bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo115: Aging Heart Failure with Preserved Ejection Fraction is Mediated by Noncoding RNAs
Authors: Chakraborty Sankalpa , Dickerson Bryce , Bounds Curren , Lemus Sophia , Hickman Caleb , Rajagopalan Viswanathan
Published: October 2024

Introduction:
Heart Failure (HF) with preserved ejection fraction (HFpEF) accounts for almost half of the HF population, and the elderly group is at higher risk. HFpEF can be life-threatening and management options are limited. We previously established the importance of thyroid hormones (THs) and inflammation in different cardiovascular disease models. However, the mechanisms of aging HFpEF pathogenesis are unclear. Long-noncoding ribonucleic acids (lncRNAs; >200 nucleotides) outnumber protein-coding counterparts by >4-fold and are clinically important. We hypothesized that lncRNAs act as mediators in aging HFpEF with thyroid dysfunction.

Methods:
Wild-type control, ZSF1 lean control, and ZSF1 obese (HFpEF) rats at 5-, 13-, ± 20-month (mo) age groups were studied (males and females; maximum 14/group). We employed cardiac ultrasonography, morphometric analyses, lncRNA microarray, real-time qPCR, TH enzyme-linked immune-sorbent, inflammation, and epigenetic assays. Statistical analyses were performed using one-way or two-way ANOVAs and t-tests wherever applicable.

Results:
Echocardiography showed that ZSF1 obese rats (HFpEF) had unaffected fractional shortening compared to both controls (51% to 53.8%; p>0.05). Morphometrics showed significant increases in weights of the body, kidney, liver, and/or left ventricle in the ZSF1 groups (worse in obese) at 5-, 13-, ± 20-mo time-points (p<0.05). Serum TH levels were significantly decreased at 5- and/or 13-mo in HFpEF vs. controls (p<0.05). LncRNA microarray showed several significant differentially expressed lncRNAs and mRNAs in HFpEF compared to controls. In addition, key lncRNAs were validated via real-time qPCR at 5-, 13-, ± 20-mo time-points (p<0.05; fold change ±1.5). Significantly enriched pathways including inflammation were also identified using GO and KEGG analyses. Correspondingly, inflammatory cytokines were significantly altered in HFpEF both in serum and cardiac tissues at 5- and 13-mo (p<0.05).

Conclusions:
These results indicate that lncRNAs play important roles in HFpEF associated with hypothyroidism and inflammation. These studies serve as significant steps in the identification of key targets in HFpEF offering potentially valuable translational opportunities.
Abstract Mo120: Overexpression of Prostaglandin E2 EP3 Receptor Subtype Alters Calcium-Handling Proteins in Mouse Hearts
Authors: Bhat Shaheen , Bryson Timothy , Taube David , Harding Pamela
Published: October 2024

Prostaglandin E2 (PGE2) is an autacoid that acts through G-protein coupled receptors (EP1-EP4) in various cell types within the heart, including cardiomyocytes in which EP3 and EP4 predominate. We previously reported that mice with cardiomyocyte-specific overexpression of the EP3 receptor (EP3 TG) demonstrate reduced cardiac function and diminished cardiomyocyte contractility in isolated cardiomyocytes. We therefore hypothesized that PGE2 via the EP3 receptor regulates calcium handling proteins to reduce contractility. To test our hypothesis, we performed RNA sequencing (RNA seq) on the left ventricle of fifteen-week-old male EP3 TG mice along with their WT controls. Western blots were used to confirm changes in the expression of calcium-handling proteins such as Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Ryanodine receptor 2 (RYR2), Phospholamban (PLB), Troponins (C, I, T) and voltage-gated calcium channel (CACNB2). For RNA seq, all data is reported as Log2Fold change, adjusted P value, and western blot data is reported as mean ± standard error (SE) of the mean. Additionally, isolated cardiomyocyte contractility was assessed using the IonOptix system, and data is reported as the mean ± SE. RNA seq data shows that SERCA2a and RYR2 expression were significantly downregulated in TG mice (SERCA2a -0.44, p=4.72 x 10
-6
; RYR2: -0.86, p=4.08 x 10
-5
) in addition to significant reductions in Troponin I (-0.43, p=0.038) and the Voltage-gated calcium channel (CACNB2: -0.71,p=0.0055). Western blot analyses confirmed the reductions in SERCA2a, RYR2, and CACNB2 although these changes were only significant for CACNB2 (1.0 ± 0.03 for WT vs 0.59 ± 0.04 for TG, p = 0.016). Expression of Troponins did not change in TG mice. Consistent with our previous data, IonOptix analysis shows reduced contractility of myocytes from EP3 TG mice (bl% peak h is 3.47 ± 0.20 for WT vs 1.0 ± 0.14 for TG, p<0.0001) despite a similar increase in the Ca
2+
transients (Ca
2+
peak h for WT= 0.86 ± 0.04 and TG=0.86 ± 0.07) suggesting potential changes in the sensitivity of the myofilament to Ca
2+
. In conclusion, the reduced contractility observed in EP3 TG mice may be due to alterations in calcium handling proteins and/or changes in calcium sensitivity at the level of the myofilament.
Abstract Mo124: New Rho-kinase Inhibitor Reduces Diastolic Dysfunction induced by Estrogen Depletion in Spontaneously Hypertensive Rats
Authors: Zapata-Sudo Gisele , Carvalho Juliana , Montagnoli Tadeu , Dematte Bruno , Rocha Bruna , Cunha Rebeca , Tolomeu Heber , Fraga Carlos
Published: October 2024

