bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo139: PDE1, 3 and 4 Inhibition and GPCR Agonism in the Regulation of cAMP Signaling
Authors: Fitch Michael , Kelly Evan , Muller Grace
Published: October 2024

Modulators of cardiac cyclic adenosine 3’5’-monophosphate (cAMP) and/or its effector protein kinase A (PKA) are used to treat heart disease. Elucidating the mechanisms of endogenous and pharmacological modulators of cAMP, including phosphodiesterase (PDE) inhibitors, is key to predicating alternative drug therapies for heart failure (HF). PDEs regulate confined cAMP/PKA signaling pathways, depending on PDE isoform and localization. PDE3 regulates the SR and sarcolemmal membrane cAMP pools, controlling calcium influx and efflux at these domains. PDE1 regulates sarcolemmal cAMP/PKA signaling. Despite these differences, inhibitors of either increase heart contractility. However, inhibition of PDE3 is pro-arrhythmic, unlike that of PDE1. The regulation of localized cAMP signaling is key to controlled regulation of cell function, with ramifications for HF, and which GPCR lies upstream of PDE1 remains unknown. We hypothesized that the three cAMP-hydrolyzing cardiac PDEs (1, 3, 4) that are found in the human heart would regulate distinct cAMP signaling pathways.

Using the cAMP biosensor EPAC-SH187, we performed fluorescence resonance energy transfer experiments in healthy guinea pig adult ventricular myocytes. Cells were treated with inhibitors of PDEs 1, 3, and 4 along with GPCR agonists isoproterenol, glucagon (gcg), and amthamine (amt), respectively for β-adrenergic, gcg and histamine H2 receptors at sub-maximal doses. Cell function and calcium data were obtained using IonOptix.

We first asked if PDEi would synergistically regulate pools of cAMP, and found that PDE4i does at pools of cAMP generated by beta-agonism. PDE3i regulates that generated by amt at the H2R, with comparable effects observed in simultaneous or prior H2R stimulation. The combination of these sub-saturating doses exerted strong inotropic and lusitropic effects in paced myocytes. In neither of these domains, PDE1i changed cAMP levels. However, PDE1i intriguingly attenuated the level of gcg-stimulated cAMP production, suggesting a feedback loop mechanism.

We conclude that PDEs 1, 3, and 4 regulate distinct pools of cAMP. Importantly, we demonstrate this in a species where the myocardial expression of these PDEs match that of humans as opposed to smaller rodent models.
Abstract Or116: One Carbon Metabolism Defect In Failing Hearts
Authors: Jung Jae Woo , Flam Emily , Bedi Kenneth , Jang Cholsoon , Rabinowitz Joshua , Margulies Kenneth , Arany Zoltan
Published: October 2024

Aberrant cardiac metabolism has long been hypothesized to contribute to heart failure (HF). However, understanding of cardiac metabolism in HF remains incomplete, especially in humans. To fill this gap, we performed metabolomic and RNA sequencing analysis of 48 non-failing (NF) and 39 dilated cardiomyopathy (DCM) hearts. Among many differences, metabolites from one-carbon metabolism were significantly reduced, especially in methionine cycle; methionine (Met) and S-adenosylmethionine (SAM), a universal methyl donor of methylation. We next studied the mechanism leading to decreased Met/SAM in DCM hearts. We first quantitatively defined methionine cycle in the heart: studies in neonatal rat ventricular myocytes (NRVMs), and
in vivo
steady-state infusions with isotope tracing, showed that Met was mostly imported or degraded from protein rather than recycled from homocysteine (~10%), suggesting that a defect in recycling is unlikely to explain low Met/SAM in DCM hearts. We next explored the impact of low SAM on cardiac function by generating mice with cardiac specific deletion of SAM synthase. Constitutive KO mice were largely embryonic lethal, and the few survivors showed dramatic systolic dysfunction and died within 12 weeks. Inducing loss of SAM synthase in adults led to 50% mortality after 2 weeks, again with marked cardiac dysfunction. Finally, we explored potential mechanisms by which low SAM can drive heart failure. In human DCM, numerous methylation products were low, including most notably creatine, a SAM methylation product and essential metabolite for cardiac energetics. Creatine is not thought to be synthesized in the heart, but we found that, to our surprise, NRVMs synthesized ~50% of their creatine pool when external supply was low, and synthesis was dependent on SAM availability. Moreover,
in vivo
steady state infusions revealed that in intact animals, even in the absence of stressors, ~10% of creatine in the heart is synthesized locally. In summary, we find that: (1) the majority of Met in cardiomyocyte is imported or derived from protein degradation, with rapid turnover; (2) Met and its product SAM are reduced in human DCM; (3) loss of SAM in murine heart leads to profound heart failure; (4) creatine is surprisingly synthesized from SAM in cardiomyocytes, and its levels are reduced in human DCM. Together, these findings suggest that a defective Met cycle may contribute to low creatine and ultimately HF.
Abstract Or118: Transcription Factor Gene Therapy for Treatment of Junctional Bradycardia in a Porcine Model of Complete Atrioventricular Block Following His Bundle Ablation
Authors: Ramirez Rafael , Bergman Samuel , Masri Jamal , Weltz Alexander , Kumar Naman , de la Uz Caridad , Kolandaivelu Aravindan , Barth Andreas , Barcelon Bernadette , Fan Jinqi , Cho Hee Cheol
Published: October 2024

Background:
Pacemaker implantation is standard treatment for patients with complete atrioventricular (AV) block. However, contraindications for device implantations create a need for alternative non-hardware cardiac pacing solutions. We developed a porcine model of complete heart block with junctional bradycardia to test a novel transcription factor-based gene therapy consisting of Tbx18, Shox2 and Tbx3 (TST). Somatic reprogramming of ventricular cardiomyocytes by TST induces pacemaker-like activity and provides biological pacing
in vitro
.

