bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo129: Elevation in male sex hormone metabolites with increased risk factors for inflammation and clotting in female kidneys following chronic binge drinking
Authors: Puthanveetil Prasanth
Published: October 2024

Background:
Binge drinking is a major systemic health concern. Binge drinking could alter renal metabolome and induce markers of injury as per our previously published work. Further investigation into the top regulated pathways revealed alterations sex hormone biosynthesis pathway was altered in a sex specific manner following 6 weeks of binge drinking. Our goal is to unveil individual metabolite mediators belonging to sex hormone biosynthesis pathway and to investigate the connecting link to cardiorenal health in both sexes following chronic binge drinking.

Materials and Methods:
For this work, n=3 male and female mice belonging to either control (water fed) or ethanol (20% fed in water) treatment groups were included. At the age of 8- 10 weeks, mice underwent water or binge (20% ethanol) drinking from Monday to Thursday for 4 hours, in dark cycle. This cycle was repeated for 6 weeks. Renal tissues from our experimental groups were collected at the end of 6 weeks and subjected to untargeted metabolomics. In brief, Ultra Performance Liquid Chromatography (UPLC) with Time-of-flight Mass Spectrometry (ESI-TOF-MS) followed by Mass spectrometry based separation was used to identify significantly altered metabolites.

Results:
Steroid hormone biosynthesis was the one among the top regulated pathways. Metabolites belonging to male hormone biosynthesis were the significantly altered one but specifically in females. Dehydroepiandrosterone, and it’s downstream 11-hydroxytestosterone were upregulated only in females and not in males. Female binge drinking group demonstrated an increase of 4.24 (fold) and 2.21 (fold) for dehydroepiandrosterone, and 11-hydroxytestosterone respectively as per the Log 2 Fold change values with a statistical significance of
P<0.05
. Interestingly, this increase in male hormone metabolites we observed in females were also accompanied by an increase in metabolites of proinflammatory and prothrombotic mediators, thromboxane B3 and 5- Oxo- 12 HETE, of 2.3 (fold) and over 1.6 (fold) respectively as per Log 2 Fold change values and with a statistical difference of
P<0.05
.

Conclusion:
Our data clearly demonstrate a sex specific effect following chronic binge drinking, where females have an increase in male hormones accompanied by an increase in prothrombotic and proinflammatory factors.
Abstract Mo132: Pouring Salt in the Wound: Sex-specific Cardiometabolic Responses to Increased Dietary Fat, Sugar, and Salt.
Authors: Schulman Emily , Kendall Avery , Collins Helen
Published: October 2024

Introduction:
The general population is becoming more obese, and diabetes rates continue to rise, with overnutrition and consumption of processed foods likely contributing factors. Many people consume diets high in fat, sugar, and salt; however, this triad of factors is rarely examined in unison in studies. In addition, studies that examined diets high in fat and sugar in the context of heart failure with preserved ejection fraction and cardiometabolic outcomes have often performed short dietary interventions, with many studies performed in only male mice, and dietary salt content is often not considered or manipulated.

Study Goal:
This study aimed to assess the long-term impact of high-fat, high-sucrose diets with differing levels of salt (NaCl) on systemic and cardiovascular physiology in male and female mice.

Methods and Results:
12-week-old C57BL/6J male and female mice (
n
= 4/group) were fed one of the following diets for 25 weeks: normal chow (NC), low-fat diet (LFD; 10 kcal%), high-fat high sucrose (HFHS; 60 kcal%) with 0.33% NaCl, HFHS with 1% NaCl (HFHS1%), and HFHS with 8% NaCl (HFHS8%). Mice were subjected to basal tail blood metabolite assessment and weighed weekly. In week 24, mice were subjected to glucose tolerance testing, dual-energy X-ray absorptiometry followed by postmortem gravimetric, blood metabolite, histological, and biochemical analyses at 25 weeks. We found that irrespective of sex, body weight was significantly greater in mice receiving HFHS (
p
<0.001) and HFHS1% (
p
<0.001) versus the other diets. By 25 weeks, male mice were significantly larger than female mice with all diets except HFHS1% (
p
<0.05), which correlated with increased percent body fat (
p
<0.01), impaired glucose tolerance (
p
<0.05) and increased ketone levels (
p
<0.05) in female mice subjected to HFHS1% versus males on the same diet. There was also increased heart weight and atrial weights in female mice subjected to HFHS and HFHS1% versus the other females, which was not observed in male mice.

Conclusions:
Although previous short-term dietary studies have shown that males have altered cardiometabolic responses more than females, our findings together show that long-term dietary interventions with HFHS diets with salt may adversely impact female systemic and cardiovascular physiology.
Abstract Mo140: Macrophage Piezo1 exacerbates cardiac dysfunction through inhibiting clearance of apoptotic cardiomyocytes after myocardial infarction
Authors: Peng Lu , Guo zhen , han xue , Wang Shiyue , Luo Zhaoyi , Xia Yunlong , Tan huilan , Xu Xiaoming , liu quanchi , Ding Fengyue , Zhao Huishou , Wang Yajing , Tao Ling , Yan Wenjun
Published: October 2024

Background:
Phagocytic clearance of dying cells by efferocytosis is pivotal for the inflammation resolution and heart repair after myocardial infarction (MI). Macrophages are considered key phagocytes involved in efferocytosis, however, the molecular mechanisms underlying macrophage efferocytosis during MI remain vastly unknown. Here, we aimed to identify the role of mechanosensor Piezo1 in macrophage efferocytosis during MI.