Heart failure with preserved ejection fraction (HFpEF) observed in women with estrogen depletion is a consequence of the reduction of cardioprotection and endothelial dysfunction. Rho-kinase (ROCK) signaling pathway plays a role in cardiac dysfunction consequent to female aging. This work investigated the effects of a new synthetic ROCK inhibitor (iROCK) after oral administration in spontaneously hypertensive rats (SHR) submitted to oophorectomy (OVX). Experimental protocols were approved Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Under ketamine (80 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.) anesthesia, female SHR submitted to OVX had cardiac function assessed after 8 weeks and compared to SHR-Sham. Eight weeks after OVX, SHR were randomly divided in groups, which were orally treated with either vehicle (DMSO) or iROCK (10 mg/kg) during 2 weeks. Novel iROCK displays potent inhibitory activity for ROCK1 and ROCK2 isoforms, with IC50 of 1.3 μM and 1.7 μM, respectively. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and LV catheterization. Cardiac tissues collected for determining the expression of mitogen-activated protein kinases p38MAPK and extracellular signal-regulated kinase (ERK-1/2) using Western blot technique. After 8 weeks of estrogen depletion, SHR displayed HFpEF because ejection fraction (EF) was 85.2 ± 0.9%, while a reduction in EF from 83.0 ± 2.7 to 64.9 ± 0.9% was only detected after 12 weeks of OVX. Altered filling pressure confirmed the diastolic dysfunction after OVX, because the ratio of rapid mitral flow (E) to rapid wave of mitral annulus tissue Doppler (e') increased from 12.1 ± 1.3 in SHR-Sham to 19.3 ± 0.5. LV end diastolic pressure (LVEDP) increased from 13.4 ± 1.3 to 21.1 ± 4.9 in SHR after 8 weeks post-OVX. Oral administration of iROCK (10 mg/kg) reduced both E/e’ to 16.2 ± 0.5 and LVEDP to 6.4 ± 0.7 mmHg. OVX in SHR increased cardiac p38 MAPK and ERK-1/2 contents by 2.3 and 1.3 fold from SHR-Sham values. In contrast, iROCK reduced p38 overexpression but not altered ERK-1/2 content. Since pro-inflammatory cytokines and oxidative stress activate p38, the recovery of its expression after treatment, indicates that iROCK could reduce the inflammatory component of HF.

In conclusion, the new iROCK recovered LV diastolic function induced by estrogen depletion in SHR, through the reduction of myocardial inflammation.
Abstract Mo135: Correlation of Leptin Resistance With Attenuation of Cardiac Vagal Activation in Type 2 Diabetes
Authors: Evans Anthony , Tu Huiyin , Whitney Lauren , Carpenter Kassidy , Li Yulong
Published: October 2024

Type 2 diabetes mellitus (T2DM) is one of the most prominent diseases impacting individuals worldwide. A leading cause of death for T2DM patients is MI-related ventricular arrhythmia, which is associated with cardiac vagal nerve dysfunction. As T2DM patients exhibit leptin resistance, and leptin signaling has been implicated in heart rate regulation, we set out to characterize changes in cardiac vagal ganglionic neuron activity during development of T2DM and determine leptin signaling’s role in cardiac vagal dysfunction. In this study, T2DM was induced in Sprague-Dawley rats by feeding a high fat diet (HFD) plus a low dose streptozotocin injection. To characterize cardiac dysfunction, we measured arterial baroreflex sensitivity and found that 12 wks post-T2DM induction, rats experienced a diminished baroreflex response (gain max 0.62 ± 0.04 bpm/mm Hg T2DM vs. 2.21 ± 0.14 bpm/mm Hg sham). To determine if the change in baroreflex sensitivity is due to altered cardiac vagal nerve activity, we measured nAChR currents and cell excitability in intracardiac ganglion (ICG) neurons and found that T2DM rat cardiac vagal neurons exhibited reduced nAChR current density (213.9 ± 25.6 pA/pF T2DM vs. 375.5 ± 44.3 pA/pF sham) and action potential frequency (9.1 ± 0.5 spikes/s T2DM vs. 19.9 ± 1.4 spikes/s sham). Furthermore, acetylcholine release from cardiac vagal nerve terminals was attenuated in T2DM rats (0.4 ± 0.16 nM T2DM vs. 1.07 ± 0.12 nM sham). To determine the role of leptin signaling in the decrease of cardiac vagal nerve activity, we examined leptin and leptin receptor levels in ICG at 4, 8, and 12 wks post-T2DM induction. Western blots revealed elevated ICG leptin levels at 4, 8, and 12 wks post-T2DM induction (8.9 ± 0.7, 9.1 ± 1.3, and 11.7 ± 2.1 times sham level, respectively). qRT-PCR revealed reduced levels of leptin receptor transcript at 12 wks post-T2DM induction (48 ± 12% of sham). A reduction of leptin receptor protein in ICG neurons at 12 wks post-T2DM induction was observed using IF microscopy. These data demonstrate that T2DM reduces cardiac vagal nerve activity resulting in diminished control of cardiac functions such as the baroreflex. Furthermore, leptin resistance may play a role in this diminished cardiac function through reductions in leptin receptor levels.
Abstract Mo138: Renal NPFFR2-mediated increase in blood pressure is associated with downregulation of cAMP signaling and upregulation of Na
+
/K
+
-ATPase protein expression in the renal proximal tubule
Authors: Amatya Bibhas , Polzin Jacob , Feranil Jun , Asico Laureano , Armando Ines , Felder Robin , Jose Pedro , Lee Hewang
Published: October 2024

