has been shown that DLK1-/- mice have increased abdominal adiposity. Indeed Dlk1 plays an essential role in regulating adipocytes differentiation and proliferation. Recently, it has been shown that there is an association between fat tissue accumulation and an increased risk of aortic stenosis. We hypothesize that dysregulation of DLK1 stimulates adiposity, increases circulating lipids, enhances valve lipids retention, and stimulates calcification. Herein, our objective is to examine the expression of DLK1 in calcified aortic valves and its impact on lipid retention.

Method:
We examined the effect of Dlk1 loss of function on lipid retention in aortic valve tissue in ApoE-/- Dlk1-/- mice fed with a high-fat diet and in human explanted aortic valves using immuno-staining.

Results:
The complete deletion of DLK-1 in ApoE-/- mice were associated with an increased oxidized-LDL deposit in aortic valve tissue. In addition, circulating cholesterol (420 ± 8 mg/dl vs 250± 12 mg/dl, P=0.002) and triglyceride concentration (200 ± 3 mg/dl vs 50± 10 mg/dl, P<0.001) were higher in ApoE-/- DLK1-/- compared to apoE-/- mice. An increased valvular calcification accompanied these changes, as shown by the over-expression of RUNX2 and the increased aortic jet velocity in ApoE-/- DLK1-/- mice. Interestingly the overexpression of DLK1 in isolated human valve cells decreased calcification and lipid retention in vitro.

Conclusion:
This study showed the significant role of Dlk1-derived abdominal adiposity in valve-lipid retention and calcification.
Abstract P3034: Placental Lymphatics In Pregnancy And Preeclampsia
Authors: Niclou Benoit , Ginn Sydney , Wang Lanfang , Joseph Giji , Levit Rebecca D
Published: October 2023

Background:
Preeclampsia is a disease of pregnancy that carries significant maternal morbidity and mortality worldwide with 16% of maternal deaths globally attributed to gestational hypertension. The pathogenesis of preeclampsia is thought to result from inadequate vascular remodeling during placentation with many research efforts focused on studying the anti-angiogenic and pro-inflammatory processes that occur in response to placental ischemia.

Hypothesis:
Given the known role of lymphatics in mediating inflammation, we hypothesize that insufficient lymphatic proliferation contributes to abnormal placentation and that augmenting lymphangiogenesis may reduce the severity of the disease.

Methods:
To test this hypothesis, we performed a systematic analysis of human placentas, as well as parietal and basal deciduas. Using immunohistochemistry we stained for lymphatic-specific markers including podoplanin, VEGFC and pan-endothelial marker CD31 and quantified their distribution and morphology.

Results:
Placentas revealed extensive podoplanin staining within the reticular networks of chorionic villous stromal cells. These same, podoplanin-positive villi, revealed the presence of VEGFC-positive structures. A similar staining pattern was observed in the basal deciduas, where podoplanin-positive decidual cells were found in close proximity to VEGFC-expressing cells. Parietal deciduas on the other hand revealed large lymphatic vessels in the vicinity of the fetal membranes.

Conclusion:
The abundance of lymphatic specific markers in the placenta and basal decidua hint at the potential existence of a lymphatic-like network within these structures but further functional analyses need to be conducted to understand their role. The dilated lymphatics found in the parietal decidua represent a concrete opportunity to compare lymphatic vessels between preeclamptic and healthy parietal deciduas. An overall better understanding of lymphatics in these tissues may offer new therapeutic strategies for a disease that is in desperate need for adequate treatment options.
Abstract P3040: Development Of A Protein Synthesis Network At The Sarcoplasmic Reticulum In Adult Cardiomyocytes
Authors: Hofmann Christoph , Brandenburg Sören , Uhlenkamp Dennis , Meyer Bianca , Sandmann Clara , Herzog Nicole , Groß Julia , Sramek Viviana , Renz Marc , Katus Hugo A , Lehnart Stephan E , Völkers Mirko , Doroudgar Shirin
Published: October 2023

Introduction:
The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. In striated muscle cells, a specialized membrane system known as the sarcoplasmic reticulum (SR) sequesters Ca
2+
and carries key functions in excitation-contraction coupling. Textbooks often define the SR as the ER of muscle, but surprisingly, the biochemical and physiological relationship between the ER and SR and even whether they are distinct membrane networks or refer to the same structure remain unknown.

Methods:
To characterize the relationship between the ER and SR, we applied biochemical and imaging techniques, including immunofluorescence and election microscopy, fluorescence
in situ
hybridization, and STimulated Emission Depletion (STED) microscopy to ventricular myocytes, which allowed us to follow the differentiation of these membrane compartments in developing cardiomyocytes.

Results:
Using localization and expression levels of SERCA2, RyR2, KDEL and different markers of ribosomes, mRNA, and the secretory pathway, we found that the membrane compartment that exists in the early postnatal stage, resembles mostly the ER, and this membrane system decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiomyocytes, replacing the ER, in all but a small perinuclear region. Both sites which appear to be interconnected, perform specialized tasks, with protein secretion through the classical ER-Golgi secretory pathway originating mainly from the perinuclear ER and Ca
2+
-dependent excitation-contraction coupling originating exclusively from the SR. Other functions, such as Ca
2+
intake or posttranslational protein processing occur in both compartments. Importantly, we show that in adult cardiomyocytes ribosomes are not only attached to the ER but also to the SR, where they actively perform mRNA translation and are major sites of protein synthesis.