Methods:
Myeloid cell-specific Piezo1 deficient mice (Piezo1
ΔLyz2
) and their littermate controls (Piezo1
fl/fl
) underwent MI or sham operation to investigate whether Piezo1 in macrophages plays a functional role during ventricular remodeling. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) were utilized to perform efferocytosis assays with or without Piezo1 activator Yoda1. RNA sequencing (RNA-seq) analysis was used to explore the possible molecular mechanisms.

Results:
Piezo1 expression was significantly increased in BMDMs isolated from mice 3 days after MI compared to sham groups. Moreover, double immunofluorescent staining indicated that Piezo1 was more likely to colocalize with macrophages in the infarcted area 1 week post-MI. Piezo1
ΔLyz2
mice exhibited significant ameliorations in left ventricular remodeling and function after MI, accompanied with the less accumulation of apoptotic cardiomyocytes and an increased index of in vivo efferocytosis in the border area. We also found the enhanced uptake of apoptotic cells by Piezo1
ΔLyz2
mice derived BMDMs compared with Piezo1
fl/fl
control. Furthermore, Piezo1 activation by Yoda1 induced defective efferocytosis in BMDMs and PMs. RNA-seq analysis revealed that Yoda1 significantly increased the mRNA level of solute carrier family 7a member 11 (SLC7A11), a negative regulator of efferocytosis. SLC7A11 knockdown in BMDMs by siRNA effectively restored the defective efferocytosis induced by Piezo1 activation. Finally, we identified that Piezo1 activation induced SLC7A11 upregulation in macrophage through a Ca
2+
/ATF4 dependent manner.

Conclusions:
Taken together, our results demonstrated a novel mechanism by which Piezo1 activation impairs macrophage efferocytosis ability and results in excessive cardiomyocyte death after MI, providing a previously unknown insight into Piezo1-mediated mechanotransduction in the regulation of macrophage efferocytosis.
Abstract Or101: Phospho-Mimic Mutation in Beta Tubulin Suppresses Arrhythmias and Improves Cardiac Connexin 43 Localization in Duchenne Muscular Dystrophy Mice
Authors: Zhou Delong , Nouet Julie , Mesa Elam , Geukgeuzian Geovanni , Li Hong , Liu Tong , Yehia Ghassan , Romanienko Peter , Rodney George , Wehrens Xander , Lampe Paul , Gourdie Robert , Xie Lai-Hua , Fraidenraich Diego
Published: October 2024

Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. There is no cure for DMD. Its associated dystrophic cardiomyopathy is the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. Cx43 localizes at the intercalated disc of the cardiomyocyte (CM), forming an intercellular channel with adjacent CMs; In DMD, Cx43 hemichannel remodels to the lateral side of the cardiomyocyte. Our previous research indicated that phospho-mimic Cx43 carrying serine to glutamic acid substitutions at the triplet regulatory site S325, S328, S330 in mdx hearts suppressed Cx43 lateralization and improved cardiac rhythm. The phospho-mimic Cx43 also reduced pathological hyperdensity and transverse directionality of microtubules in mdx cardiomyocytes. Colchicine (Colch) treatment, known to inhibit microtubule polymerization, also improved cardiac rhythm and Cx43 localization in mdx mice. To phenocopy the effect observed with Colch, we generated a mouse model with a beta tubulin mutation at serine 172, which was replaced by glutamic acid (S172E). We found reduced arrhythmias and improved localization of Cx43 in mdx hearts carrying the S172E phospho-mimic mutant tubulin by electrocardiography and immunofluorescent staining, respectively. Moreover, the proteomics data showed that the level of phospho-Cx43 was also upregulated in mdx hearts with S172E tubulin mutation. Further investigation will explore how this mutation impacts protein regulation and electrophysiology, and elucidate its mechanism of action in microtubule polymerization. These studies investigate a regulatory link between microtubule dynamics and localization of Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.
Abstract Or103: Troponin I Serine 150 Phosphorylation as a Novel Cardiac Inotrope Without Detrimental Effects
Authors: Salyer Lorien , Sturgill Sarah , Zanella Helena , Brundage Elizabeth , Lane Cemantha , Hund Thomas , Ziolo Mark , Biesiadecki Brandon
Published: October 2024

Heart failure with systolic dysfunction is characterized by insufficient contractility. Standards of care for heart failure treat symptoms, however there are currently no approved therapies to increase contractility to directly improve the ability of the heart to pump blood. Previously tested positive inotropes increased heart function through mechanisms that increased intracellular calcium. Unfortunately, these early inotropes were associated with detrimental effects and worsened outcomes and therefore are not approved for long-term use. There remains a need for an alternative mechanism to increase contractility without increasing intracellular calcium. We previously demonstrated that phosphorylation of the inhibitory subunit of the troponin complex, troponin I (TnI) at serine residue 150 (S150) increases force development in
ex vivo
muscle by increasing calcium sensitivity. Increasing the sensitivity of the myofilament to calcium is an alternative mechanism to increase contractility without increasing intracellular calcium. We therefore hypothesize that increasing TnI-S150 phosphorylation
in vivo
would improve systolic function without harmful effects. To determine the effects of TnI-S150 phosphorylation
in vivo
, we generated a phosphorylation-mimetic mouse with TnI-S150 mutated to aspartic acid (TnI-pS150). Structural and functional measurements derived from echocardiography and hemodynamics demonstrate that TnI-pS150 mice have increased cardiac systolic function and contractility
in vivo
. We confirm that the mechanism for increasing
in vivo
function is through increased myofilament calcium sensitivity. Detrimental effects commonly observed with the use of inotropes (e.g. hypertrophy, hypertension, severe diastolic dysfunction, increased arrythmia susceptibility, increased mortality) were not observed in TnI-pS150 mice. Additionally, we did not observe any adverse long-term detrimental effects on cardiac structure and function in aged TnI-pS150 mice. These results support the phosphorylation of TnI-S150 as a novel signaling mechanism to increase systolic function without detrimental effects and is therefore a novel target for systolic heart failure therapies.
Abstract Or106: DNA Double-stranded Breaks, a Hallmark of Aging, Defined at the Nucleotide Resolution, Are Increased and Associated with Transcription in the Cardiac Myocyte Genome in LMNA-Cardiomyopathy
Authors: Cathcart Benjamin , Marreddy Sirisha , Rouhi Leila , Zhao Zhongming , Gurha Priyatansh , Marian Ali
Published: October 2024

