tbuckley/cabot-search huggingface dataset:
Abstract Tu012: Innate Immune Pathway cGAS-STING in Macrophage Function in Atherosclerosis
Authors: MariaSanta Mangione, Dian Cao
Journal: Circulation Research (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 Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats
Authors: Syed Anees Ahmed, Abdel Abdel-Rahman
Journal: Circulation Research (2024)

Background: Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear. Aim: This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction. Methods: Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies. Results: Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p&lt;0.05) body weight gain and fat mass; (2) higher (p&lt;0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p&lt;0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p&lt;0.05) redox enzymes; (2) increased (p&lt;0.05) ROS level and glutathione depletion (p&lt;0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p&lt;0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p&lt;0.05) compared to findings in OVX+EtOH rats in the absence of E2. Conclusion: This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.
Abstract Tu078: Mechanistic basis of protein phosphatase 2A inhibition by I2PP2A dimerization: A key step in βAR resensitization
Authors: Anushruti Ashok, Maradumane Mohan, Kate Stenson, Yu Sun, Sathyamangla Naga Prasad
Journal: Circulation Research (2024)

Dysfunction of beta-adrenergic receptor (βAR) is a key hallmark of heart failure, wherein mechanisms of phosphorylation-mediated desensitization of βARs are well understood. However, less is known about dephosphorylation-mediated resensitization of βARs that is regulated by protein phosphatase 2A (PP2A) in heart failure. Our previous studies have shown that agonist stimulation of βAR activates phosphoinositide 3-kinase γ (PI3Kγ) which phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine residues 9 &amp; 93. Phosphorylated I2PP2A (pI2PP2A) binds to PP2A inhibiting its activity thereby, impairing βAR resensitization. Although structural studies have shown I2PP2A to be a dimer, yet little is understood about the mechanistic basis of PP2A inhibition by I2PP2A. We therefore hypothesized that phosphorylation of I2PP2A promotes dimerization driving robust interaction with PP2A resulting in inhibition of PP2A and impairment in βAR resensitization. To test for dimerization, hemagglutinin (HA)-tagged I2PP2A and Myc-tagged I2PP2A were generated and co-expressed in HEK 293 cells. Immunoprecipitation of HA-I2PP2A led to significant co-immunoprecipitation of Myc-I2PP2A reflecting the ability of I2PP2A to dimerize. To determine whether phosphorylation is key for dimerization, phospho-mimetic mutants of I2PP2A (S9,93A (inactive) or S9,93D (active)) were generated. Immunoblotting of cellular lysates with expression of these phospho-mimetics showed that S9,93D exclusively formed dimers, while S9,93A I2PP2A only formed monomers suggesting that phosphorylation is key facilitator of dimerization. The role of I2PP2A phosphorylation in its dimerization and PP2A interaction with consequences on βAR resensitization will be presented. Determining the mechanistic basis of this understudied pathway is critical as human heart failure is associated with increased PI3Kγ activity, elevated expression of I2PP2A and reduced βAR function. Key words: Desensitization, Resensitization, I2PP2A, PP2A, Dimerization
Abstract Tu085: The β <sub>IV</sub> -spectrin/STAT3 Complex regulates the orientation of cardiac hypertrophic growth
Authors: Drew Nassal, Shivangi Mohta, Rebecca Shaheen, Benjamin Buck, Simon Lococo, Nicholas Leahy, Alex Winkle, Xianyao xu, Thomas Hund
Journal: Circulation Research (2024)