Conclusion:
The ER and SR are two distinct coexisting membrane networks in cardiomyocytes that undergo molecular differentiation and structural organization during cardiac muscle development. Significant amounts of protein synthesis occur in adult cardiomyocytes at the SR and may serve localized mRNA translation near the sarcomere.
Abstract P3064: Cytotoxic T-cells Drive Doxorubicin Induced Cardiac Fibrosis And Systolic Dysfunction
Authors: Bayer Abraham L , Zambrano Maria , Elwell Hannah , Smolgovsky Sasha , kaur Kuljeet , Asnani Aarti , Alcaide Pilar
Published: October 2023

Background:
Doxorubicin (DR), the most commonly prescribed chemotherapy, results in dose-dependent cardiotoxicity and heart failure (HF), and patients respond less frequently to standard HF treatments. DR causes oxidative stress and myocyte death, both potent inducers of a T-cell immune response and cardiac inflammation in other causes of HF. However, mechanistic depth on the immune response elicited by DR is limited. We
hypothesized
that an aberrant T-cell immune response secondary to cardiac damage by DR contributes to DR-induced HF and cardiac remodeling.

Methods and Results:
We treated wild type (WT) and T-cell deficient (

Tcra
-/-
)

mice with 5.0 mg/kg DR or PBS weekly by intraperitoneal injection for 4 or 8 weeks. We found at both time points

Tcra
-/-

mice had preserved ejection fraction (EF) by echocardiography and decreased cardiac fibrosis by picrosirius red staining, compared to WT. At 8 weeks,

Tcra
-/-

mice had fewer TUNEL
+
cardiac cells than WT mice. DR treated mice had increased frequency of activated effector CD8
+
CD62L
lo
CD44
hi
cells in the cardiac draining lymph nodes, increased circulating CD8
+
T-cells, and cardiac CD8
+
T-cell infiltration at both time points. Histologic analysis demonstrated localization of CD8
+
T-cells adjacent to cardiac fibroblasts (CFB) in the hearts of DR treated mice. Targeted antibody depletion of CD8
+
but not CD4
+
cells prior to 4 week DR treatment resulted in improved EF and decreased cardiac fibrosis. Moreover, cardiac CD8
+
T-cells expressed higher IFNγ
in vivo
in DR compared to PBS treated mice, and DR hearts had increased gene expression of the IFNγ inducible chemokines
Cxcl9
and
Cxcl10
.
Ex vivo
DR (0.1 μg/mL) treatment of CD8
+
T-cells during TCR activation with αCD3/CD28 increased IFNγ expression at the protein and transcript level. The same dose of DR also increased surface expression of ICAM-1 on CFB, and CD8
+
T-cells adhered to DR treated CFB
in vitro
in greater numbers than untreated CFB, and induced more αSMA expression. Lastly, in patients treated with DR, plasma levels of CXCL9 and CXCL10 correlated with decreases in EF after 3 months of treatment.

Conclusion:
These data outline a novel CD8 T-cell-driven inflammatory mechanism in DR cardiotoxicity, driven by IFNγ-inducible chemokines in animal models and patients.
Abstract P3082: Loss Of Myeloid Cell-specific Beta2-adrenergic Receptor Improves Cardiac Function After Acute Ischemia
Authors: Nayak Tapas K , Bajpai Anamika , Carter Rhonda , Patwa Viren , Gao Erhe , Tilley Douglas G
Published: October 2023

Heart failure (HF) is one of the leading causes of global mortality, which is often caused by acute myocardial infarction (MI). This type of acute cardiac injury promotes robust inflammatory cascades by regulating spatiotemporal myeloid cell (neutrophil, monocyte and macrophage) responses in the infarct area, which are essential in the orchestration of post-infarct tissue remodeling and repair. We previously reported that deletion of β2-adrenergic receptor (β2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact of myeloid cells in particular. To investigate this, we crossed floxed β2AR (β2
f/f
) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific β2AR knockout mice (LB2) and subjected them to MI. M-mode echocardiography indicated a preservation of ejection fraction (%EF) and fractional shortening (%FS) in the LB2 mice versus either β2
f/f
or LysM-Cre mice at 3 days post-MI, which was maintained at 28 days post-MI, concurrent with reduced infarct size and interstitial fibrosis, suggesting a cardioprotective role of myeloid cell-specific β2-AR deletion. While immune cell profiling by FACS analysis showed similar CD45
+
CD11b
+
Ly6C
+
monocyte, CD45
+
CD11b
+
Ly6G
+
neutrophil and CD45
+
CD11b
+
F4/80
+
macrophage accumulation in LB2 and β2
f/f
mouse hearts at 1 day post-MI, neutrophil frequency was significantly reduced in LB2 mouse hearts by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for efferocytosis of dead cells. Notably, RT-qPCR analysis revealed a significant upregulation of pro-efferocytosis genes in LB2 bone marrow cells, including annexin A1 (ANXA1). Additionally, ANXA1 protein expression was shown to be upregulated in LB2 versus β2
f/f
cardiac CD11b
+
myeloid cells, including macrophages, at 1-day post-MI. In vitro efferocytosis assay data suggested that there was significant increment in %-efferocytosis of LB2 neutrophils as compared to β2
f/f
. Taken together, our data suggest that β2AR is a crucial regulator of myeloid cell-dependent efferocytosis in the heart following injury and suggests a crucial timepoint at which β2AR-selective therapeutics may promote the clearance of dead cells to promote repair.