Background:
An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction.

Hypothesis and Aims:
We posit that DSBs would be more prevalent at the genomic sites associated with gene expression. As Lamin-A (LMNA) regulates active transcription through Lamin associated domains (LADS) we predict for DSBs to be more common in non-LAD regions. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes.

Methods:
We identified the genome-wide DSBs in ~ 1 million cardiac myocytes per heart in 3 wild-type and 3 myocyte-specific LMNA-deficient (
Myh6-Cre:Lmna
F/F
) mice by END-Sequencing, the LADs were defined in cardiac myocytes via LMNA CUT&RUN assay (N=5).

Results:
The prevalence of DSBs was 0.8% and 2.2% in the wild-type and
Myh6-Cre:Lmna
F/F
myocytes, respectively. The END-Seq signals for 8 DSBs were enriched in the wild-type and 6,764 in
Myh6-Cre:Lmna
F/F
myocytes, respectively (q<0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the LADs, we defined the LADs in cardiac myocytes and found an average of 818 LADs per myocyte. LADs that were shared by at least three mice (N= 2,572) were considered constitutive LADs (cLADs), which comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N=3,975) were ~ 16-fold lower than those at the non-LAD regions (N=~17,778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of LAD regions, defined as the juxtaposed genomic regions in the
Myh6-Cre:Lmna
F/F
that corresponded to the LAD regions in the wild-type myocytes.

Conclusions:
To our knowledge, this is the first identification of DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various disease states and aging.
Abstract Or110: Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific peptidase 20
Authors: Roy Bipradas , Wu Jiao-Hui , Freedman Neil , Shenoy Sudha
Published: October 2024

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser
334
augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in

Ldlr
-/-

mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both
in vitro
and
ex vivo
models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from

Usp20
-/-

mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in

Usp20
-/-

compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3

Usp20
-/-

, P<0.0001; females, N=2 WT; N=3,

Usp20
-/-

P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser
334
diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.
Abstract Or112: Cardiac Macrophage are Key Immune Drivers of Cardiometabolic HFpEF
Authors: Goel Mehak , Ismahil Ameen , SONKAR RAVI , Bansal Shyam , Zhu Yujie , liu wei , Hamid Tariq , Rokosh Gregg , Katsnelson Michael , Prabhu Sumanth
Published: October 2024

Heart failure with preserved ejection fraction (HFpEF) can develop with obesity, hyperinsulinemia, and glucose intolerance or type 2 diabetes, a phenogroup designated cardiometabolic HFpEF. While inflammation considered an important underlying mechanism, the role of innate immunity is poorly define. We investigated the role of macrophages in pathogenesis of cardiometabolic HFpEF. C57BL/6 mice were fed either high-fat diet (HFD, 45% kcal fat, 35% carbohydrate, 20% protein, 4.73 kcal/g) or control diet (CD, 10% kcal fat, 70% carbohydrate, 20% protein, 3.85 kcal/g) for 24-28 weeks. HFD mice exhibited significantly increased body weight (BW) at equivalent calorie consumption, increased white adipose tissue (WAT) mass, glucose intolerance, and hyperinsulinemia. HFD mice also exhibited HFpEF, evidenced by increased relative wall thickness (RWT), E/e’ ratio, LA area, LVEDP, and lung weight and decreased exercise tolerance with preserved EF. CD64
+
MHCII
+
LyVE1
+
CCR2
–
resident and CD64
+
MHCII
+
CCR2
+
infiltrating macrophages expanded significantly in both cardiac and visceral fat tissue. To evaluate the role of macrophages in HFpEF, we used Macrophage Ablation Fas-Induced Apoptosis (MaFia) mice that allow for conditional depletion of mononuclear phagocytes with the Fas ligand dimerizer AP20187. AP20187 treatment after 24 weeks of HFD feeding in MaFia mice depleted macrophages and normalized BW, and reversed cardiac hypertrophy (decreased RWT, histological myocyte cross-sectional area [CSA]), and cardiac dysfunction (improved E/e’ ratio and global longitudinal strain [GLS]), as compared to vehicle-treated HFD-induced HFpEF MaFia mice. To establish sufficiency of cardiac macrophages, we FACS-sorted CD45
+
CD64
+
F4/80
+
cardiac macrophages from HFD and CD mice, and adoptively transferred (10K cells/mouse) by intramyocardial injection into naïve recipient mice, with evaluation 4 w later. Mice with HFD-heart cardiac macrophage transfer evidenced significant increases in left atrial area (P=0.008, E/e’ ratio (P=0.016), cardiomyocyte CSA (P=0.015) and cardiac fibrosis (P<0.05), and reduced |GLS|, as compared to recipient mice with CD-heart cardiac macrophage transfer. There were no differences in body weight between the two groups.
We conclude
: cardiac macrophage expansion is central to the pathogenesis of cardiometabolic HFpEF, and are both necessary and sufficient for the cardiac phenotype. These results raise the potential for macrophage-targeted interventions to mitigate HFpEF.
Abstract Or121: Druggable Genome CRISPRi/a Screens in iPSC-Derived Cardiomyocytes Identify Therapeutic Targets for Doxorubicin-Induced Cardiotoxicity
Authors: Liu Chun , Shen Mengcheng , Manhas Amit , Zhao Shane , Belbachir Nadjet , Nishiga Masataka , Thomas Dilip , Zhang Angela , Zhang Mao , Zhang Joe , Caudal Arianne , Yang Huaxiao , Sayed Nazish , Rhee June , Qi Stanley , Wu Joseph
Published: October 2024