Neuropeptide FF (NPFF), an octapeptide originally found in the brain, participates in the regulation of blood pressure because it is present with its receptors, NPFFR1 and NPFFR2, in the cardiovascular regulatory center in the hypothalamus. However, the role of NPFF and its receptors in the kidney on blood pressure regulation is not known. In both the human and mouse renal proximal tubule, NPFFR2, rather than NPFFR1, is the predominant NPFF receptor determined by RNA
in situ
hybridization and immunofluorescence microscopy. In mouse renal proximal tubule cells, AC263093 (10
-6
M, 15 min), a specific NPFFR2 agonist and NPFFR1 antagonist, inhibited the forskolin-stimulated cAMP production (Vehicle: 100±6.14%, n=6; AC263093: 72.4±6.53%, n=6, p<0.05), which was reversed by pretreatment with RF-9, an antagonist of both NPFFR1 and NPFFR2 (98.2±9.10%, n=6); RF-9, by itself, had no significant effect (100.8±11.44%, n=6), indicating linkage of NPFFR2 to the inhibitory G protein Gai in renal proximal tubule cells. In human renal proximal tubule cells, NPFF also increased Na
+
/K
+
-ATPase protein expression, determined by immunoblotting, in a time- and concentration-dependent manner. Protein expression of Na
+
/K
+
-ATPase was decreased in
NPFFR2
-deficient human renal proximal tubule cells caused by specific
NPFFR2
siRNA (Mock: 100±1.71%,
NPFFR2
siRNA: 58.4±4.93%, n=4; P < 0.05). Furthermore, blood pressure, measured by telemetry, was increased and sodium excretion was decreased in conscious C57Bl/6 mice infused with NPFF (9.25 mmol, 0.5 mL/hr/7 days) underneath the renal capsule. The effect was probably via NPFFR2 because the blood pressure of mice was increased by the intraperitoneal injection of AC263093 (20 mg/kg/day, 7 days) and fed a high salt diet (4% NaCl), which is consistent with the decrease in blood pressure (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) by the renal subcapsular infusion of
Npffr2
siRNA in C57Bl/6 mice also fed the high salt diet. Taken together, NPFFR2 activation increases blood pressure and decreases sodium excretion that is associated with downregulation of cAMP signaling and upregulation of Na
+
/K
+
-ATPase protein expression in the renal proximal tubule.
Abstract Or107: MyBP-HL nonsense mutations fail in sarcomere incorporation leads to modifications in myocyte contraction trough calcium modifications.
Authors: Alvarez-Arce Alejandro , Fritzmann Geena , Araujo Kelly , Pena Alexandra , Wittenkeller Lucas , Barefield David
Published: October 2024

Introduction:
Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. AF is the most common cardiac arrhythmia and is associated with ischemic stroke, heart failure, and substantial morbidity and mortality. Recently, our group described a novel sarcomere component, myosin binding protein-H like (MyBP-HL), that is mainly expressed in the cardiac atria. MyBP-HL is composed of two immunoglobulin domains and one fibronectin domain, with homology to last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations prevent his incorporation into the sarcomere. C-MyBP-C truncated peptides are identified by cells as poisoning peptides, and they are rapidly degraded. cMyBP-C nonsense mutations are causative of hypertrophic cardiomyopathy (HCM). The MyBP-HL Arg255stop variant has been described in humans and has been linked to ventricular conduction system abnormalities, atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. The gnomAD database reports 9 nonsense mutations for MYBPHL in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307).

Hypothesis:
MYBPHL nonsense mutations fail to incorporate into the sarcomere will lead to contractile disfunction.

Results:
We demonstrated that MyBP-HL overexpression showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. Nevertheless, nonsense mutations disrupt normal sarcomere localization. MyBP-HL nonsense mutations promote an increase in contraction amplitude and slower relaxation kinetics. Also, we observed an increase in sarcomere length. Similarly, MyBP-HL nonsense mutations lead to increase in amplitude peak of calcium transients, and reduction in velocity to maximal calcium peak. In accordance with calcium modifications, we observed through mass spectrometry an increase in calpain 6 abundance promoted by MyBP-HL nonsense mutations.

Conclusion:
Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament. The failure of MyBP-HL truncating peptides to incorporate into the sarcomere leads to a deficiencies in myocytes contraction patterns, changes in calcium patterns and increase in degradation proteins abundance, which are related to AF prevalence.
Abstract Or108: The role of SARM1 in regulation of immune response in heart failure with preserved ejection fraction
Authors: Nasuhidehnavi Azadeh , Minor Keaton , Light Christine , Lee Chi Fung
Published: October 2024

Heart failure with preserved ejection fraction (HFpEF) is an unmet medical need. Recent studies have shown that HFpEF is associated with immune dysregulation and decline in nicotinamide adenine dinucleotide (NAD
+
) levels. However, the molecular mechanisms linking these factors in HFpEF are not completely understood. Sterile alpha and TIR motif-containing 1 (SARM1) is a NAD
+
consuming enzyme that may regulate immune response and NAD
+
metabolism. Here, we investigated the role of SARM1 in HFpEF pathophysiology.