Methods:
Eight domestic swine (Yorkshire/Landrace) received catheter ablation of the AV node/His Bundle resulting in complete AV block, confirmed by dissociation of P and R waves on ECG. An implanted device pacemaker (VVI mode) maintained cardiac output during occurrences of junctional bradycardia below 50 bpm. Telemetric 3-lead ECG was recorded continuously. Subjects received TST gene therapy or control gene (GFP) via catheter-based intramuscular injection into the high right ventricular septum. Pacemaker usage and ECG were continuously monitored for 8 weeks.

Results:
Following AV node ablation, junctional escape rhythm decelerated into junctional bradycardia. After 3 weeks of AV block, 49.9 ± 13.1% of heartbeats were initiated by the device pacemaker. Upon reaching pacemaker dependence of 95% in an average of 27.9 ± 5.5 days, subjects received TST gene therapy or control injection. In subjects receiving TST therapy, daily pacemaker reliance decreased to 50% dependency in an average of 14.7 ± 2.3 days, while in GFP controls, daily pacemaker dependency at 50 bpm remained unchanged at >98% throughout follow-up. In TST-treated subjects, reductions in pacemaker dependence were associated with increases in ventricular rates exceeding 70 bpm. Efficacy of gene therapy waned after about one month; an improvement of >10% pacemaker dependency over two consecutive days was still observed on average 29.0 ± 4.1 days after TST injection.

Conclusion:
In a porcine model of complete AV block, ventricular junctional escape rhythm decelerated into bradycardia in less than one month. Subsequent gene therapy by TST injection into the high ventricular septum alleviated pacemaker dependency and enhanced heart rate for 29.0 ± 4.1 days. Somatic reprogramming of cardiomyocytes by TST gene delivery may provide a biological pacemaker alternative to hardware device implantation for therapeutic cardiac pacing.
Abstract Or126: Targeting ER stress can attenuate arrhythmia in catecholaminergic polymorphic ventricular tachycardia
Authors: Veress Roland , Terentyeva Radmila , Quiggle Sage , Perger Fruzsina , Belevych Andriy , Terentyev Dmitry , Hamilton Shanna
Published: October 2024

Gain-of-function mutations of the sarcoplasmic reticulum (SR) Ca
2+
release channel, the ryanodine receptor (RyR2), are linked to the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Increasing evidence suggests that RyR2 gain-of-function not only disturbs intracellular Ca
2+
homeostasis but drives remodeling of cell signaling and ultrastructure that markedly contributes to the arrhythmogenic phenotype. It is well established that disturbed SR Ca
2+
homeostasis can activate the endoplasmic reticulum (ER) stress response, yet whether RyR2 gain-of-function in CPVT drives ER stress is yet to be explored. The goal of our study was to determine the contribution of ER stress evoked by RyR2 gain-of-function to cardiac arrhythmogenesis. To test this, we created a new rat model of CPVT induced by RyR2-S2222L
(+/-)
mutation. Simultaneous whole cell patch clamp and Ca
2+
imaging demonstrated that under β-adrenergic stimulation, CPVT ventricular myocytes (VMs) exhibit a high propensity to spontaneous Ca
2+
waves (SCWs) and delayed afterdepolarizations. Importantly, CPVT VMs showed increased XBP1 splicing as a marker of ER stress, as well as increased intra-SR redox stress measured using biosensor ER_roGFPiE. Assessment of ER stress proteins that contribute to SR redox status revealed upregulation of H
2
0
2
-producer enzyme ERO1α, with no compensatory change in expression of H
2
O
2
-degrader Peroxiredoxin-4 (PRDX4). Adenoviral overexpression of PRDX4 in CPVT VMs not only normalized SR redox status but attenuated Ca
2+
mishandling, reducing RyR2 activity and the incidence of proarrhythmic spontaneous Ca
2+
waves. Of note, immunofluorescence and biochemical studies suggest a direct interaction between RyR2 and PRDX4. To test whether targeting ER stress was protective at the whole heart level, we delivered AAV9-αMHC-PRDX4 to CPVT rats and performed ex vivo optical mapping. While CPVT hearts exhibited triggered activity and increased incidence of VT, sustained VT was prevented in PRDX4-injected hearts. Collectively, these data strongly suggest that ER stress contributes to the arrhythmogenic phenotype of CPVT. Targeting ER stress protein PRDX4 has promising therapeutic potential to normalize SR homeostasis, stabilize RyR2 activity and attenuate Ca
2+
-dependent arrhythmogenesis.
Abstract Tu001: MiR-21 mitigated pulmonary hypertension induced right ventricular dysfunction through pulmonary circulating exosomes
Authors: Chang Wei-Ting , Fisch Sudeshna , Hsu Chih-Hsin
Published: October 2024

Background:
Patients with pulmonary arterial hypertension (PAH) often experience adverse outcomes primarily due to right ventricular (RV) dysfunction. However, the intricate relationship between pulmonary circulation and RV remodeling remains largely unexplored. Exosomal microRNAs (miRNAs) function as paracrine signaling mediators in diverse diseases.

Aims:
To investigate the ability of exosomal miRNAs derived from pulmonary endothelial cells to transmit signals affecting cardiomyocytes.