Introduction:
Doxorubicin is a highly effective chemotherapy drug whose therapeutic utility is significantly hampered by its life-threatening adverse cardiac effects. The need for strategies to mitigate these cardiotoxic effects without diminishing its efficacy against cancer is critical in cardio-oncology.

Hypothesis:
We hypothesize that through an integrated drug discovery pipeline, utilizing patient iPSC-derived cardiomyocytes (iCMs) and CRISPRi/a bidirectional pooled screens combined with small molecule screening and molecular docking, we can identify therapeutic targets that ameliorate doxorubicin-induced cardiotoxicity (DIC) while preserving its anticancer efficacy.

Methods:
Our approach included patient iPSC-derived cardiomyocytes (iCMs), CRISPRi/a bidirectional pooled screens targeting druggable genome (~2000 genes) to identify candidate genes implicated in DIC, and small molecule screening alongside molecular docking to find potential inhibitors. Notably, carbonic anhydrase 12 (CA12) was identified as a primary hit for its role in DIC.

Results:
The CRISPRi/a screens revealed several novel genes, including CA12, as potential contributors to DIC. Genetic inhibition of CA12 in iCMs enhanced cell survival, preserved sarcomere structure, and improved contractile function and calcium handling, effectively protecting against DIC. Furthermore, we discovered that a CA12 antagonist significantly attenuates DIC in both iCMs and mouse models. Mechanistic studies suggested that doxorubicin triggers CA12 activation, leading to a metabolic change towards glycolysis in cardiomyocytes, thereby exacerbating DIC by disrupting cellular metabolism and cardiac functions.

Conclusions:
Our findings propose a novel therapeutic avenue to safeguard against doxorubicin-induced cardiotoxicity through the targeting of CA12, offering new direction for the development of more refined anticancer therapies that minimize cardiotoxic risk. This study sets the stage of combining CRISPRi/a screen with iPSC technology for future drug discovery endeavors aimed at eliminating off-target cardiotoxicities of chemotherapy agents.
Abstract Tu023: The VEGF-A splice variants as a mechanism of impaired Inflammatory Angiogenesis in the context of advanced aging.
Authors: Mantsounga Chris , Morrison Alan , Caballero Olivya , Parry Crystal , Sharma Sheila , Butler Celia , Neverson Jade , Carley Rachel
Published: October 2024

Background and Objectives:
Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairment of angiogenesis responses to injury. Critical ischemic limb and wound injury animal models have shown impact of monocytes/macrophages as a major source of angiogenic mediators leading to new arterial growth. Aging has also been associated with impairment of blood flow recovery and reduced VEGF-A expression in animals. However, recombinant VEGF-A administration or clinical trials involving VEGF-A have not fully overcome the foot perfusion and prevent limb/leg amputation. We sought to understand molecular mechanisms of reduced VEGF-A expression which remain incomplete in the context of advanced aging.

Results:
Firstly, in hindlimb ischemia mouse model, our data from liposomes containing-clodronate treated mice (12-weeks C57Bl6 with
macrophage depletion model
) (
Fig. 1a
) demonstrate impairment of blood flow recovery compared to Control group. On day 3 post-surgery, VEGF-A and VEGF-A isoforms expression (VEGF-A
165
a and VEGF-A
165
b, respectively) were reduced in ischemic muscle. Fluorescence staining showed a reduction of macrophages (CD68
+
) was also associated with reduced endothelial cells (CD31
+
). Using a myeloid-specific and inducible deleted VEGF
fl/fl
mice, we then have shown a key role of early inflammatory macrophages stage as a major source of VEGF-A required for angiogenesis in young mice (Fig. 1b).

Secondly, bone marrow-derived-macrophages (BMDMs) from C57Bl6 104-weeks mice also demonstrate reduced VEGF-A expression. Aged BMDMs demonstrate reduced VEGF-A
165
a while VEGF-A
165
b was highly increased. Polarized BMDMs from 12-weeks demonstrate increased VEGFR1 in “M2" macrophages while VEGFR2 was increased in “M1” macrophages. On the other hand, BMDMs from 104-weeks demonstrate reduced VEGFR2 expression while VEGFR1 was unaffected compared to young.

Lastly, 104-weeks mice also demonstrate severe impairment of blood flow recovery (
Fig. 2
) after surgery and reduced VEGF-A and VEGF-A
165
a in ischemic muscle tissue. VEGF-A
165
b was increased and both receptors, VEGF-R1/R2 levels (
Fig. 3
) were also reduced. Fluorescence staining on day 3 post ligation demonstrates reduced endothelial cells recruitment in muscle tissue while macrophage (CD68+) number was similar.