Background: Cardiac hypertrophy is a major risk factor for adverse cardiovascular events including heart failure and arrhythmia. However, the precise orientation of cell and organ growth varies considerably depending on stress type and duration with important implications for cardiac function. Despite this, little is known regarding the mechanisms that regulate hypertrophic orientation. Here we evaluate the role of the cytoskeletal protein β IV -spectrin and signal transducer and activator of transcription (STAT3) in directing the orientation of pressure overload-induced hypertrophy. Methods: Transgenic mouse models with altered STAT3 signaling through modified interaction with its scaffolding partner β IV -spectrin, or phospho-regulation of STAT3 directly, were evaluated at baseline and transaortic constriction (TAC) for its role in promoting concentric versus eccentric morphologies and resulting impact on systolic function. Unbiased screening of gene expression from these structurally divergent states were evaluated for pathways responsible for directing myocyte length/width. These pathways were tested in vitro using primary mouse myocytes and in vivo to tune growth patterns for therapeutic intervention. Results: Loss of β IV -spectrin or direct STAT3 activation promoted a preferential increase in myocyte length over width, resulting in dilation of the left ventricular (LV) chamber (eccentric hypertrophy) and decreased systolic function. Conversely, preservation of β IV -spectrin favored an increase in myocyte width without LV dilation (concentric hypertrophy) and preserved systolic function in response to TAC. Differential expression of genes associated with microtubule dynamics were identified in concentric vs. eccentric hypertrophic states. In vitro assays revealed a relationship between β IV -spectrin/STAT3 signaling and microtubule stability which impacted spatial distribution of mRNA for the sarcomeric gene actc1 . Finally, intervention with pharmacologic STAT3 inhibition following chronic 6-week TAC successfully recovered concentric growth with improved systolic function. Conclusions: These data identify a novel and pivotal role for β IV -spectrin/STAT3 to modify microtubule dynamics and sarcomeric transcript distribution to direct myocyte geometry and therapeutically serve in the prevention or recovery from HF. These mechanisms further illustrate the unique separation of hypertrophic drivers from growth orientation as distinct pathways in cardiac remodeling.
Abstract Mo109: Importance of Myocyte Serine De Novo Biosynthesis and its Downregulation in Heart Failure
Authors: Mohammad Keykhaei, Navid Koleini, Mariam Meddeb, Masih Tajdini, Farnaz Farshidfar, Kejun Liu, David Kass
Journal: Circulation Research (2024)

Introduction: The failing heart exhibits metabolic inflexibility and dysregulated metabolism. Serine is a non-essential amino acid that contributes to purine synthesis, redox, and amino acid synthesis. It is transported into cells from the circulation or formed by de novo synthesis (SdS). We have found human failing myocardium has reduced serine (Circ 2023;147:1147) and lower expression of SdS genes (Circ 2021;143:120). Yet, little is known about the role of SdS or its depression in myocardium. Hypothesis: Reduced serine biosynthesis impairs myocyte energy and redox balance that could contribute to heart failure. Methods/Results: Immunoblots of human HF myocardium found phosphoglycerate dihydrogen-ase (PHGDH, rate limiting SdS enzyme) and other SdS enzymes were reduced in HF, and moreso in HF with preserved ejection fraction. Expression of plasmalemmal serine transporters SLC1A4, SLC1A5, SLC12A4 were consistently depressed in HFpEF. To assess the relative contribution of exogenous serine versus SdS, we inhibited PHGDH by siRNA or an inhibitor (NCT503). The former reduced serine by 71% despite exogenous sources, and both induced greater cytotoxicity than from solely depleting exogenous sources. Inhibiting PHGDH in serine/glycine-free media induced cytotoxicity after 1hr, whereas with exogenous serine, this occurred by 24 hours. 24 hr PHGDH knockdown reduced ATP and GTP levels, mitochondrial DNA. In response to endothelin-1 stimulation, basal and maximal mitochondrial O 2 consumption increased along with PHGDH gene expression. These changes in mitochondrial respiration were essentially prevented by gene silencing of PHDGH coupled to a decline in TCA metabolites. PHGDH inhibition also increased oxidative stress (increased GSSG/GSH) and reduced NAD levels. The cytotoxicity induced by 24 hr SdS inhibition by PHGDH knockdown could be offset by supplementing myocytes with ribose (purine precursor) and DTT (antioxidant). Conclusion: This study identifies SdS as a pivotal metabolic pathway in myocyte energy metabolism homeostasis, and its depression may play a role in HF, particularly HFpEF. These results support enhancing serine metabolic pathways as a therapeutic strategy for these diseases.
Abstract Tu122: Induction of Mitochondrial and Endoplasmic Reticulum Stress in Early Response to High-Fat Diet-Induced Hyperglycemia in Mouse Hearts
Authors: Subhankhi Pal, Nicole Eminhizer, Kienan ODwyer, Sakthijothi Muthu, Zinnia Tran, Saina Prabhu, Murugesan Velayutham, Paul Chantler, Venkatesh Sundararajan
Journal: Circulation Research (2024)