Methods and Results:
Utilizing next-generation sequencing, we identified several target miRNAs by analyzing exosome samples obtained through right-heart catheterization. Notably, exosomal miR-21 exhibited the highest expression in the pulmonary circulation
(Figure 1)
. Under hypoxic conditions, the expression of miR-21 increased in human pulmonary endothelial cells (HPECs) and in exosomes derived from their conditioned medium. Treatment with these exosomes enhanced miR-21 expression and concurrently mitigated apoptosis in hypoxia-exposed cardiomyocytes. Likewise, miR-21 demonstrated a protective role against hypoxia-induced injuries and apoptosis, involving the SPRY-2/p-ERK and PTEN/Akt pathways. In a murine model of sugen/hypoxia-induced PAH, mice lacking miR-21 (miR-21
-/-
) exhibited more severe RV dysfunction, altered hemodynamics, and increased fibrosis compared to wild-type (WT) mice. Employing a parabiosis model, pairs consisting of WT and miR-21
-/-
mice were shielded from RV dysfunction, whereas pairs of miR-21
-/-
mice displayed exacerbated RV function
(Figure 2)
.

Conclusion:
Hypoxia stimulates the production of exosomal miR-21 in both HPECs and their microenvironment, subsequently mitigating apoptosis in cardiomyocytes
(Figure 3)
. Our findings propose endothelial cell-derived exosomal miR-21 as a promising therapeutic target for PAH.
Abstract Tu004: Global distribution of cardiac exosomes after myocardial infarction
Authors: Wang Xinjie , Du Xiao , chen Zhishi , Yu Wei , Wang Zhongjing , Chen Yanping , Chang Daniel , Mata Isla , Jensen Garrett , Chang Jiang , Luo Weijia
Published: October 2024

Background:
Exosomes’ beneficial effects on the injured hearts show promising results. Understanding the biodistribution and kinetics of cardiac exosomes during myocardial infarction (MI) is crucial for exploring the underlying mechanisms.

Aim:
We aimed to elucidate dynamic biodistributions of cardiomyocyte-released exosomes in MI hearts at post-MI day 3 and day 14, representing the acute and remodeling stages, respectively. We employed the cardiac exosome reporter mice expressing CD63NanoLuc to track the endogenous cardiac exosomes.

Method:
The NanoLuc reporter mice underwent MI surgery (n=3-12/group). Reporter expression was induced by tamoxifen injection. To assess post-MI cardiac exosome distribution via circulation, fourteen organs, and plasma exosomes were harvested for luciferase activity quantification at each time point. Bioluminescence analysis was conducted in isolated cardiac fibroblasts, endothelial cells, and leukocytes to profile exosome-mediated paracrine signaling.

Result:
Plasma exosome luciferase activities were consistent across groups. A decline in bioluminescent levels was observed in all organs at both time points, with significant differences in the pancreas, thyroid, kidney, lung, and thymus (p<0.05). Day 3 post-MI showed increased heart endothelial cell bioluminescence in risk and remote areas (p=0.0007), whereas day 14 showed decreased bioluminescence in cardiac fibroblasts in both areas (p=0.0029 and p=0.0086). Endothelial cells and leukocytes exhibited a ten-fold higher exosome uptake than cardiac fibroblasts.

Conclusion:
There was a significant decrease in exosome uptake by major organs from the injured heart. Cardiac endothelial cells and fibroblasts demonstrated dynamic exosome uptake changes, with endothelial cells and leukocytes showing notably higher uptake compared to fibroblasts.
Abstract Tu011: Targeted chelation therapy decreases Cryopyrin/NLRP3 expression and acts as senomorphic in Chronic Kidney Disorder induced Vascular Calcification.
Authors: Arora Shivani , Vyavahare Naren
Published: October 2024

Background:
The high phosphate (P) microenvironment caused by mineral imbalance in chronic kidney disease (CKD) has been reported to induce osteogenic changes in healthy Vascular Smooth Muscle Cells (VSMCs), leading to the calcification of arteries. We have previously shown that EDTA therapy can reverse calcification of the arteries in a rat model of CKD. This study investigated whether global mineral imbalance observed during CKD can initiate senescent changes in the aorta. We focused on NLRP3 as an intracellular source of SASP and evaluated if chelation therapy with EDTA has senotherapeutic potential.

Methods:
We used an early-stage adenine diet-based rodent model of CKD to evaluate whether senescent cells accumulate prior to vascular calcification. For validation studies, we adopted an ex-vivo approach, wherein we cultured aortic rings under high phosphate conditions for different durations to simulate the early and late stages of vascular calcification. Further, to test the efficacy of chelation therapy, EDTA loaded- human serum albumin nanoparticles tagged with anti-elastin antibody- Flexibzumab (EL-EDTA -NPs) were successfully targeted to the site of calcification in the aorta in a progressive CKD rodent model.

Results:
Using a flow-cytometry-based approach to detect senescent cells based on SA-βGal activity, we evidence the presence of senescent cells that accumulate in the aorta prior to calcific changes as observed using micro-CT and histopathology in the in vivo model of vascular calcification. Further, we show that targeted chelation therapy with EDTA NPs acts as senomorphic and decreases NLRP3 inflammasome formation. The EL-EDTA-NPs decreased mineral deposits in arteries and thus altered the local immune microenvironment, secretion of growth factors, and proteases within the aortic tissue, identified as SASP.

