tbuckley/cabot-search huggingface dataset:
Abstract Tu013: ETV2 Transcriptionally Activates <i>Rig1</i> Gene Expression and Promotes Reprogramming of the Endothelial Lineage
Authors: Young Geun Choi, Xiao Ma, Satyabrata Das, Javier Sierra-Pagan, Wuming Gong, Hesham Sadek, Jianyi Zhang, Mary Garry, Daniel Garry
Journal: Circulation Research (2024)

ETV2 is a master regulator and pioneer factor for the endothelial lineage. Previous studies have established that this transcription factor was essential for embryogenesis and direct reprogramming of fibroblasts to endothelial cells. ETV2-mediated direct reprogramming holds therapeutic potential in treating ischemic tissues including ischemic heart disease, yet the underlying molecular mechanisms remain to be completely defined. In the current studies, we focused on deciphering the mechanisms that governed ETV2-mediated reprogramming, including the role of RIG1 inflammatory responses. We hypothesized that ETV2 transcriptionally activated Rig1 gene expression, increased expression of its downstream network [RIG1-like receptor (RLR) pathway] and modulated the reprogramming of fibroblasts to endothelial cells. Mouse embryonic fibroblasts (MEFs) with inducible ETV2 expression system were used to overexpress ETV2, and single-cell RNA-seq defined transcript expression involved in RLR signaling pathways. ChIP-seq, electromobility gel shift assays (EMSA), and transcriptional assays were used to verify that ETV2 was a direct upstream activator of Rig1 gene expression. Both RIG1 and RLR pathway proteins were significantly upregulated in ETV2-overexpressing MEFs. ETV2 mediated the Rig1 upregulation through direct binding to the Rig1 promoter and transcriptional activation of Rig1 expression. Knockdown of Rig1 using shRNA significantly (p &lt; 0.05) reduced the efficiency of fibroblast to endothelial cell reprogramming more than 70%. These results highlight that 1) ETV2 activates inflammatory pathways and 2) RIG1 is direct downstream target of ETV2 and together they have an essential role in the reprogramming process. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of a revascularization strategy for the treatment of ischemic heart disease.
Abstract Mo104: Cardiolipin-Metabolite Crosstalk in HFpEF
Authors: Ali Kamiar, Monique Williams, Jose Manuel Condor, Emely Robleto, Anna Kovilakath, Rosemeire Kanashiro-Takeuchi, Lauro Takeuchi, Alisson Cardoso, Marcos Alborghetti, Jose Geraldo de Carvalho Pereira, Mariane Shinzato, Kleber Franchini, Keith Webster, Gary Lopaschuk, Joshua Hare, Lauren Cowart, Lina Shehadeh
Journal: Circulation Research (2024)

Background: Transition from fatty acid oxidation to glycolysis is linked to oncometabolism and cardiac hypertrophy. We hypothesize that such a metabolic shift is pivotal in driving HFpEF in 3 murine models. The citric acid cycle (CAC) enzyme αKGDH is dysregulated in HFpEF patient biopsies and Alport hearts. Therefore, we also hypothesize more generalized CAC dysregulation at αKGDH associated with HFpEF. Methods &amp; Results: Alport and LDLR/P407 mice were generated on 129J background, and HFD/L-NAME HFpEF on C57Bl6N, (n= 5M,5F/group). Two groups of Alport and LDLR/P407 mice were fed a 2% αKG diet, and a 3rd subgroup of Alport mice injected with AAV9-CMV-αKGDH. PET-CT of 18 F-FDG showed significant increased cardiac glucose uptake in Alport and LDLR/P407 mice up to 8 wk of treatment. αKG diet conferred significantly increased cardiac glucose uptake and worse diastolic dysfunction in LDLR/P407 mice. αKGDH protein expression was reduced by ~40% (p&lt;.05) in the HFD/L-NAME hearts. Targeted metabolomic analysis by LC-MS/MS indicated normalization of intermediate metabolites in LDLR/P407 hearts by αKG diet and significantly shortening life spans (p&lt;.001). The αKG diet exacerbated adverse remodeling of cardiolipin (CL) in LDLR/P407 hearts. Expression of CL remodeling enzymes, CL Synthase (p&lt;.05) and Tafazzin (p&lt;.0001), were altered in the LDLR/P407 hearts. NADH increased in Alport and LDLR/P407 hearts suggesting decreased ATP availability and impaired oxidative phosphorylation. HFD/L-NAME HFpEF mice showed a possible block in CAC flux at αKGDH, with increased αKG and reduced succinyl-CoA. Acetyl-CoA and αKG were increased in Alport hearts. Serum metabolic signatures in 273 patients by 1 H NMR stratified into non-HF, pre-HFpEF, and HFpEF by the H2FPEF algorithm, indicated elevated serum αKG levels associated with patients with higher H2FPEF scores. Leucine is increased in LDLR/P407 and HFD/L-NAME hearts consistent with published findings in patients with HFpEF. These 2 models also have increased levels of purines. Increased G-6P in LDLR/P407 hearts supports increased glucose uptake. Glutamine is increased in HFD/L-NAME, consistent with published findings in patients with HFpEF. Conclusions: Dysregulated αKG and CL remodeling modulate oncometabolism and energy transduction that exacerbate HF in Alport, LDLR/P407, and HFD/L-NAME mice. Circulating αKG may constitute a marker of CAC dysfunction in HFpEF patients and αKGHD activation as a potential therapeutic goal.
Abstract Tu011: Targeted chelation therapy decreases Cryopyrin/NLRP3 expression and acts as senomorphic in Chronic Kidney Disorder induced Vascular Calcification.
Authors: Shivani Arora, Naren Vyavahare
Journal: Circulation Research (2024)