We utilized a two-hit diet model to induce HFpEF in mice (HFpEF diet: high-fat diet and L-NAME). Wild-type (WT) mice on HFpEF diet showed progressive decline in diastolic function (e.g. decreased E’/A’, increased relaxation time, IVRT) at 5- and 15-week, associated with unchanged systolic function and increased cardiac hypertrophy. Despite decline in NAD
+
levels as previously reported in HFpEF hearts, qPCR analysis showed no significant differences in the expression of enzymes involved in NAD
+
biosynthesis including the salvage, Preiss-Handler and
de novo
pathways. We employed RNA-sequencing to identify cardiac gene signatures that are associated with HFpEF. Gene ontology analysis showed activation of various immune pathways in HFpEF hearts. Using flow cytometry analysis, we observed around 66% increase in total leukocytes (CD45+) number, along with infiltration of both T cells (CD3+ CD4+/CD8+) and myeloid cell populations (CD11b+) in HFpEF hearts. To understand the role of SARM1 in HFpEF pathogenesis, we subjected global SARM1 knock out (KO) mice to HFpEF diet. SARM1 KO hearts had improved diastolic function and reduced hypertrophy after 15-week HFpEF diet. RNA-sequencing of SARM1 KO-HFpEF hearts showed downregulations of transcripts in immune activation pathways, compared to WT-HFpEF hearts. In summary, our findings suggest that SARM1 may regulate immune activation pathways and promote HFpEF pathogenesis. We plan to determine the intricate relationships between immune dysregulation, metabolic stress, and cardiac dysfunction in HFpEF pathogenesis. The future direction may identify potential avenues for targeted therapeutic interventions aimed at modulating immune responses for HFpEF.
Abstract Or117: JNK2-mediated gut-heart crosstalk in aging-associated AF pathogenesis.
Authors: Carrillo Elena , Khanal Saugat , Richardson Abigail , Ricchiuti Nikola , Yan Jiajie , Ai Xun
Published: October 2024

Introduction:
Aging is an independent risk factor for atrial fibrillation (AF). We recently revealed a critical role of activated cardiac stress kinase JNK2 in AF risk. Yet, the mechanisms underlying JNK2 activation in the aged heart remain unclear. Emerging evidence suggests that organ crosstalk critically contributes to the development of heart diseases. Interestingly, increased AF episodes were clinically observed in patients with active gastrointestinal (GI) diseases (with a hallmark of enhanced intestinal epithelial barrier permeability (‘leaky gut’), but reduced AF during remission. While both GI dysfunction and enhanced AF risk are seen in aged humans and animal models, whether aging-associated leaky gut leads to AF pathogenesis through activated JNK2 remains unknown.

Methods:
To assess the leaky gut and AF link, young (Yg, 2 mo) wildtype (WT) and JNK2dn mice (cardiac-specific overexpression of inactive dominant negative (dn) JNK2) were used for aged fecal matter transplant (FMT), while aged (Ag, 24 mo) mice were transplanted with Yg fecal matter to rescue aging-associated AF. Atrial tachycardia/fibrillation (AT/AF) incidence was measured using our well-established protocol, and GI permeability was measured using a serum dextran (4-kDa) FITC fluorescence assay 7 days post-FMT.

Results:
Aged WT mice had an elevated FITC level (4.9±1.5 µg/ml vs 0.98±0.04 µg/ml; n=11,8; p<0.01) with increased AT/AF inducibility compared to Yg mice (n=7,10; p<0.05). Interestingly, Yg WT recipients subjected to aged FMT (Ag-to-Yg) exhibited an ~3-fold increase in GI permeability (n=14; p<0.01) and significantly enhanced AT/AF inducibility relative to Yg sham controls (8 of 13 vs 0 of 10; p<0.05), while Yg-to-Yg FMT did not alter GI permeability and AT/AF inducibility in Yg recipients (0 of 5, p=NS). Strikingly, cardiac-specific JNK2 inhibition prevented aged-FMT-caused AT/AF in Yg JNK2dn recipient mice (0 of 10; p<0.01), suggesting that activated cardiac JNK2 underlies leaky gut-evoked AF in aging. Further, Yg-to-Ag FMT eliminated aged-associated AT/AF inducibility in aged recipients (0 of 6).

Conclusion:
Aging-associated GI dysfunction enhances cardiac JNK2 activation, which in turn drives AF risk. Targeting cardiac JNK2 could be a novel anti-AF therapeutic strategy.
Abstract Or123: Increasing ketone supply to the heart has beneficial effects on the source of cardiac ATP production in heart failure with preserved ejection fraction (HFpEF)
Authors: Sun Qiuyu , Wagg Cory , Wang Nathan , Ketema Ezra , Zhang Liyan , Lopaschuk Gary
Published: October 2024

Introduction:
There is considerable confusion as to what changes in cardiac energy metabolism occur in heart failure with preserved ejection fraction (HFpEF). In HFpEF mice hearts, we found a marked decrease in cardiac glucose oxidation, which is accompanied by an increase in fatty acid oxidation. Ketones have been proposed as an extra source of fuel for the failing, heart, although it remains unclear if ketones may adversely affect glucose and fatty acid oxidation.

Methods:
13-month-old C57BL/6N female mice were administered a 60% high-fat diet and L-NAME (0.5g/L/day) for 6 weeks to induce HFpEF. Control mice were fed with regular low-fat diet and regular drinking water. Hearts of the mice were excised and perfused in the isolated working mode aerobically with 5 mM glucose, 0.8 mM palmitate, 100 µU/ml insulin, with either low (0.6 mM) or high (1 mM) levels of β-hydroxybutyrate. Metabolic rates of the hearts were measured with radiolabelled [U-
14
C] glucose, [9,10-
3
H] palmitate, and [3-
14
C] β-hydroxybutyrate.