Conclusion:
For the first time, the current study reports that the accumulation of senescent cells occurs before calcification and provides an opportunity to analyze the potential role of senescence in the pathological progression of vascular calcification. Additionally, these findings emphasize the role of altered Ca and Pi microenvironment observed during CKD in initiating senescent changes via NLRP3 activation in the aorta. Furthermore, these findings highlight the potential of a targeted EDTA delivery approach to modulate tissue micro-environment selectively by decreasing Ca deposition and SASP in the aorta.
Abstract Tu013: ETV2 Transcriptionally Activates
Rig1
Gene Expression and Promotes Reprogramming of the Endothelial Lineage
Authors: Choi Young Geun , Ma Xiao , Das Satyabrata , Sierra-Pagan Javier , Gong Wuming , Sadek Hesham , Zhang Jianyi , Garry Mary , Garry Daniel
Published: October 2024

ETV2 is a master regulator and pioneer factor for the endothelial lineage. Previous studies have established that this transcription factor was essential for embryogenesis and direct reprogramming of fibroblasts to endothelial cells. ETV2-mediated direct reprogramming holds therapeutic potential in treating ischemic tissues including ischemic heart disease, yet the underlying molecular mechanisms remain to be completely defined. In the current studies, we focused on deciphering the mechanisms that governed ETV2-mediated reprogramming, including the role of RIG1 inflammatory responses. We hypothesized that ETV2 transcriptionally activated
Rig1
gene expression, increased expression of its downstream network [RIG1-like receptor (RLR) pathway] and modulated the reprogramming of fibroblasts to endothelial cells. Mouse embryonic fibroblasts (MEFs) with inducible ETV2 expression system were used to overexpress ETV2, and single-cell RNA-seq defined transcript expression involved in RLR signaling pathways. ChIP-seq, electromobility gel shift assays (EMSA), and transcriptional assays were used to verify that ETV2 was a direct upstream activator of
Rig1
gene expression. Both RIG1 and RLR pathway proteins were significantly upregulated in ETV2-overexpressing MEFs. ETV2 mediated the
Rig1
upregulation through direct binding to the
Rig1
promoter and transcriptional activation of
Rig1
expression. Knockdown of
Rig1
using shRNA significantly (p < 0.05) reduced the efficiency of fibroblast to endothelial cell reprogramming more than 70%. These results highlight that 1) ETV2 activates inflammatory pathways and 2) RIG1 is direct downstream target of ETV2 and together they have an essential role in the reprogramming process. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of a revascularization strategy for the treatment of ischemic heart disease.
Abstract Tu016: Alleviating Aortic Valve Calcification By Blocking TNFα Receptors
Authors: Thent Zar Chi , Butcher Jonathan , Zhou Bin
Published: October 2024

Background:
Tumor Necrosis Factor-alpha (TNFα) is a key inflammatory cytokine involved in calcific aortic valve disease (CAVD). Following mechanical stress, activated monocytes, macrophages induce TNFα signaling in aortic valve (AoV). The activated TNFα binds to its own receptors, TNFR1 and TNFR2, in which TNFR1 has a death domain likely to cause cell death in AoV. Subsequently, the release of extracellular vesicles (calcium and inorganic phosphates) following inflammatory process results in valve calcification and stenosis.

Hypothesis:
Blocking TNFα-mediated inflammation by genetic knockout of Tnfr1/Tnfr2 slowdown CAVD progression in mice.

Methods and Results:
Using a mouse model of CAVD with deletion of
Ldlr,
low-density lipoprotein receptor, we compared the disease progression in mice fed with standard and high fat diet. To get better understanding on the role of TNFα in CAVD,
Ldlr
-/-
mice (3 months old) on the TNFR1 and TNFR2 knockout background, were also fed with high fat diet for 3 months (

Ldlr
-/-

;

Tnfr1
-/-

:

Tnfr2
-/-

). Aortic valve function was analyzed by using echocardiogram and it showed decreased AoV velocity and increased ejection fraction in

Ldlr
-/-

;

Tnfr1
-/-

:

Tnfr2
-/-

compared to

Ldlr
-/-

;

Tnfr1
+/-

:

Tnfr2
+/-

group. The pathological changes such as fibrosis, calcium and collagen deposition seen under routine and specialized histological staining methods were reduced in TNF receptors knockout group. Next, using customized designed for PCR arrays, we examined the expression of 62 candidate genes involved in inflammation, apoptosis, extracellular matrix remodeling or aortic valve stenosis pathways. The top downregulated genes in the

Ldlr
-/-

;

Tnfr1
-/-

:

Tnfr2
-/-

group compared to

Ldlr
-/-

;

Tnfr1
+/-

:

Tnfr2
+/-

group are (Col3a1, MMp7, Klk1) whereas the top upregulated gene are (Igf1r and MMp14). We are currently validating these findings by using qPCR and immunofluorescence.

Conclusion:
Our findings reveal blocking TNFα signaling receptors downregulate the genes related to inflammatory cytokine activation and aortic valve stenosis in the Ldlr knockout mice fed with high fat diet. Blockage of TNFα likely reduces apoptosis and inflammation while improves extracellular matrix remodeling to slowdown CAVD progression. Our findings thus suggest a new therapeutic strategy for CAVD.
Abstract Tu017: MerTK inhibition aggravates partial carotid ligation-induced atherosclerosis
Authors: Ding Zufeng
Published: October 2024

Introduction:
Partial carotid ligation (PCL) is a widely used model to generate acute disturbed flow (d-flow) in left carotid artery (LCA), leading to rapid endothelial dysfunction and atherosclerosis formation. While the right carotid artery (RCA) without PCL is a built-in control characterized by steady flow (s-flow). Endothelial cells (ECs) form a single cell layer that is constantly exposed to blood flow patterns and play important roles in atherosclerosis. MER proto-oncogene tyrosine kinase (MerTK) is the major receptor for efferocytosis, a process of the efficient clearance of apoptotic cells. MerTK is mainly expressed in macrophages and there are very limited reports focusing on MerTK function in ECs. This study was designed to investigate the role of blood flow patterns in regulating MerTK expression in aortic ECs and its contributions to the development of atherosclerosis.