Background: The high phosphate (P) microenvironment caused by mineral imbalance in chronic kidney disease (CKD) has been reported to induce osteogenic changes in healthy Vascular Smooth Muscle Cells (VSMCs), leading to the calcification of arteries. We have previously shown that EDTA therapy can reverse calcification of the arteries in a rat model of CKD. This study investigated whether global mineral imbalance observed during CKD can initiate senescent changes in the aorta. We focused on NLRP3 as an intracellular source of SASP and evaluated if chelation therapy with EDTA has senotherapeutic potential. Methods: We used an early-stage adenine diet-based rodent model of CKD to evaluate whether senescent cells accumulate prior to vascular calcification. For validation studies, we adopted an ex-vivo approach, wherein we cultured aortic rings under high phosphate conditions for different durations to simulate the early and late stages of vascular calcification. Further, to test the efficacy of chelation therapy, EDTA loaded- human serum albumin nanoparticles tagged with anti-elastin antibody- Flexibzumab (EL-EDTA -NPs) were successfully targeted to the site of calcification in the aorta in a progressive CKD rodent model. Results: Using a flow-cytometry-based approach to detect senescent cells based on SA-βGal activity, we evidence the presence of senescent cells that accumulate in the aorta prior to calcific changes as observed using micro-CT and histopathology in the in vivo model of vascular calcification. Further, we show that targeted chelation therapy with EDTA NPs acts as senomorphic and decreases NLRP3 inflammasome formation. The EL-EDTA-NPs decreased mineral deposits in arteries and thus altered the local immune microenvironment, secretion of growth factors, and proteases within the aortic tissue, identified as SASP. Conclusion: For the first time, the current study reports that the accumulation of senescent cells occurs before calcification and provides an opportunity to analyze the potential role of senescence in the pathological progression of vascular calcification. Additionally, these findings emphasize the role of altered Ca and Pi microenvironment observed during CKD in initiating senescent changes via NLRP3 activation in the aorta. Furthermore, these findings highlight the potential of a targeted EDTA delivery approach to modulate tissue micro-environment selectively by decreasing Ca deposition and SASP in the aorta.
Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy
Authors: PRERNA GIRI, Magda Zaoralova, Giovanni Fajardo, Paul Heinrich, Sean Wu, Peter Dahlberg, Alexander Dunn, Daniel Bernstein
Journal: Circulation Research (2024)

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in individual thick filaments in HCM hiPSC-CMs, distinct from the classical disarray at the level of whole myofibrils and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.
Abstract Tu076: N4-acetylcytidine-regulated Cardiomyocyte Ferroptosis Mediates Cardiac Benefits of Exercise in Aging
Authors: Jiayi Kang, Chunyan wang, Aya Sugai-Munson, Zipeng Liu, James Rhee, Haobo LI
Journal: Circulation Research (2024)