Results:
In HFpEF mouse hearts, glucose oxidation was significantly decreased with a parallel increase in fatty acid oxidation. As a result, the HFpEF hearts showed overall preserved ATP production rates (79 ± 12 vs. to 106 ± 13 μmol
.
g dry wt
-1.
min
-1
control vs. HFpEF). Increasing β-hydroxybutyrate levels from 0.6 mM to 1 mM resulted in a rise in ketone oxidation rates in HFpEF hearts (from 692 ± 47 to 952 ± 90 nmol
.
g dry wt
-1.
min
-1
). Interestingly, this increase in ketone oxidation did not further compromise the rates of glucose oxidation in HFpEF hearts, but actually increased glucose oxidation rates (from 95 ± 13 to 190 ± 17 nmol
.
g dry wt
-1.
min
-1
), and had minor effects on fatty acid oxidation rates (from 796 ± 123 to 678 ± 92 nmol
.
g dry wt
-1.
min
-1
). This resulted in an increase in the contribution of ketone and glucose oxidation to ATP production from 15 % to 28 % in HFpEF hearts.

Conclusion:
In HFpEF mice hearts overall ATP production is not impaired, but rather the heart switches from glucose oxidation to fatty acid oxidation as the main source of ATP production. Increasing ketone supply to the heart increases ketone oxidation and results in a favourable shift in ATP production in HFpEF.
Abstract Or125: Numb Family Proteins regulate left ventricular compaction by modulating autophagic process
Authors: Zhao Luqi , li jun , Miao Lianjie , Lu Yangyang , Nusrat Anika , Jung Sung Yun , Evans Sylvia , Wu Mingfu
Published: October 2024

Background:
Left ventricular noncompaction (LVNC) is a type of cardiomyopathy, which patients manifest clinical features of heart failure, arrhythmias, thrombotic events, and sudden death. We previously found that the ablation of
Numb(Nb)
, and its homologue
Numbl(Nl)
at early stage, causes LVNC defects resulting in embryonic lethality. In this study, we found that mice with
Nb
and
Nl
deletion at later stage survive to adulthood and develop toLVNC cardiomyopathy.

Method:
We applied
aMHC-Cre
to delete and generate
Nb
and
Nl
double knockout (ADKO); examine the structure and functions of the ADKO hearts via echocardiography (ECHO); determine the heart arrhythmia via telemetry; determine the
Nb
subcellular localization via a
Nb
overexpression line (R26-Flag:mCherry:Numb); and determine the mechanistic function of NFPs in autophagy via autophagic reporter, electron microscopy, and molecular and biochemical assays.

Results:
The
cTnT-Cre
mediated
Nb
and
Nl
deletion causes LVNC and embryonic lethality, while the ADKO can survive to adulthood and died between eight- and twelve-month-old. The ADKO hearts display LVNC by two months of age, a reduced systolic function by four months old, and left ventricular remodeling and dilatation starting at six months old. ADKO hearts manifest major clinical features of LVNC, including arrhythmias and thrombosis. ADKO hearts exhibit significant accumulation of damaged mitochondria, autophagosomes and autophagolysosomes compared to controls. Biochemical analyses reveal elevated levels of p62 and a higher ratio of LC3II/LC3I in ADKO hearts. NFP null cells and ADKO hearts exhibit abnormal autophagic flux, characterized by arrested at autophagolysosome, increased expression of Lamp1 and Lamp2, but reduced levels of cathepsin D. Mass spectrometry analyses reveal Numb's interaction with proteins involved in endocytosis and autophagy. Consistently, Numb localizes to autophagosomes and autophagolysosomes.

Conclusion:
NFPs regulate left ventricular compaction at both embryonic and postnatal stages, suggesting that LVNC can be both congenital and acquired. As LVNC mice progress, they develop dilated cardiomyopathy (DCM) at later stage, suggesting that LVNC can occur independently at early stage, and is associated with DCM at later stage. Deletion of NFPs in cardiomyocytes disrupts endocytosis and autophagic flux. Our studies suggest that NFPs mediated endocytosis and autophagy are essential for left ventricular compaction.
Abstract Tu010: Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes
Authors: Su liping , Liu Bingli , Loo Szejie , Gao Yu , Khin Ester , Kong Xiaocen , Dalan Rinkoo , Su Xiaofei , Lee Kok-Onn , Ma Jianhua , Ye Lei
Published: October 2024

Aims:
To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs)
in vitro
, early beginning of atherosclerosis
in vivo
in diabetic mice, and patients with diabetes.

Methods:
Active hMMP9
(
act-hMMP9) was added to HCASMCs and markers of inflammation were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to d
i
abetic KK.Cg-

A
y

/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques were determined.

Results:
Act-hMMP9 induced inflammatory response in HCASMCs
in vitro
, including increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM.