Background: Diabetic cardiomyopathy (DCM) is the leading cause of mortality in diabetic patients, accounting for about 80% of the death. Mitochondria and endoplasmic reticulum (ER) play a critical role in cardiomyocyte function, yet their interaction and function in diabetic hearts remains unclear. Hypothesis: We hypothesize that mitochondria and ER stress response are essential in maintaining cardiac function and preventing cardiac remodeling during early diabetic insult in the heart. Methods: Utilizing 6-month-old male and female C57BL/6 mice, we divided them into groups receiving a standard chow diet (N=8) and a 60% high-fat diet (HFD, N=8) for four months. Post-diet, we conducted glucose tolerance tests to verify hyperglycemia. We analyzed heart samples for mitochondrial stress (LonP1, ClpX, ClpP, Afg3l2, spg7, GRP75) and ER stress (XbP1, IRE1a, CHOP, PDI, Hspa5, Dnajb9, Hsp90) markers, along with mitochondrial biogenesis (mtDNA, TFAM, Mt-ND4, and Mt COII levels), and whole heart homogenate oxidative potential by electron paramagnetic resonance (EPR) spin probe (CMH). Between the groups, a p-value &lt; 0.05 was considered significant calculated by one-way ANOVA. Results: HFD significantly increased body weight (p&lt;0.0001) and fasting glucose levels (p&lt;0.0001), confirming hyperglycemia. Among the mitochondrial stress response markers, LonP1 was significantly (p&lt;0.05) elevated in HFD hearts compared to the control. However, the ER stress-associated transcription factors, CHOP and XbP1, were significantly upregulated (p&lt;0.05) in female HFD hearts compared to the control and male groups. Further, EPR analysis showed a significant (p&lt;0.0001) four- and two-fold increase in oxidative potential in the heart homogenate of female and male HFD groups, respectively. However, mitochondrial biogenesis was notably higher, observed only in HFD females compared to other groups. Conclusion: Our study indicates that pre-diabetic hyperglycemia enhances gender-specific mitochondrial biogenesis and triggers mitochondrial and ER stress responses, highlighting the importance of mitochondrial-ER interaction in early DCM and suggesting potential therapeutic avenues.
Abstract Tu134: Oncostatin M favors cardiomyocyte survival by promoting glycolysis
Authors: Ruopu Li, Xue Bai, Yunxiaoxiao Wu, Jie Feng, Shengshou Hu, Yu Nie
Journal: Circulation Research (2024)

Introduction: Cardiomyocytes is in high demand of energy and is susceptible to low oxygen environment. Timely switch of energy metabolism after cardiac ischaemia is helpful for cardiomyocyte preservation. We previously identified a critical role of Oncostatin M (OSM) in cardiomyocyte proliferation. Since the level of OSM quickly rises in myocardium after myocardial injury, OSM may play a protective role in early phase after myocardial injury through metabolic regulation. Hypothesis: We hypothesized that Oncostatin M regulates cardiomyocyte energy metabolism to promote its resistance to hypoxia. Methods and Results: We cultivated cardiomyocytes under hypoxic condition observed that in OSM-treated group, the percentage of TUNEL positive cardiomyocytes significantly decreased compared with that in vehicle-treated group. Published RNA sequencing data from OSM-treated neonatal cardiomyocytes revealed alterations in genes related to energy metabolism. Our Seahorse metabolic stress assays demonstrated that OSM-treated cardiomyocytes exhibited higher glycolytic metabolism and lower mitochondrial respiration than vehicle-treated cells. Pyruvate kinase M2 (PKM2), among the regulatory enzymes, was significantly upregulated in OSM-treated cardiomyocytes as was detected via western-blotting. Interestingly, PKM2 has lower catalytic activity compared to its isotype PKM1, suggesting that the augmented glycolysis might be mediated by PKM2. The mutually exclusive splicing of the gene Pkm between exons 9 and 10 led to the switch between PKM1 and PKM2. qPCR results indicated a 1.5-fold increase in Pkm transcripts and a 2-fold increase in the ratio of Pkm2 to Pkm1 , suggesting that OSM concomitantly promoted Pkm transcription as well as alternative splicing of Pkm2 . Furthermore, we performed RNA interference to knock down Stat3 , the canonical downstream signal of OSM, in cardiomyocytesand observed that OSM failed to upregulate Pkm transcription. ChIP-qPCR analysis on cardiomyocyte cell line HL-1 confirmed an increased STAT3 binding signal upstream of Pkm after OSM treatment. Additionally, RIP-qPCR assays revealed a 3-fold increase in PTBP1-binding signal to Pkm transcripts in cardiomyocytes following OSM treatment. Conclusions: Our results suggest that OSM promotes glycolysis and suppresses mitochondrial respiration in cardiomyocytes by upregulates PKM2 through STAT3 mediated transcription of Pkm and PTBP1-mediated alternative splicing of Pkm transcripts.
Abstract Mo033: Circulating Long Noncoding RNA Expression Signature Predicts Adverse Cardiovascular Outcomes in Patients with Coronary Artery Disease
Authors: Wei Lun Song, Chih-Fan Yeh, Kai-Chien YANG
Journal: Circulation Research (2024)