Methods:
Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet (HFD) were used to set up acute atherosclerosis within 4 weeks with WT and

MerTK
-/-

mice. Proteomics and molecular experiments were applied to reveal the potential mechanism pathways.

Results:
Our proteomics data showed that, compared with WT group,

MerTK
-/-

aggravates atherosclerosis in LCA through upregulation of endothelial dysfunction markers (e.g. IL-1β, NF-κB, TLR4, MAPK signaling) and mitochondrial dysfunction. Our immunostaining data further validates the proteomics data, showing that

MerTK
-/-

induces impaired endothelial efferocytosis and expression of endothelial dysfunction markers (e.g., Caspase-3, NF-κB, TLR4, vWF, VCAM-1, and P22
phox
) and promotes the development of atherosclerosis.

Conclusion:
Our data suggests that endothelial MerTK is sensitive to d-flow and determines endothelial efferocytosis in vivo. MerTK inhibition promotes endothelial dysfunction and atherosclerosis. Our data may provide novel evidence and reference opinions for the application of endothelial MerTK- targeted therapeutics to treat atherosclerosis.
Abstract Tu020: Contrasting effect of Ox-PAPC and PC-KLH in atherosclerotic plaques or peripheral blood T cell, macropahges and dendritic cell activation
Authors: Frostegard Johan , Ahmed Sabbir , Steuer Johnny , Gillgren Peter , Rahman Mizanur
Published: October 2024

Background:
Atherosclerosis is a chronic inflammation characterized by accumulation of immune cells, oxidized low density lipoprotein (OxLDL) and dead cells in the lesions, both of the latter have the hapten phosphorylcholine (PC) as an important antigen. PC is also exposed on microorganisms and other compounds and needs a carrier to be antigenic. Antibodies against PC (anti-PC) ameliorates deleterious effects of both OxLDL and dead cells. Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) is a major compound in the lipid moiety of OxLDL exposing PC.

Methods:
We studied effect of Ox-PAPC and PC with keyhole limpet cyanid (KLH) on T cells, dendritic cells (DC) and macrophages (MQ). We examined the influence of Ox-PAPC and PC conjugated to KLH (PC-KLH) on T cells, DC, and MQ from peripheral blood or atherosclerotic plaques.

We investigated phospholipid and glutathione (GSH) levels in the blood plasma of healthy controls (n=30) and patients (n=30).

Results:
Ox-PAPC, in contrast to PAPC and PC-KLH, independently of DC, induced proinflammatory T cell activation, which was inhibited by mito tempo, inhibitor of reactive oxygen species (ROS). Ox-PAPC triggered inflammatory activities while reducing metabolic activities in T cells. PC-KLH, on the other hand, reduced CD86 and CD11C, proinflammatory cytokines, and induced anti-inflammatory IL-10 in DC. Ox-PAPC induced ROS, GSH, and proinflammatory MQ activation, inhibited by Mito tempo or GSH inhibitor. Additionally, Ox-PAPC, in the presence of PCSK9, elevated CD86 expression and lipid peroxidation. Patients exhibited higher GSH levels comapre to control groups, associated with phospholipid levels.

Conclusions:
The study reveals the differential effects of Ox-PAPC on various immune cell types, showcasing T cell activation independent of DC and heightened macrophage activation in the presence of PCSK9. Ox-PAPC-induced redox imbalance regulates T cell activation, apoptosis, and MQ activation, potentially rendering plaques more vulnerable and contributing to lesion formation and thus plaque rupture. In contrast, PC-KLH had anti-inflammatory effects, inducing anti-inflammatory cytokines from T cells, DC and MQ. PC-effects thus vary from proinflammatory to anti-inflammatory depending on presentation. Inhibition of OxPAPC effects by agents such as anti-PC could thus be anti-atherogenic, while on the other hand PC presented on a protein as KLH could be anti-atherogenic and anti-inflammatory itself.
Abstract Tu021: Genome-wide CRISPR Screen In Vivo Decodes Monocyte Infiltration Associated With Abdominal Aortic Aneurysm
Authors: Lu Haocheng , Zhao Yang , Xue Chao , Chen Eugene
Published: October 2024

Introduction:
Aortic aneurysm is the second most common aortic disease following atherosclerosis and accounts for the ninth-leading cause of death overall. The inflammatory response has a crucial role in the Abdominal Aortic Aneurysm (AAA) development. The infiltration of monocyte into aortic wall is a hallmark of aortic inflammation and it still requires further understanding of this complicated process. CRISPR screening is a powerful tool to investigate biological process, but it requires large pool of mutant cells and reliable selection procedure, which can usually only be performed in vitro or in xenograft models. To overcome this problem, we developed an unbiased genome-wide CRISPR-Cas Screen for Monocyte Infiltration in vivo (CRISPR-MI) and employed this new method in mouse AAA model to systematically study key genes regulating monocyte infiltration into aortic wall.

Hypothesis:
Monocyte infiltration is an important process in AAA development and CRISPR-MI could be utilized to study this process in vivo.

Methods:
To acquire enough monocytes for screen, we used the adoptive transfer method to transplant in vivo differentiated monocytes (bone marrow derived macrophage, BMDM) back into mouse circulation via tail vein injection. We optimized the lentivirus infection protocol, so that sgRNA library delivered by lentivirus can effectively knock out target genes in BMDMs. The BMDMs were infected with sgRNA library before adoptive transfer into AAA-model mice. After transfer, by comparing the sgRNA enrichment in different organs of receipt mice, we could identify key genes inhibiting monocyte infiltration.