Conclusion:
Our investigations will contribute to define mechanisms whereby vascular injury is associated with VEGF-A isoforms specific switch in the context of advanced aging.
Abstract Tu044: Cardiac Mitochondrial Dysfunction Induces Region-Specific Mitochondrial Stress Response In The Brain To Adapt Neuronal Changes
Authors: Tran Zinnia , Muthu Sakthijothi , Karelina Kate , ODwyer Kienan , Pal Subhankhi , Sundararajan Venkatesh
Published: October 2024

Background:
The hippocampus and cortex are susceptible to changes in blood supply, metabolites, and oxygenation. However, how disrupted cardiac function affects these critical areas of the brain, leading to the cognitive and neurological consequences of heart failure, remains unclear.

Hypothesis:
We hypothesize that disrupted cardiac function cross-talks with the brain by inducing region-specific mitochondrial stress responses, leading to adaptive changes.

Methods:
We utilized cardiac-specific LonP1 knockout (LonP1cKO) mice to induce heart failure within 21 days of birth. Alongside cre- control mice, we assessed cerebral blood flow using Laser speckle contrast imaging, locomotor activity through a comprehensive laboratory animal monitoring system (CLAMS), and gene expression profiles in the hippocampus and cortex by RT-PCR. Student’s ‘t’ test determined statistical significance with a p-value<0.05.

Results:
At 21 days, LonP1cKO mice demonstrated a significant (p<0.0001) reduction in cerebral blood flow (587.1±33.68, n=7) compared to the control (1034±31.26, n=5). Additionally, there were notable decreases in both X and Y-axis ambulatory and total locomotor activities in LonP1cKO mice [XAMB (100.3±6.6 vs 224±13.6 n=16), YAMB) (67.9±4.7 vs 150±8.5 n=16), XTOT (351.8±18.1 vs 534.4±26.3 n=16) and YTOT (190.7±9.6 vs 354.9±17.07 n=16). RT-PCR analysis revealed significant upregulation of genes related to hypoxia and cellular stress (Hif1a, FGf2, Atf6), mitochondrial biogenesis (mtDNA, Tfam, mt-COI, mt-COII), mitochondrial stress response proteases (LonP1, Afg3l2, Spg7, Yme1l1), and downregulation in neurotrophic factors (Bdnf, Ngf, Gfap) in the hippocampus and cortex.

Conclusion:
The study highlights that cardiac dysfunction in LonP1cKO mice leads to mitochondrial and cellular stress response in the brain to adapt neuronal changes, suggesting a critical link between cardiac health and brain function regulated through mitochondrial stress response pathways.
Abstract Tu048: Viscoelastic remodeling of the left ventricular myocardium in myocardial infarction
Authors: Neelakantan Sunder , Mehdi Rana Raza , Xiang Qian , Zhang Xiaoxiao , Vanderslice Peter , Avazmohammadi Reza
Published: October 2024

Introduction.
Myocardial infarction (MI) leads to cardiac myocyte death and the formation of a fibrotic scar in the left ventricular myocardium (LV). LV free wall (LVFW) remodeling in MI is known to significantly alter the biomechanical response of the LV and, in turn, LV function. While changes in the elastic behavior of the LVFW in MI have been reported, changes in the viscoelastic behavior of the LVFW remain understudied. Our objective in this study was to determine the changes in the stress relaxation behavior of the LVFW in MI.

Methods.
MI was induced in rats via the ligation of the LAD artery, and hearts were harvested for three groups: sham, 2-week (2wk), and 4wk post-MI (n=3 for each group). Prior to sacrifice, echocardiography was performed to estimate ejection fraction (EF). LVFW specimens were harvested and subjected to viscoelastic stress relaxation tests. The specimens were stretched to 15% along the circumferential and longitudinal directions and allowed to relax. The stress was normalized against the peak (initial) stress for each specimen for proper comparison across the groups.

Results.
The EF at 2wk and 4wk specimens was reduced compared to the sham specimens (Fig. 1A). The 4wk post-MI specimens showed slower stress relaxation compared to that in the sham and 2wk specimens in both directions (Figs. 1B, C). Considering a 2-minute stress relaxation span, the normalized stress in the 4wk specimens was significantly higher than the sham specimens in both directions (Fig. 1D).

Conclusions.
Our results suggest that the viscoelastic relaxation rate of the LVFW diminishes at late MI timepoints. A larger and more mature scar in the LVFW is expected to contribute to slower relaxation at late MI. Future studies can determine the effect of LVFW viscoelastic remodeling on LV diastolic dysfunction and impaired relaxation in MI.
Abstract Tu053: Activin Signaling Inhibition Enhances Cardiac Functional Recovery After Aortic Debanding
Authors: Singh Anand , Guseh James Sawalla , Xiao Chunyang , Babbs Keith , Seehra Jasbir , LI Haobo , Rosenzweig Anthony
Published: October 2024

Background:
Human aortic stenosis (AS) induces adverse cardiac remodeling and heart failure by pressure overload. Reducing pressure overload (e.g., Trans-catheter aortic valve replacement (TAVR) mitigates or reverses adverse remodeling but these benefits are limited in many TAVR patients. Here we established a murine transverse-aortic-constriction (TAC)-debanding model to mimic TAVR and identify ways to enhance reverse remodeling.