Conclusions:
MMP9 contributes to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes
Abstract Tu012: Innate Immune Pathway cGAS-STING in Macrophage Function in Atherosclerosis
Authors: Mangione MariaSanta , Cao Dian
Published: October 2024

Macrophages are key cells in atherosclerotic cardiovascular disease. Macrophages within a single plaque are heterogenous: some are lipid-laden “foam cells” with varying ability to recycle lipoproteins or clear dead cells while others secrete traditional “pro-inflammatory” signals. Determining what signals bias macrophages to one phenotype versus another could identify a therapeutic target. Our lab showed that after acute myocardial infarction, macrophages are skewed to a reparative phenotype by loss of cGAS-STING signaling. cGAS-STING are a pattern recognition receptor-adaptor pair that are activated by double stranded DNA in a sequence-independent manner. Global deletion of
Sting
and
Cgas
have been shown to protect against atherosclerosis in mice. We hypothesize that hematopoietic-specific loss of
Cgas
will reduce plaque burden by biasing macrophages to a less inflammatory phenotype and upregulating pro-resolving functions. To test this, B6.129S7-

Ldlr
tm1Her

/J (
Ldlr
-/-) mice (female, age 8-12 weeks, 9 per group) underwent total body irradiation followed by bone marrow transplant from C57BL6/N-
Cgas
-/- (
Cgas
-/-) or wildtype donor mice (female, age 6-8 weeks). After bone marrow reconstitution, recipient mice were started on Western diet. Mice will continue Western diet for 12 weeks (through April 24, 2024). Then, aortas, peripheral blood, spleen, and bone marrow will be collected for further analysis including plaque burden, fibrous cap thickness, and macrophage phenotype. We will also perform in vitro studies using primary macrophages to determine how
Cgas
-/- affects macrophage effector functions critical for atherosclerosis. To date, mice that received
Cgas
-/- or wildtype donor bone marrow had similar baseline cell count, triglyceride and total cholesterol levels. Bone marrow derived macrophages (BMDM) from
Cgas
-/- mice have increased expression of fibronectin. Thus, at baseline
Cgas
-/- BMDM express high levels of a marker associated with pro-reparative macrophages. More studies are needed to characterize this phenotype and determine the consequence for in vivo atherosclerosis.
Abstract Tu014: Endoplasmic reticulum stress exacerbates atherosclerosis via microRNA-181a by inhibiting mitophagy
Authors: Wang Lingjun , Cao Yanhong , chen xin
Published: October 2024

Introduction:
Atherosclerosis is a chronic and progressively advancing vascular disease, which ultimately leads to the hardening and narrowing of blood vessel walls, significantly increasing the risk of cardiovascular events such as heart attacks and strokes. Within plaques, macrophages undergo endoplasmic reticulum stress(ERS) in response to lipid stimulation, directly impacting mitochondrial function. Mitophagy serves as the primary pathway for macrophages to clear damaged mitochondria. However, the relationship between endoplasmic reticulum stress in macrophages and mitophagy remains unclear.

Hypothesis:
there exists a regulatory relationship between endoplasmic reticulum stress and mitophagy in macrophages.

Objective:
To study the effect of endoplasmic reticulum stress on mitophagy and its mechanism in atherosclerosis.

Methods:
To investigate the effect of ERS on mitochondrial autophagy, we specifically inhibited IRE1α activity in vivo, followed by testing atherosclerosis plaque and mitophagy. To explore the mechanism by which endoplasmic reticulum stress acts on mitophagy, we detected miRNAs by qPCR. The Binding of Inositol-requiring enzyme 1(IRE1α) to miRNA was detected by RNA Binding Protein Immunoprecipitation Assay (RIP). We verified miRNA’s effect on regulating mitophagy by knocking down or over expressing miRNA in vivo. To find the downstream mechanism, we predicted and verified the target gene and testified the signaling pathway by rescue experiments.

Results:
IRE1α RNase activity inhibition lightened atherosclerosis but upregulated miRNA181a and mitophagy level. In vivo, overexpressing miRNA181a reduced atherosclerosis plaque and enhanced mitophagy; results were reversed when knocking down miRNA181a. Overexpressing or silencing target gene in vitro, mitophagy is correspondingly inhibited or promoted.

Conclusion:
In atherosclerosis, endoplasmic reticulum stress increases, resulting in inhibition of mitophagy. IRE1 α further increases its target gene JNK by shearing pre-miRNA181, thereby inhibiting the expression of miRNA181a. The over expression of JNK eventually upregulated the JNK-c-JUN-Parkin signaling pathway, aggravating atherosclerosis. These results provided new data for the mechanism of endoplasmic reticulum stress on atherosclerosis.
Abstract Tu015: Angiopoietin-like 4 Stabilizes Atherosclerotic Plaques by Modulating the Phenotypic Transition of Endothelial Cells and Vascular Smooth Muscle Cells
Authors: Cho Dong-Im , Cho Hyang , Cho Meeyoung , Kang Bogyeong , Kim YongSook , Ahn Youngkeun
Published: October 2024