Results:
CRISPR-MI identified novels genes regulating monocyte infiltration into aorta in AAA model, including Trem2 gene. In vitro experiments confirmed that Trem2 knockout increased adhesion molecules and chemokine receptors expression.

Conclusions:
CRISPR-MI can be used to identify new therapeutical targets for cardiovascular diseases.
Abstract Tu042: Fatty Acid Dysregulation Drives Cardio-Hepatic Crosstalk in Heart Failure with Preserved Ejection Fraction (HFpEF)
Authors: Mushala Bellina , Stoner Michael , McMahon Brenda , Vandevender Amber , Mullett Steven , Gelhaus Stacy , Jurczak Michael , Scott Iain
Published: October 2024

Introduction:
Heart failure with preserved ejection fraction (HFpEF) is a heterogenous clinical syndrome that accounts for nearly half of all heart failure cases world-wide. The pathophysiological mechanisms underlying HFpEF include a variety of extracardiac abnormalities such as metabolic derangements, arterial hypertension, and microvascular endothelial dysfunction, that all drive left ventricular remodeling and dysfunction. The phenotypic diversity presented in HFpEF involves functional and structural changes in several organs including the liver. As a highly vascularized organ, the liver is highly sensitive to any hemodynamic changes in the heart and clinical studies have established the presence of liver injury in HFpEF patients. However, there is limited evidence of this relationship in preclinical mouse models, thereby hindering the exploration of the pathological mechanisms linking the two organs. Cardio-hepatic interactions are poorly understood yet play a critical role in the pathophysiology of HFpEF where aberrant metabolic signaling pathways such as insulin resistance, inflammation and oxidative stress significantly contribute to disease progression and further perpetuate a vicious cycle of cardio-hepatic deterioration.

Hypothesis:
With limited preclinical data in the field, we sought to elucidate the potential pathophysiological mechanisms driving cardio-hepatic crosstalk in HFpEF.

Methods:
We used a two-hit preclinical mouse model that recapitulates cardiometabolic HFpEF to characterize the coexistence of cardiac and hepatic dysfunction in HFpEF. Male mice were kept on the HFpEF model for 15 weeks and subjected to echocardiography, glucose tolerance tests, histopathology, and multi-omics analyses.

Results:
We report that markers of HFpEF severity such as diastolic dysfunction and left ventricular hypertrophy, significantly correlated with hepatic injury and dysfunction that exacerbated metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Impaired energy substrate utilization due to fatty acid dysregulation was a major mechanism underlying cardio-hepatic crosstalk in HFpEF.

Conclusion:
Our findings highlight fatty acid metabolism as a potential target to mitigate inter-organ dysfunction in HFpEF, representing a promising opportunity to create new treatment paradigms and drive positive outcomes in HFpEF.
Abstract Tu043: Gut Microbial Metabolite Phenylacetylglutamine Leads to Cardiomyocyte Hypercontractility and Vascular Endothelial Cell Activation
Authors: Li Zhen , Zaparte Aline , Fu Xiaoming , Li Xinmin , Wong David , gibson aaron , Gorse Kiersten , Wong Zeneng , Hazen Stanley , Lefer David , Quijada Pearl , Makarewich Cat , Sharp Thomas
Published: October 2024

Background:
Numerous gut microbial metabolites including phenylacetylglutamine (PAGln) are associated with increased cardiovascular disease risk and mortality. However, little is known about the cellular-specific perturbations by which PAGln may drive cardiovascular dysfunction.

Aims:
To understand if elevated PAGln leads to cardiovascular dysfunction and what direct cardiac- and vascular-specific pathophysiology is occurring.

Methods:
Herein, we subject C57Bl/6N male mice to pharmacological increases in circulating PAGln (50mg/kg) or vehicle twice daily for 20 days. We performed echocardiography, invasive hemodynamics, and vascular reactivity assays. We also performed
ex vivo
cardiomyocyte cell physiology experiments in the presence or absence of PAGln. Moreover, to understand vascular effects we subjected endothelial cells to PAGln and looked at expression of proinflammatory and cell adhesion molecules.

Results:
We first performed LC-MS/MS analysis demonstrating significant elevation in PAGln levels in multiple tissues compared to vehicle. There was no significant alteration in cardiac structure and function through echocardiographic analysis. Invasive hemodynamic assessment of systemic blood pressure showed no change, however left ventricular filling pressures were significantly elevated in PAGln group. Aortic rings subjected to varying concentrations of acetylcholine in the PAGln-treated animals were significantly impaired compared to vehicle controls. Given the elevated filling pressures and aberrant vascular endothelial relaxation we measured nitrite levels. Nitrite was significantly reduced in the plasma, cardiac, and liver tissue in PAGln group. Cardiomyocyte fractional shortening was significantly increased compared to baseline and equivalent to b-adrenergic agonism. PAGln hypercontractility was not inhibited with adrenergic antagonism. Vascular endothelial cells which were exposed to PAGln had an increase in expression of proinflammatory and cell adhesion molecules versus vehicle.

Conclusion:
Our data demonstrates that PAGln drives cardiovascular dysfunction through cardiomyocyte hypercontractility and inducing vascular coronary endothelial cell activation
in vitro
.
Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair
Authors: Wong David , Tran Matthew , Thome Helen , Cheng Jenny , Rathbun Elle , Yang Xia , Quijada Pearl
Published: October 2024

Background:
In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability.