Methods:
10-week-old wild-type C57BL/6 mice were subjected to TAC surgery and debanding by suture removal, or sham-debanding, between 4 (short duration) and 10 weeks (long duration) after TAC. Echocardiography was performed to assess cardiac function. RNA sequencing (RNAseq) of TAC and debanded hearts demonstrated activation of activin signaling with TAC that was relieved by debanding. Based on this, TAC-debanded animals were treated with an investigational modified activin receptor type IIB ligand trap, RKER-012 (10 mg/kg IP, bi-weekly), to inhibit signaling downstream of activin and related ligands.

Results:
Serial echocardiography of TAC-debanded animals showed greater dysfunction with longer duration TAC (Fractional shortening [FS]=39.2±3.84 for 4 weeks-TAC vs Sham: 61.2±0.94, p<0.05; FS=18.2±1.80 for 10 weeks-TAC vs Sham-TAC: 64.7±0.43, p<0.05). Cardiac functional recovery was slower and less complete in animals with longer duration TAC (10 weeks after debanding with TAC for 4 weeks, FS=57.8±1.41 vs 46.6±3.4 TAC for 10 weeks). RNAseq analysis of hearts post-debanding (10 weeks TAC+10 weeks debanding) showed a 5.73-fold decrease (p=2.57x10
-16
) in FSTL3 expression, a biomarker of activin pathway signaling, suggesting a decrease in activin signaling accompanied functional recovery. To assess functional contribution of activin signaling to incomplete recovery and therapeutic potential of pathway inhibitors, we treated debanded animals (debanded 8 weeks post-TAC) with RKER-012 or placebo (TBS) for 4 weeks. RKER-012-treated mice showed significantly increased ejection fraction (54.12±1.50 vs placebo control 42.88±3.21; p=0.001) and reduced heart weight (p=0.01).

Conclusions:
Debanding after TAC mitigates adverse remodeling that is slower and less complete with longer duration TAC. A ligand trap (RKER-012) that was designed to inhibit signaling downstream of activin enhanced recovery after debanding and warrants further study as a potential therapeutic intervention in patients with incomplete functional recovery after TAVR.
Abstract Tu057: Renal denervation enhances right ventricular function, reduces myocardial damage, and restores myocardial sympathetic signaling in rats with heart failure induced by volume overload
Authors: Miklovic Matus , Gawrys Olga , Honetschlagerova Zuzana , Kala Petr , Huskova Zuzana , Kikerlova Sona , Vanourkova Zdenka , Maxova Hana , Puertas-Frias Guillermo , Kitamoto Misuzu , Adamova Marketa , Jichova Sarka , Kvasilova Alena , Mracek Tomas , Sedmera David , Melenovsky Vojtech
Published: October 2024

Introduction:
Heart failure (HF) is associated with chronically increased sympathetic nervous activity with depleted norepinephrine (NE) in the myocardium. Despite the known anti-hypertensive effect of renal denervation (RDN), its influence on the function of the right ventricle (RV) and the cardiac sympathetic nervous system in HF is not well comprehended.

Hypothesis:
RDN improves RV function by reducing central sympathetic drive and changes of a sympathetic nervous branch in the heart.

Aims:
To investigate the effect of RDN on RV function, HF markers, myocardial NE, and its metabolism in RV in HF induced by aorto-caval fistula (ACF).

Methods:
HF was induced by ACF in Ren-2 transgenic rats (TGR). One week later, the RDN was performed by phenol application. Two weeks after RDN, RV function was measured by pressure-volume analysis and RV tissue was collected. Echocardiography was done once a week, during the entire experiment. NE levels were measured using an ELISA kit, protein expression was assessed by western blotting, gene expression by TaqMan PCR gene expression assay, and ROS by oxidation of Amplex Red assay.

Results:
The ACF increased RV weight, RV dimensions (RVD1, RVD2, RVD3), end-systolic (ESP) and end-diastolic pressure (EDP), reduced RV systolic function (end-systolic elastance - Ees, fractional area change - FAC) and ventricular-arterial coupling (Ees/Ea ratio), increased gene expression of HF markers collagen I/III ratio, natriuretic peptide A (Nppa), myosine heavy chain 7/6 (Myh7/6) ratio, transglutaminase 2 (Tgm2), and decreased superoxide dismutase 2 (SOD2) protein expression. ACF increased plasmatic NE levels, depleted NE in RV, decreased tyrosine hydroxylase protein expression in RV, and increased gene, protein expression, and activity of monoamine oxidase A (MAO-A).

RDN decreased RV hypertrophy and dilatation (RVD1, RVD2, RVD3), decreased RV ESP (-7 mmHg, p=0.004) and EDP (-2.2 mmHg, p=0.003), improved RV function (Ees +45%, p=0.03, FAC +20%, p=0.005, Ees/Ea ratio +46%, p=0.02), decreased HF markers collagen I/III ratio, Nppa, Tgm2, Myh7/6 ratio, and increased SOD2 protein expression. RDN decreased plasmatic NE levels, increased NE in RV (+32%, p=0.03), and decreased MAO-A in RV.

Conclusions:
RDN decreased RV ESP and EDP, improved RV systolic function, restored NE levels, and decreased RV gene expression of selected HF markers, probably by reduction of central sympathetic nerve drive and alterations of the cardiac sympathetic nervous system.
Abstract Tu061: Pericyte-mediated perivascular fibrosis in the pressure-overloaded heart is dependent on TGF-β signaling and is restrained by ITGB1.
Authors: Tuleta Izabela , Hanna Anis , Hernandez Silvia , Huang Shuaibo , Humeres Claudio , Kubota Akihiko , Venugopal Harikrishnan , Frangogiannis Nikolaos
Published: October 2024

Background:
Pericytes exhibit remarkable phenotypic plasticity and have been implicated in the pathogenesis of fibrosis. Cardiac pericytes are involved in repair and fibrotic remodeling of the infarcted heart; however, their role in the cardiomyopathy induced by pressure overload is not known. We hypothesized that cardiac pericytes may contribute to remodeling of the pressure-overloaded heart by converting into fibroblasts and we examined the molecular signals mediating pericyte activation.