Atherosclerosis, the leading cause of death, is a vascular disease of chronic inflammation. We recently showed that angiopoietin-like 4 (ANGPTL4) promotes cardiac repair by suppressing pathological inflammation. Given the fundamental contribution of inflammation to atherosclerosis, we assessed the role of ANGPTL4 in the development of atherosclerosis and determined whether ANGPTL4 regulates atherosclerotic plaque stability. We injected ANGPTL4 protein twice a week into atherosclerotic
Apoe-/-
mice and analyzed the atherosclerotic lesion size, endothelial dysfunction, inflammation, and plaque stability. In atherosclerotic mice, ANGPTL4 reduced the atherosclerotic plaque size, endothelial dysfunction, and vascular inflammation. Furthermore, ANGPTL4 was associated with maintenance of vascular integrity and attenuated endothelial-to-mesenchymal transition (EndMT) in both HUVEC and atherosclerotic plaque of
Apoe-/-
mice. In the atherosclerotic lesions and fibrous caps, the number of α-SMA(+), SM22α(+), and SM-MHC(+) cells was higher, while the number of CD68(+) and Mac2(+) cells was lower in the ANGPTL4 group. Most importantly, the fibrous cap was significantly thicker in the ANGPTL4 group than in the PBS group. Smooth muscle cells (SMCs) isolated from atherosclerotic aortas showed significantly increased expression of CD68 and Krüppel-like factor 4 (KLF4), a modulator of the vascular SMC phenotype, along with downregulation of α-SMA, and these changes were attenuated by ANGPTL4 treatment. Furthermore, ANGPTL4 reduced TNFa-induced NADPH oxidase 1 (NOX1), a major source of reactive oxygen species, resulting in attenuation of KLF4-mediated SMC phenotypic changes. Our results reveal that ANGPTL4 treatment inhibits atherogenesis and suggest that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. Even more importantly, ANGPTL4 treatment inhibited the phenotypic changes of SMCs into macrophage-like cells by downregulating NOX1 activation of KLF4, leading to the formation of more stable plaques.
Abstract Tu024: Nitric oxide-dependent Vasodilation in Preeclampsia is impaired due to dysregulated L-arginine pathways and reduced cyclic Guanosine Monophosphate activity
Authors: Valdivia-Silva Julio
Published: October 2024

To demonstrate the relationship between nitric oxide-dependent vasodilation and the expression of several enzymes from L-arginine pathway such as protein arginine N-methyltransferase-1, arginase-2, and nitric oxide synthase 2 -3, the cyclic guanosine monophosphate expression, and their activity in preeclamptic women, we employed a non-invasive technique to compare endothelial vasomotor function in 60 nonpregnant women, 60 healthy pregnant women, 55 preeclamptic women, and 40 pregnant women with essential hypertension. The concentrations of cGMP were measured in samples of platelets from all groups. RNA and protein expression for the enzymes were examined by reverse transcription-polymerase chain reaction and western blot analysis in umbilical vein endothelial cells that previously were isolated from all groups of women. Nitrite/nitrate in plasma levels were measured spectrophotometrically by Griess reaction.

The percentage increase of arterial diameter during reactive hyperemia in nonpregnant women, normal pregnant women, essential hipertensive pregnant women, and preeclamptic women was 7.8 ±2.2%; 16.93 ±2.86%; 9.94 ±1.51% and 6.48 ±1.96% respectively. Vasodilation in preeclampsia and essential hypertensive women was significantly less than that in normal pregnant women. The concentration of platelet cGMP was higher in the normotensive pregnant women than in nonpregnant and essential hypertensive women but did not differ from that in preeclamptic groups. Relative expressions of messenger RNA and protein for eNOS were decreased significantly in preeclamptic and essential hypertension endothelial cells compared with cells from normal pregnancies (P<0.001). Nevertheless, relative messenger RNA for iNOS, Arginase-2, and protein arginine N-methyltransferase-1 was significantly increased in cells from preeclampsia (P<0.001) versus cells from normal pregnancies. Preeclamptic women had significantly higher plasma levels of nitrite/nitrate (49.7 ±7.1 µmol/L) compared with normal pregnant, non-pregnant, and essential hypertensive women (34,2 ±3.1 µmol/L, 29.0 ±8.21 µmol/L, and 17,4 ±2.7 µmol/L respectively).

Our results indicate that reduced NO-dependent vasodilation in preeclampsia may be due to the dysregulated expression and activity of enzymes from L-arginine pathway rather than the reduction of cGMP production. Furthermore, increased production of nitrite/nitrate in preeclampsia might be a redirection of NOS 2 and 3 activities to the peroxinitrite production.
Abstract Tu029: Effects of the phosphodiesterase inhibitors, cilostazol and ensifentrine, on alveolar-capillary dysfunction caused by MRSA
Authors: AL Matni Mohammed Yaman , Ha Alison , Dudek Steven , Letsiou Eleftheria
Published: October 2024

Background:
Acute lung injury (ALI) is characterized by excessive inflammation and dysfunction of the alveolar-capillary barrier, which consists of lung vascular endothelial and alveolar epithelial cells. Therapeutic interventions that protect this barrier are promising strategies for ALI treatment. Phosphodiesterase (PDE) inhibitors are potent modulators of inflammation. These include cilostazol, a PDE3 inhibitor, and ensifentrine, a dual PED3/4 inhibitor, which are both clinically available compounds that exhibit cytoprotective and anti-inflammatory properties. However, their roles in regulating alveolar-capillary function in ALI are unknown. The aim of this study is to investigate the effects of cilostazol and ensifentrine on lung endothelial and epithelial dysfunction caused by methicillin-resistant
Staphylococcus aureus
(MRSA), a common bacterial pathogen involved in ALI pathogenesis.

Methods:
Human primary lung endothelial cells (EC) and A549 (alveolar epithelial cell line) were pre-treated with cilostazol (CZ; 15 μM) or ensifentrine (Ens; 10 μΜ) for 1 hour and then challenged with heat-killed MRSA (2.5 x10
8
/ml). The Electrical Cell Impedance Sensing (ECIS) assay was used to assess EC barrier integrity. Pro-inflammatory signaling markers were measured by western blotting (WB) and qPCR. Cell conditioned media was analyzed for extracellular vesicle release by flow cytometry and WB.