Methods:
We used the
Wt1
(Wilms Tumor 1)

CreERT2

;

R26
tdTomato

mouse line for tamoxifen-inducible epicardial-specific lineage tracing and

Pdgfra
nGFP

to label cardiac fibroblasts (

Wt1
CreERT2
; R26
tdT
;

Pdgfra
nGFP

). Tagged epicardial-derived cells (tdTomato
+
) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed
in vitro
studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI.

Results:
We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with
Wnt
signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in
Wt1
-expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition.

Conclusions:
Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.
Abstract Tu047: Carbonic anhydrase inhibitors (CAIs) mitigate cardiac amyloid β pathology, attenuating neuro-signaling adverse remodeling and improving cardiac function in the Tg2676- AD mouse model.
Authors: Elia Andrea , Parodi-Rullan Rebecca , Vazquez Rafael , Carey Ashley , Fossati Silvia
Published: October 2024

FDA-approved carbonic anhydrase inhibitors (CAIs) have been shown to attenuate Aβ pathology, neurodegeneration, and cerebrovascular dysfunction in models of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA), suggesting a key role for CAs as a novel and previously unexplored target for AD therapy. Amyloid-β accumulation severely impairs the cerebral neuro-signaling pathway with a progressive loss in neurotrophic factors (like BDNF), potentially also affecting the cardiac nervous system and culminating in lethal heart dysfunction. Yet, no studies have proposed the use of CAIs for treating and preventing the systemic and cardiac effects of amyloidosis and AD. Here, we explored for the first time, the protective effects of a chronic CAIs therapeutic regimen against cardiac amyloid pathology, neuronal fibers’ loss, and heart neurotrophic dysregulation in Tg2576 mice, a widely used model of AD and cerebral amyloidosis. Cardiac fibrosis enhancement along with progressive Aβ deposition in the heart seriously affected the cardiac neuro-signaling pathway culminating in a robust decline of BDNF expression associated with myocardial adrenergic nerve fibers and regenerated nerve endings impoverishment (stained with TH and GAP-43 respectively) in 13-month-old Tg2576 mice compared to age-matched WT littermates. These degenerative mechanisms severely compromised heart function, evidenced by a decline of both ejection fraction and fraction shortening percentage in Tg2576 animals. Accordingly, we observed that human Aβ40 or Aβ42 oligomers’ treatment drastically reduced BDNF and GAP-43 expression in human neuronal cells and human cardiomyocytes. Notably, long-term CAIs treatment in Tg2576 mice reduced Aβ accumulation in the heart tissue and reverted cardiac neuro-signaling pathway adverse remodeling, thus ameliorating heart function, as evidenced through speckle tracking-based strain echocardiography analysis. These in vivo results were validated by in vitro experiments both in neuronal and cardiac cells. These findings suggest a new beneficial role of CAIs in preventing the Aβ induced cardiovascular remodeling and neuro-signaling deregulation for the first time in the Tg2576 model of AD. Taken together with the positive effects of CAIs on cerebrovascular function and cognition, this study paves the way for future clinical trials aimed at repurposing FDA-approved CAIs to prevent and correct both central and peripheral pathologies induced by AD and amyloidosis.
Abstract Tu050: Deficiency Of Mitochondrial Disulfide Relay Carrier Leads To Cardiac Hypertrophy
Authors: Karius Alexander , Ma Jin , Son Annie , Syed Abu , Wang Pingyuan , Hwang Paul
Published: October 2024

Background:
Innate immune activation is critical for cardiac hypertrophy; however, its upstream regulation is less well understood. Coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4) is an essential carrier for the import of various mitochondrial proteins including
TP53
-regulated inhibitor of apoptosis 1 (TRIAP1). CHCHD4 was recently identified as a dysregulated gene in patients with dilated cardiomyopathy and was separately found to mediate innate immune signaling in skeletal muscle. Therefore, we hypothesized that CHCHD4 may play a role in the development of cardiac hypertrophy by regulating innate immunity.

Methods:
Genetically modified C57BL/6 mice overexpressing CHCHD4 (CHCHD4 Tg) or haploinsufficient in CHCHD4 (

CHCHD4
+/-

) were utilized. Mice were injected either with low dose (2 mg/kg) or high dose (30 mg/kg) isoproterenol (ISO) daily for up to 3 wk to induce hypertrophy.

Results:

CHCHD4
+/-

mice developed cardiac hypertrophy (HW/BW (mg/g): 3.62 ± 0.15

CHCHD4
+/-

vs 2.70 ± 0.13 wild-type (WT) or 2.55 ± 0.16 CHCHD4 Tg, p<0.0001) when allowed to age to 28 wk and showed reduced left ventricular ejection fraction (EF) at 1 year when compared with wild-type or CHCHD4 Tg mice (EF (%): 50.94 ± 0.94

CHCHD4
+/-

vs 58.33 ± 0.78 wild-type or 57.88 ± 1.32 CHCHD4 Tg, p<0.0001). After low dose ISO treatment,

CHCHD4
+/-

mice had reduced EF at 2 wk compared to WT or CHCHD4 Tg mice (EF (%): 44.60 ± 1.74

CHCHD4
+/-

vs 54.28 ± 0.86 wild-type, p<0.0001; or vs 52.76 ± 1.33 CHCHD4 Tg, p=0.0006). After high dose ISO treatment, WT mice developed cardiac hypertrophy by 1 wk and impaired EF by 3 wk, while CHCHD4 Tg mice remained normal. ISO treatment caused reduction of CHCHD4 levels in both the 24 hr and 1 wk states. This reduction was associated with decreased TRIAP1 and activation of the cGAS-STING-NFkB pathway in wild-type, but not CHCHD4 Tg mice. Further, plasma mitochondrial DNA (mtDNA) was 2.3 times higher in WT mice treated with high dose ISO for 1 wk compared to WT control (n=8-11/group, p=0.011), while that of CHCHD4 Tg mice was not significantly changed by ISO treatment.