Methods and Results:
Transverse aortic constriction (TAC) protocols were performed to study the role of pericytes in the pressure-overloaded heart. Using fibroblast:pericyte dual reporter mice and lineage tracing experiments with the inducible NG2
CreER
mice (to track pericytes) combined with PDGFRa
EGFP
reporter mice (to reliably label fibroblasts), we found no pericyte to fibroblast conversion in the pressure-overloaded heart. scRNA-seq demonstrated that pericyte lineage cells do not express fibroblast identity genes after TAC but acquire a fibrogenic phenotype expressing high levels of matricellular genes, growth factors, adhesion molecules and integrins. Bioinformatic analysis identified transforming growth factor (TGF)-b and integrin beta-1 (ITGB1) as important upstream regulators of the transcriptional profile of pericytes after TAC. Pericyte-specific loss of transforming growth factor beta receptor 2 (TGFBR2) attenuated dysfunction and perivascular fibrosis after TAC. In contrast, pericyte-specific ITGB1 loss had deleterious effects increasing mortality, promoting dysfunction, inducing microvascular rarefaction and stimulating a pro-inflammatory and fibrogenic pericyte phenotype.

Conclusions:
In the pressure-overloaded heart, pericytes acquire a fibrogenic phenotype without converting to fibroblasts. Pericyte-specific activation of TGF-β pathways mediates perivascular myocardial fibrosis after TAC. In contrast, pericyte-specific ITGB1 signaling exerts protective actions, preserving microvascular structure, attenuating inflammation and inhibiting fibrosis.
Abstract Tu063: Acute sleep deprivation induces cardiac remodeling via activation of AT1R/ERK/GSK-3β signaling
Authors: Luo Tao , Liu Haiqiong
Published: October 2024

Background:
Sleep deprivation (SD) has an adverse effect on cardiovascular system. However, whether acute SD contributes to the morphological and functional changes of the heart remains unclear.

Approach and Results:
The modified multiple platform method (MMPM) was used to establish the SD model in Sprague-Dawley rats. Acute SD significantly enhanced cardiac ejection fraction and fractional shortening in rats as observed by echocardiography. The elevated ratio of heart weight to body weight (HW/BW) and enlarged cross-section area of cardiomyocytes by WGA staining were detected in SD groups (3, 7,10 days). The upregulation of ANP and BNP was also detected by western blotting. Masson-trichrome staining showed significant fibrosis in the SD groups, which was confirmed by western blotting of the fibrosis markers α-SMA and collagen 1. Furthermore, SD increased the level of angiotensin II (Ang II) and angiotensin type 1 receptor (AT1R) in the heart tissue of SD groups accompanied by the activation of the ERK/GSK-3β pathway. Administration of valsartan and benazepril dramatically ameliorated SD-induced cardiac fibrosis and hypertrophy by downregulating Ang II/AT1R and phosphor-ERK/GSK-3β levels.

Conclusion:
Our results indicated that cardiac remodeling was induced by the acute and short duration of SD, in which the AT1R/ ERK/GSK3β signal pathway was involved. Our study demonstrates a novel complementary phenomenon induced by SD and provides detailed evidence for the risk of SD on the cardiovascular system.
Abstract Tu080: Unique Physiology of Fibroblast and Immune Cells in the Right Ventricle
Authors: Burghaze Madeline , Olverson Zianne , Sandstrom Trent , Gorr Matthew
Published: October 2024

Introduction:
Failure of the right ventricle (RV) is a main component of the morbidity and mortality of pulmonary hypertension (PH). There exists no specific treatment for RV dysfunction and failure and the only cure for many patients (especially in the case of pulmonary arterial hypertension) remains transplantation. This is partially due to the critical lack of understanding of RV biology; therapeutic strategies that are beneficial for LV failure have worse outcomes when applied to those with RV failure. RV dysfunction is characterized by exaggerated fibrosis and divergent contractile and hypertrophic responses to several drugs when compared to LV dysfunction. Thus, the RV may contain distinct cellular physiology compared to the LV that has high significance for the treatment of PH.

Methods:
Tissue digests from the RV and LV of Sprague Dawley rats were collected. We utilized digests to collect RNA for RNA-seq, and prepared cells for flow cytometry and fibroblast isolations. Fibroblasts were cultured for proliferation assays and cell signaling studies.

Results:
We found that the RV and LV have significant differences in expression of immune-related and fibrotic genes. The RV of control rats had an average of four-fold higher density of immune cells, namely macrophages/monocytes, compared to the LV (P<0.05 via paired t-test). Fibroblasts from the RV of control rats, which had a higher proliferation rate and an alteration of metabolic genes than those from the LV (P<0.05 via two-way ANOVA), also had a higher fold-change increase in periostin and CTGF expression upon treatment with TGFβ.