Introduction: Advanced age is a major risk factor for heart diseases. Exercise training has emerged as an effective intervention to improve functional outcomes in age-related cardiac dysfunction. N-acetyltransferase (NAT10), the only known N4-acetylcytidine (ac4C) ‘writer’ enzyme, has recently been implicated in heart failure. However, the role of NAT10 in cardiac aging and the cardiac benefits of exercise on age-related cardiac dysfunction are unclear. Hypothesis: NAT10 is necessary and sufficient for cardiac benefits of exercise in aging. Methods: Ten-week-old (young) or twenty-month-old (aged) mice received sedentary (Sed) or voluntary wheel running (Run) for eight weeks. AAV9 encoding cardiac troponin-T-driven NAT10 was tail vein-injected to overexpress NAT10 in cardiomyocytes (CMs). Results: Compared with young mice, aged mice exhibited increased E/e’ (Young: -14.68±1.09 vs Aged: -20.16±1.61, n=8, p=0.03) and decreased global longitudinal strain (GLS, Young: -20.04±1.47 vs Aged: -15.88±0.84, n=8, p=0.04) with preserved ejection fraction. Aged hearts also displayed increased ferroptosis (reduced GPX4 and FSP1, p&lt;0.05 vs Sed) and senescence (increased gene expression of p16, p21, and PAI, all p&lt;0.05). In aged mice, running improved diastolic function (E/e’, Sed: -20.16±1.61 vs Run: -15.4±1.06, n=8, p=0.04; GLS, Sed: -15.88±0.84 vs Run: -20.66±1.02, n=8, p=0.02), reduced cardiac ferroptosis (increased GPX4 and FSP1, p&lt;0.05 vs Sed), and decreased senescence gene expression. Cardiac NAT10 was downregulated in aged mice and was upregulated by exercise (all p&lt;0.05). In aged mice, AAV9 injection for eight weeks led to ~6-fold increase in cardiac NAT10, resulting in reduced E/e’ (AAV9-NAT10: -22.67±1.03 vs Control: -26.33±1.12, n=7, p=0.03) and increased GLS (AAV9-NAT10: -19.23±0.93 vs Control: -15.26±1.01, n=7, p=0.04) as well as reduced senescence gene expression (reduced p16, p21, and PAI, p&lt;0.05 vs AAV9-control). Mechanistically, NAT10 functioned by increasing ferritin ac4C deposition and promoting its translation, thereby inhibiting CM ferroptosis. Conclusions: NAT10 regulates cardiac aging through ferritin, and it mediates cardiac benefits of exercise in aging. NAT10 is a promising therapeutic target for combating cardiac aging.
Abstract Tu088: Fibroblast Activation, Collagen Secretion, and Migration Occurs Independently of <i>Has2 Expression</i>
Authors: Danielle Little, Daniel Nguyen, Kenneth Brittian, Robert Brainard, Steven Jones
Journal: Circulation Research (2024)

Background: Hyaluronan (HA) accumulates after tissue injury and may regulate the wound-healing process. We found that HA is increased in and around the scar following myocardial infarction (MI). Fibroblasts are the main producers of the collagen that comprises the scar, and we have shown that they are responsible for HA accumulation. Given that these two extraordinarily high-volume synthetic processes occur in the same place and simultaneously, we queried whether alterations in Has2 -dependent HA synthesis impacted collagen secretion and other fibroblast functions. Goal: To determine the impact of HA synthesis on fibroblast function. Methods and Results: We isolated cardiac fibroblasts from Has2 fl/fl mice and treated with Cre adenovirus to delete Has2 expression or Has2 adenovirus to overexpress Has2. We assessed cell number and found no differences when Has2 was deleted or overexpressed in both naïve fibroblasts (n=6); activation with TGF-b did not change this response . We used a scratch assay to assess migration and found no change between treatments in naïve fibroblasts (n=6). Hydroxyproline assessment of deposited collagen and immunoblotting for soluble collagen secretion in the media indicated no differences between treatments in naïve fibroblasts (n=3). Conclusion: Genetic manipulation of HA production did not impact proliferation, migration, or collagen secretion in fibroblasts. Considering the enormous metabolic requirement of fibroblasts to produce HA and collagen, these results suggest that the regulation of these processes may be distinct or that the substrate is not limiting in our experimental conditions. In the intact heart, substrate could become limiting and a relationship between these two biosynthetic processes may emerge
Abstract Tu090: P21-activated kinase 1 safeguards cardiac function from impairment during fasting
Authors: John Yarbro, Lionel Chong, Caleb Sooknanan, Chris Mathew, Pritpaul Singh, Michelle Kim, Kinza Ahmed, Rohan Francis, Tamayo Kobayashi, Yuan Huang, Satoru Kobayashi, Qiangrong Liang
Journal: Circulation Research (2024)