Conclusions:
Our findings suggest that CHCHD4 may be a key upstream regulator of innate immune activation mediating cardiac hypertrophy and that cell-free mtDNA in blood could be a potential biomarker of pathogenesis.
Abstract Tu051: ROR2 Drives Right Ventricular Failure Via Proteostatic Imbalances
Authors: Edwards Jonathan , Uy Genevieve , Hartman Hali , Uchida Keita , Scarborough Emily , Yang Yifan , Barr Eric , Brandimarto Jeff , Li Li , Eaton Deborah , Conn Crystal , Bedi Kenneth , Margulies Kenneth , Prosser Benjamin , Arany Zoltan
Published: October 2024

Background:
No therapies exist to treat right ventricular failure (RVF), in part because RVF molecular drivers have been incompletely studied. We recently identified that the developmentally restricted noncanonical WNT receptor ROR2 is re-expressed in a severity-dependent manner in human RVF. Here we test in mice if, and how, the re-expression of
Ror2
causes RVF.

Methods/Results:
We find in neonatal rat ventricular myocytes (NRVMs) that
Ror2
overexpression (Ror2
OE
) both activates protein translation and reduces
Hspa1a/b
mRNA and Hsp70 protein, resulting in increased protein turnover, fragmented sarcomeres, intercalated disc disruption, reduced cell major axis, impairment of contractile and relaxation kinetics, and ectopy.
Ror2
knockdown (Ror2
KD
) results in opposing phenotypes. Hsp70 overexpression or proteasome inhibition rescues many of the Ror2
OE
phenotypes, demonstrating their causal involvement. NRVM proteomic analysis indicates that Ror2
OE
activates an ERK/p90RSK/Eef2 pathway that is known to regulate translation extension. In vivo, cardiac Ror2
OE
in mice causes RV dilation and dysfunction, intercalated disc disruption, reduced
Hspa1b
(encoding for Hsp70), proteasome activation, and reduced cardiomyocyte ubiquitin staining. The mouse pulmonary artery banding (PAB) model of RVF reveals similar
Ror2
induction and proteasome activation. Ror2
KD
by AAV9 in PAB reduces RV dilation and improves RV systolic and diastolic function. Finally, in cardiac samples from human RVF, we find evidence for a similar ROR2-responsive proteostatic imbalance with increased proteasome activity and ubiquitin as well as markers of increased ERK and p90RSK.

Conclusions:
ROR2 is induced in human and mouse RVF, and mechanistic studies in NRVMs and mice show that the induction of ROR2 causes RVF by disrupting the proteostatic balance of translation, folding, and turnover. Knockdown or suppression of ROR2 may serve as a novel RVF therapeutic target.
Abstract Tu052: RPN6-Serine14 Phosphorylation by PKA Protects Against Systolic Overload-Induced Cardiac Remodeling and Heart Failure
Authors: Ahammed Md Salim , Wu Penglong , Yang Liuqing , Sternburg Jack , Liang Huiyun , Li Faqian , liu Jin , Wang Xuejun
Published: October 2024

The ubiquitin-proteasome system is the main proteolytic pathway for quality control (PQC) and regulatory degradation of proteins in living cells. Systolic overload, as seen in hypertension and aortic stenosis, is a leading cause of heart failure (HF). Proteasome functional insufficiency is implicated in HF caused by systolic overload. Previous studies have established that Ser14-RPN6 phosphorylation (pS14-RPN6) mediates the activation of 26S proteasomes by protein kinase A. However, the pathophysiological significance of pS14-RPN6 in systolic overload remains to be established.

We hypothesize that pS14-RPN6 helps maintain proteostasis and protects the heart during systolic overload.

We observed a significant increase in myocardial pS14-RPN6 and ubiquitin conjugates in human HF of ischemic or non-ischemic etiologies (p=0.020, p=0.034) and similarly in wild type (WT) mice after 2 weeks of transverse aortic constriction (TAC) (p<0.0001, p=0.001).

To investigate the role of pS14-RPN6 in vivo, our lab created

Rpn6
S14A

(S14A) knock-in mice where Ser14-Rpn6 phosphorylation is blocked. Here we have compared the responses to TAC between WT and S14A mice.

We found that TAC-induced increases in myocardial proteasome peptidase activities were significantly attenuated, and accumulation of ubiquitin conjugates exacerbated in S14A mice (p<0.0001; p<0.0005). Serial echocardiography revealed that S14A mice displayed significantly greater left ventricular (LV) end-diastolic posterior wall thickness and LV mass to body weight ratios at 1- and 12-week post-TAC (p=0.024, 0.0003; p=0.005, 0.01), and lower ejection fraction at 12-week post-TAC than WT mice (p=0.035). S14A mice also showed greater heart weight to tibial length (TL) and ventricular weight to TL ratios at 1 and 12-week post-TAC (1-week, p=0.043, 0.039; 12-week, p=0.007, 0.006). Wheat germ agglutinin and picrosirius red staining indicated a greater cardiomyocyte cross-sectional area and cardiac fibrosis in 12-week post-TAC S14A mice vs. WT mice (p<0.0001; p=0.005).

These novel findings demonstrate compellingly that myocardial pS14-RPN6 helps maintain proteostasis, curtails cardiac hypertrophy and delays heart failure occurrence during systolic overload