Conclusions:
The results demonstrate that in comparison to the LV, the RV is made up of a unique population of cells and has a unique capacity for fibrotic response that is reflected in its molecular makeup, which may contribute to its distinct response to PH. Future experiments will focus on determining the molecular mechanisms driving these differences. This knowledge is expected to yield new therapeutic approaches for RV dysfunction and failure occurring with PH, which currently have no specific treatment.
Abstract Tu084: Endothelial Cell STING contributes to Systolic Dysfunction by modulating cardiomyocyte hypertrophy and capillary density in pressure overload-induced heart failure
Authors: Sanders Erin , Wagner Noah , Bayer Abraham , Smolgovsky Sasha , Theall Brandon , aronovitz mark , Blanton Robert , kaur Kuljeet , Alcaide Pilar
Published: October 2024

Introduction:
Cardiomyocyte (CM) hypertrophy, endothelial cell (EC) dysfunction and cardiac inflammation are hallmarks of adverse cardiac remodeling that contribute to heart failure (HF) progression. The “Stimulator of Interferon Genes” (STING) is highly expressed in ECs, the dominant non-CM cell in the heart and contributes to vascular inflammation, yet its role in adverse cardiac remodeling is unknown. We hypothesized that EC STING contributes to systolic dysfunction by modulating adverse remodeling in HF.

Methods:
Wildtype (WT), global deficient (STING-/-) and inducible EC STING-/- mice or

EC STING+/+ littermate controls (Cad5ERTCre2+/–Stingfl/fl treated with Tamoxifen or oil, respectively) were subjected to transverse aortic constriction (TAC) or Sham surgery for 2-8 weeks. Cardiac function was analyzed by echocardiography. Left ventricular (LV) sections were stained with wheat germ agglutinin and isolectin to analyze CM hypertrophy and capillary density. Cardiac leukocytes were analyzed by flow cytometry and gene expression by qPCR. Bulk RNA Sequencing was performed on sorted CD45- CD31+ mouse heart ECs (MHEC) from EC STING+/+ and EC STING-/- after 4 weeks of TAC or sham surgery. ELISA was performed on MHEC supernatant.

Results:
STING-/- mice did not show a decline in fractional shortening (FS), capillary density or develop LV hypertrophy and had decreased intramyocardial CD45+ leukocytes in response to TAC, compared to WT mice. EC STING-/- mice also showed preserved FS, capillary density, and ameliorated CM hypertrophy over the course of 2, 4 and 8 weeks of TAC, compared to EC STING+/+ mice, despite having similar numbers of myocardial CD45+ leukocytes. RNASeq revealed decreased IL6 transcription, a pro-hypertrophic cytokine, in TAC STING-/- MHECs compared to WT MHECs. Myocardial IL6 gene expression was also reduced in EC STING-/- TAC hearts compared to EC STING+/+. In vitro, STING-/- MHECs had decreased secretion of IL6 protein compared to WT MHECs.

Conclusions:
Our data demonstrate that EC STING modulates both the EC and cardiac expression of the pro-hypertrophic cytokine IL6, suggesting a mechanism contributing to cardiomyocyte hypertrophy, capillary rarefaction, and systolic dysfunction in response to cardiac pressure overload.
Abstract Tu093: Adeno-associated virus-mediated gene delivery of PERM1 enhances cardiac contractility and mitochondrial biogenesis in mice.
Authors: SREEDEVI KARTHI , Doku Abigail Oforiwaa , James Amina , Do Sara , Zaitsev Alexey , Warren Junco
Published: October 2024

Heart failure remains the leading cause of death in the U.S., and 50% of patients with heart failure still die within 5 years after diagnosis. Mitochondrial impairment and contractile dysfunction are the hallmarks of heart failure with reduced ejection fraction (HFrEF). Yet, there is currently no therapeutic strategy targeting both mitochondria and muscle contractility. PERM1 is a striated-muscle-specific regulator of mitochondrial bioenergetics and is predominantly expressed in the heart and skeletal muscle. Our recent studies demonstrated that PERM1 is downregulated in human and mouse HFrEF hearts and that loss of PERM1 in mice leads to reduced contractility and energy reserve in the heart. However, it is largely unknown whether PERM1 positively regulates both muscle contractility and energetics in the heart. Here, we performed the gene delivery of
Perm1
to the heart in C57BL6 wild-type mice (8-12 weeks old) through retro-orbital injection of the adenovirus-associated virus (AAV)9 vector carrying
Perm1
(AAV-PERM1). The protein expression levels of PERM1 in the heart was increased by 3 fold as compared with control (mice injected with AAV9-GFP vectors), while there was no change in PERM1 expression in skeletal muscle (n=4/group). Strikingly, AAV-PERM1 mice exhibited a significant increase in ejection fraction (EF) and fractional shortening (FS) (AAV-GFP vs. AAV-PERM1: 0.93 vs. 1.43 and 0.92 vs. 1.64 in DEF and DFS, respectively, both p<0.05 in t-test). In addition, we observed a subtle, yet significant increase in the ratio of the heart weight to tibial length in AAV-PERM1 mice (AAV-GFP vs. AAV-PERM1: 4.7 vs 5.97, p<0.05 in t-test), suggestive of moderate cardiac hypertrophy development. Furthermore, PERM1 overexpression in the heart increased the mitochondrial copy number by 38% as compared to control AAV-GFP mice (p<0.05 in t-test), concurrently with an upregulation of the mitochondrial bioenergetics master regulator PGC-1α (155% of control, p<0.05 in t-test). Overall, these results showed that AAV-mediated PERM1 overexpression simultaneously enhances muscle contractility and mitochondrial biogenesis in the heart. This study further suggests that the gene delivery of PERM1 could be a potential therapeutic approach to treat HFrEF patients.