Results:
MRSA-induced EC barrier disruption was attenuated by both CZ and Ens, although Ens exhibited superior barrier protective properties. CZ and Ens suppressed MRSA-induced upregulation of the inflammatory adhesion molecules, VCAM-1 and ICAM-1, in EC. Ens, but not CZ, reduced MRSA-induced ICAM-1 upregulation in A549. MRSA caused a significant increase in the release of EC-derived extracellular vesicles, which was partially inhibited by CZ or Ens pretreatment.

Conclusions:
Both cilostazol and ensifentrine exert anti-inflammatory properties in MRSA-treated lung EC. Compared to cilostazol, ensifentrine exhibits enhanced endothelial barrier protection and inhibits inflammation in both lung endothelial and epithelial cells. Future in vivo studies will evaluate the therapeutic efficacy of these inhibitors in bacterial-induced ALI.
Abstract Tu030: High-density Lipoprotein Induces Arginase I Expression in M1 Macrophages
Authors: Lin Wei-Ling , Wu Shuan-Hui , Hung Yu-En
Published: October 2024

Macrophages play a central role in atherosclerosis. While M1 macrophages contribute to atherosclerosis progression, M2 macrophages are primarily associated with its regression. Increasing levels of high-density lipoprotein (HDL) in mice with atherosclerosis have been demonstrated to cause plaque regression; however, the underlying mechanism is poorly characterized. Our objective is to examine whether HDL would ameliorate plaque formation by counteracting M1 phenotypes. Monocyte-derived macrophages were polarized into M1 (interferon-γ plus lipopolysaccharide) and M2 (interleukin-4 plus interleukin-13), followed by treatment with HDL. The phenotype changes, including gene expression and cytokine secretion, in M1 and M2 under HDL treatment were assessed. The results demonstrated that HDL induces the expression of arginase 1 (Arg1), the anti-inflammatory M2 marker facilitating tissue repair, exclusively in M1 macrophages. Reciprocal downregulation of inducible nitric oxide synthase and tumor necrosis factor-α was observed, suggesting a reduction of inflammation. Interleukin-6, the downstream cytokine triggered by Arg1, was elevated upon HDL treatment. Gene expression of L-arginine transporters, SLC7A1, was dominantly expressed in M1 macrophages, but its levels were not altered by HDL. Moreover, we analyzed the concentration of L-arginine, a substrate of Arg1, in human serum and revealed a positive correlation with low-density lipoprotein and total cholesterol levels, but no significant relation to HDL. Subcellular groups analysis in ascending aorta from mice identified that Arg1 expression was higher in normal diet-fed than in high-fat diet-fed. The present study demonstrated that HDL reduces the inflammatory phenotypes of M1 macrophages by increasing Arg1 expression, which may contribute to atherosclerosis plaque regression.
Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis
Authors: Tranter John , Kumar Ashutosh , Brown Gavriel , Mikami Ryan , Zhao Yonghui , Abraham Nihil , Abd El-Aziz Tarek , Ajanel Gomez Jehiel Abigail , Xie Litao , Ashworth Katrina , Hong Juan , Zhang Haixia , Kumari Tripti , Balutowski Adam , Liu Alice , Bark David , Nair Vinayak , Lasky Nina , Feng Yongmei , Stitziel Nathan , Lerner Daniel , Campbell Robert , Di Paola Jorge , Cho Jaehyung , Sah Rajan
Published: October 2024

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl
3
-induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.
Abstract Tu035: Targeted Downregulation of MKI67 Mediated by miR-10b-5p Contributes to the Pathogenesis of Kawasaki Vasculitis
Authors: Yoon Somy , Eom Gwang Hyeon
Published: October 2024

Kawasaki disease is a systemic disorder of enigmatic origin, postulated to stem from an aberrant immune response possibly triggered by infectious agents. This condition prominently risks developing vasculitis, particularly in the coronary arteries, which may lead to aneurysm formation, a grave complication. To shed light on the mechanisms underpinning Kawasaki vasculitis, a thorough investigation was conducted on patients presented at emergency departments. Whole RNA sequencing was utilized to analyze samples from individuals with general infectious diseases, acting as controls, and those diagnosed with Kawasaki disease, forming the experimental group. This approach identified a set of 13 microRNAs (miRNAs) that exhibited increased levels in patients with Kawasaki disease. Following initial findings, the Lactobacillus casei cell wall extract (LCWE)-induced vasculitis animal model, a recognized analog for human Kawasaki vasculitis, was employed to validate the relevance of these miRNAs. Among them, miR-10b-5p was pinpointed as a critical regulator; its inhibition via antagomirs markedly mitigated inflammation near the aortic valve in the LCWE-induced model. To uncover the direct downstream targets of miR-10b-5p relevant to Kawasaki vasculitis, single-cell RNA sequencing was performed. This revealed alterations in human coronary artery endothelial cells (HCAECs) upon miR-10b-5p treatment. Specifically, a subpopulation of HCAECs, initially characterized by proliferation, shifted towards a pro-inflammatory cluster upon miR-10b-5p exposure. This transformation was marked by a decrease in the expression of the proliferation marker, Ki-67 (MKI67), in the pro-inflammatory cluster. Further whole RNA sequencing on HCAECs treated with miR-10b-5p confirmed a significant downregulation of MKI67. Subsequent experiments with siRNA-mediated knockdown of MKI67 resulted in an increase in C-X-C Motif Chemokine Ligand 8 (CXCL8) expression, a pivotal cytokine in inflammation. These findings suggest a novel signaling pathway involving miR-10b-5p, MKI67, and CXCL8, potentially serving as a therapeutic target for Kawasaki vasculitis management in future interventions.