Excessive calorie intake poses a heightened risk for cardiovascular disease, while caloric restriction and intermittent fasting have demonstrated benefits in enhancing cardiovascular health and improving cardiometabolic risk profiles. Despite this knowledge, the precise mechanisms underlying the cardioprotective effects of these dietary regimens remain inadequately understood. P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase known to play a crucial role in promoting cardiomyocyte survival in response to various stressors. In this study, we aimed to elucidate the role of PAK1 in maintaining cardiac function during fasting, utilizing mice with modified PAK1 activity in the heart. Under normal feeding conditions, heart function exhibited no discernible differences among wild type (WT), transgenic (TG), and knockout (KO) mice, as evidenced by consistent fractional shortening (FS), ejection fraction (EF), E/A ratio, and isovolumic relaxation time (IVRT). After a one-day fast, systolic contractile function remained robust across all groups of mice. However, the E/A ratio decreased in KO mice, and IVRT was prolonged in KO mice, while shortened in TG mice, highlighting the crucial role of PAK1 in maintaining diastolic cardiac function. Upon a two-day fast, systolic function was compromised in both WT and KO mice, whereas TG mice exhibited preserved function, as evidenced by FS and EF measurements, emphasizing PAK1's equal significance in sustaining systolic cardiac function. Furthermore, fasting accelerated mitophagy, a process eliminating dysfunctional mitochondria through lysosomal degradation, as observed by mitochondria-associated LC3-II and a dual fluorescent mitophagy reporter. Additionally, we established PAK1 as a positive regulator of mitophagy, suggesting its potential protective role in the heart during fasting via mitophagy modulation. In summary, PAK1 emerges as a critical mediator of cardiac function during fasting, presenting promising avenues for therapeutic interventions aimed at improving cardiovascular health.
Abstract Tu106: Cardiomyocyte knockout of Ceramide Synthase 5 protects against metabolic cardiomyopathy and obesity in mice
Authors: Anna Kovilakath, Jordan Dail, Adolfo Mauro, Yolander Valentine, Maryam Jamil, Mark Subler, Jolene Windle, Martin Kronke, Fadi Salloum, Lauren Cowart
Journal: Circulation Research (2024)

Introduction: The prevalence of metabolic cardiomyopathy in the absence of hypertension is on the rise. Ceramide Synthase 5 (CerS5) has been implicated in the pathophysiology of this cardiomyopathy. Hypothesis: Depletion of cardiomyocyte CerS5 protects against metabolic cardiomyopathy. Methods: Inducible cardiomyocyte-specific CerS5 knockout (CerS5 cKO) mice were generated and placed on a milk-fat diet (HFD) or control diet (CD) for 18 weeks along with control (Myh6-MCM) mice (n=6-11/group). Diet-induced metabolic syndrome was monitored through weekly weight measurements, glucose and insulin tolerance tests, and blood plasma collection for Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). Echocardiography and systemic blood pressure were analyzed at baseline and end of diet. Hearts were assessed for hypertrophy, targeted lipidomics, untargeted metabolomics, and markers associated with cardiometabolic pathophysiology. Results: CerS5 cKO mice on HFD exhibited protection from increased heart mass (Fig. 1A-B, KO:6.1±0.2 vs. control:7.6±0.3 mg/mm, P=0.0001), cardiomyocyte hypertrophy (Fig. 1C, KO:252.6±8.1 vs. control:315.2±14.0 µm 2 , P=0.0084), and reduced ejection fraction (Fig. 1D, KO:65.8±3.0 vs. control:51.7±2.4 %, P=0.0047) compared to controls on HFD. Systemic systolic (SBP) and diastolic (DBP) blood pressures at 18 weeks on diet did not vary between groups (Fig. 1E-F). KO mice on HFD also showed protection from metabolic comorbidities, including obesity (Fig. 2A-B, KO:26.1±1.1 vs. control:30.7±1.7 g, P=0.0192), increased HOMA-IR (Fig. 2C-D, KO:12.6±3.0 vs. control:26.0±3.4, P=0.0015), reduced glucose and insulin insensitivity (Fig. 2E-L) compared to control on HFD. This phenotype was associated with reduced CerS5-derived sphingomyelin C14 (Fig. 3A, KO:8.9±1.4 vs. control:164.5±13.5 pmol/mg protein, P=0.0082), C16 (Fig. 3A, KO:168.0±16.8 vs. control:812.4±287.6 pmol/mg protein, P&lt;0.0001) and ceramide C16 (Fig. 3B, KO:34.6±6.2 vs. control:112.7±16.1 pmol/mg protein, P&lt;0.0001), in the heart. Excessive autophagy (Fig. 3C-D) was observed in the hearts of control mice on HFD, but not in the KO mice on HFD. The heart metabolome of CerS5 cKO mice on HFD was more similar to control mice on CD than control mice on HFD (Fig. 3E). Conclusions: Cardiomyocyte CerS5 depletion is crucial in maintaining cardiac and systemic metabolic homeostasis in mice with cardiometabolic pathophysiology. Excessive autophagy is a novel mechanism through which this may occur.
Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.
Authors: Laura BEN DRISS, Hannah Fandl, Imane Wifak, Julian Wagner, Florian Kleefeldt, Daphne van den Berg, Lucy Xu, Richard Lee
Journal: Circulation Research (2024)

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p&gt;0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.