Background: Patients with coronary artery disease (CAD) have increased risks of subsequent major adverse cardiovascular events (MACE). Currently, there are no reliable tools to predict recurrent MACE in CAD patients. Objective: To determine if circulating long noncoding RNA (lncRNA) expression signature predicts recurrent MACE in CAD patients. Methods: As part of the Taiwan Biosignature Study, this prospective nested case-control study included 168 CAD patients who experienced MACE during the follow-up and another 108 CAD patients who remained free of MACE after a≥24-month follow-up. Plasma lncRNAs were profiled to determine their prognostic values for MACE in CAD patients. Results: Initially, RNA sequencing in 59 CAD patients (41 with recurrent MACE) revealed that a total of 124 plasma lncRNAs were dysregulated in CAD patients with recurrent MACE. Subsequent analyses showed that the expression signature of these lncRNAs classified CAD patients with vs. without recurrent MACE with an accuracy of 93%. Elevated levels of plasma lnc-NCF1-1, BRE-AS1, lnc-ZFAT-6, lnc-SLC46A3-5, lnc-CXCL3-2 and lnc-EBF3-4 were associated with increased risks of recurrent MACE. Additional studies in the remaining 217 CAD patients (127 with recurrent MACE) validated that increased plasma levels of lnc- NCF1-1, BRE-AS1, lnc-ZFAT-6, and lnc-EBF3-4 were significant predictors of recurrent MACE. In addition, lnc-SCORE, an index calculated from MACE-associated plasma lncRNA levels, was shown to be a strong independent predictor (adjusted HR, 2.70 [95% CI 1.78-4.09]) of recurrent MACE in CAD patients. BRE-AS1 and lnc-ZFAT-6 , two endothelium-enriched MACE-associated lncRNAs, were specifically dysregulated in cardiomyopathy of ischemic, but not of non-ischemic, origin, suggesting their potential contribution to the development of myocardial ischemia. Moreover, knocking BRE-AS1 reversed hypoxia-induced upregulation of VCAM-1 in human aortic endothelial cells, suggesting its functional contribution to vascular inflammation upon ischemia. Conclusions: This study shows the prognostic roles of circulating lncRNAs in predicting recurrent MACE in CAD patients. Endothelium-enriched lncRNA BRE-AS1 is involved in ischemia-induced vascular inflammation.
Abstract Tu016: Alleviating Aortic Valve Calcification By Blocking TNFα Receptors
Authors: Zar Chi Thent, Jonathan Butcher, Bin Zhou
Journal: Circulation Research (2024)

Background: Tumor Necrosis Factor-alpha (TNFα) is a key inflammatory cytokine involved in calcific aortic valve disease (CAVD). Following mechanical stress, activated monocytes, macrophages induce TNFα signaling in aortic valve (AoV). The activated TNFα binds to its own receptors, TNFR1 and TNFR2, in which TNFR1 has a death domain likely to cause cell death in AoV. Subsequently, the release of extracellular vesicles (calcium and inorganic phosphates) following inflammatory process results in valve calcification and stenosis. Hypothesis: Blocking TNFα-mediated inflammation by genetic knockout of Tnfr1/Tnfr2 slowdown CAVD progression in mice. Methods and Results: Using a mouse model of CAVD with deletion of Ldlr, low-density lipoprotein receptor, we compared the disease progression in mice fed with standard and high fat diet. To get better understanding on the role of TNFα in CAVD, Ldlr -/- mice (3 months old) on the TNFR1 and TNFR2 knockout background, were also fed with high fat diet for 3 months ( Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- ). Aortic valve function was analyzed by using echocardiogram and it showed decreased AoV velocity and increased ejection fraction in Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- compared to Ldlr -/- ; Tnfr1 +/- : Tnfr2 +/- group. The pathological changes such as fibrosis, calcium and collagen deposition seen under routine and specialized histological staining methods were reduced in TNF receptors knockout group. Next, using customized designed for PCR arrays, we examined the expression of 62 candidate genes involved in inflammation, apoptosis, extracellular matrix remodeling or aortic valve stenosis pathways. The top downregulated genes in the Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- group compared to Ldlr -/- ; Tnfr1 +/- : Tnfr2 +/- group are (Col3a1, MMp7, Klk1) whereas the top upregulated gene are (Igf1r and MMp14). We are currently validating these findings by using qPCR and immunofluorescence. Conclusion: Our findings reveal blocking TNFα signaling receptors downregulate the genes related to inflammatory cytokine activation and aortic valve stenosis in the Ldlr knockout mice fed with high fat diet. Blockage of TNFα likely reduces apoptosis and inflammation while improves extracellular matrix remodeling to slowdown CAVD progression. Our findings thus suggest a new therapeutic strategy for CAVD.