tbuckley/cabot-search huggingface dataset:
Abstract Tu023: The VEGF-A splice variants as a mechanism of impaired Inflammatory Angiogenesis in the context of advanced aging.
Authors: Chris Mantsounga, Alan Morrison, Olivya Caballero, Crystal Parry, Sheila Sharma, Celia Butler, Jade Neverson, Rachel Carley
Journal: Circulation Research (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 Tu040: A Novel Anti-Thrombotic and Anti-AF Drug without an Adverse Bleeding Problem
Authors: Nikola Ricchiuti, Maxwell Niebrzydowski, Abigail Richardson, Jiajie Yan, Xun Ai
Journal: Circulation Research (2024)

Introduction: Aging is an independent risk factor of atrial fibrillation (AF), which leads to a high mortality due to thromboembolic stroke. But available antithrombotic therapies are limited for the aged AF patients due to aging-associated bleeding risk. We recently revealed critical roles of activated cardiac stress kinase JNK2 in both enhanced AF risk and thrombogenesis in aging through a mechanism of heart-platelet crosstalk. Here, we assessed anti-thrombotic potentials of JNK2 inhibition. Methods: Young (Yg) and aged (2 vs 24 mo) wildtype (WT) mice with and without a JNK2-specific inhibitor (JNK2I, 5 mg/kg i.p.; every other day/4 doses) or meloxicam (0.5 mg/kg/day, i.p.; five days) treatment were used for in vivo FeCl 3 -induced carotid artery occlusion (thrombus formation) and tail bleeding assays. The effects of cardiac JNK2-specific inhibition in thrombogenesis and bleeding were assessed in our unique cardiac-specific mice of MKK7D (inducible overexpression of active MKK7D, a JNK upstream activator) and MKK7D-JNK2dn (overexpressed inactive dominant negative (dn) JNK2 to competitively inhibit endogenous JNK2 in MKK7D mice). Results: Aged mice had a shortened artery occlusion time compared to Yg (5.8±0.2 min vs. 7.1±0.2 min, p&lt;0.001, n=12,7). JNK2I reversed the occlusion time in aged mice to that seen in Yg (p=NS, n=8,7). Yg MKK7D mice also showed a shortened occlusion time compared to WT-controls (p&lt;0.001, n=9,8) that was rescued by cardiac-specific JNK2 inhibition in MKK-JNK2dn mice (p=NS, n=5,8), suggesting a key role of cardiac JNK2 in thrombus formation. Next, we found that 40% of aged WT mice had continuous bleeding compared to its absence in Yg WT. However, meloxicam (an anti-platelet NSAID drug) increased continuous bleeding risk in both aged (83% had it) and Yg (20%) and prolonged the initial clotting time in aged mice (385±56s vs 84±18s in aged controls; p&lt;0.001, n=8,6). In contrast, JNK2I eliminated this bleeding issue, evidenced from the absence of continuous bleeding and shortened initial clotting time compared to aged controls (p=NS, n=8,8). Conclusion: Heart JNK2 enhances thrombotic risk in the blood. JNK2 inhibition could be a promising anti-thrombotic and anti-AF therapeutic approach without adverse bleeding for aged AF patients.
Abstract Tu021: Genome-wide CRISPR Screen In Vivo Decodes Monocyte Infiltration Associated With Abdominal Aortic Aneurysm
Authors: Haocheng Lu, Yang Zhao, Chao Xue, Eugene Chen
Journal: Circulation Research (2024)

Introduction: Aortic aneurysm is the second most common aortic disease following atherosclerosis and accounts for the ninth-leading cause of death overall. The inflammatory response has a crucial role in the Abdominal Aortic Aneurysm (AAA) development. The infiltration of monocyte into aortic wall is a hallmark of aortic inflammation and it still requires further understanding of this complicated process. CRISPR screening is a powerful tool to investigate biological process, but it requires large pool of mutant cells and reliable selection procedure, which can usually only be performed in vitro or in xenograft models. To overcome this problem, we developed an unbiased genome-wide CRISPR-Cas Screen for Monocyte Infiltration in vivo (CRISPR-MI) and employed this new method in mouse AAA model to systematically study key genes regulating monocyte infiltration into aortic wall. Hypothesis: Monocyte infiltration is an important process in AAA development and CRISPR-MI could be utilized to study this process in vivo. Methods: To acquire enough monocytes for screen, we used the adoptive transfer method to transplant in vivo differentiated monocytes (bone marrow derived macrophage, BMDM) back into mouse circulation via tail vein injection. We optimized the lentivirus infection protocol, so that sgRNA library delivered by lentivirus can effectively knock out target genes in BMDMs. The BMDMs were infected with sgRNA library before adoptive transfer into AAA-model mice. After transfer, by comparing the sgRNA enrichment in different organs of receipt mice, we could identify key genes inhibiting monocyte infiltration. Results: CRISPR-MI identified novels genes regulating monocyte infiltration into aorta in AAA model, including Trem2 gene. In vitro experiments confirmed that Trem2 knockout increased adhesion molecules and chemokine receptors expression. Conclusions: CRISPR-MI can be used to identify new therapeutical targets for cardiovascular diseases.
Abstract Tu146: GJA1-20k Promotes Formation of Mitochondrial Actin Cages
Authors: Vu Nguyen, Rachel Baum, Mario Maalouf, JENNIFER HUNTER, Daisuke Shimura, Robin Shaw
Journal: Circulation Research (2024)

Introduction: GJA1-20k, an N-terminus truncated isoform of the gap junction protein Connexin 43, is stress responsive and mimics ischemia preconditioning, reducing ischemia-reperfusion (IR) injury. GJA1-20k localizes to the outer membrane of mitochondria, stabilizes actin, and mediates an actin mediated mitochondrial protection. The mechanism is not known how GJA1-20k and actin work towards mitochondrial protection. Methods: Cell-free TIRF imaging was performed to study direct interactions between actin and purified GJA1-20k. High-resolution confocal microscopy was applied to visualize GJA1-20k and actin organization around mitochondria in intact HEK cells and cell-free suspensions wherein rhodamine-labeled actin, purified GJA1-20k, and isolated mitochondria were incubated together. Results: Cell-free TIRF imaging establishes that GJA1-20k directly binds to actin , forming both actin clusters and stabilized actin filaments. As expected, live-cell imaging reveals that GJA1-20k is enriched at the mitochondrial outer membrane. The direct binding of GJA1-20k and actin results in a rich collection of actin around mitochondria, as evidenced in both live-cell imaging and cell-free suspensions containing only purified actin, GJA1-20k, and isolated mitochondria. 3D reconstruction reveals that mitochondrial localization of GJA1-20k causes formation of dense actin sheets enveloping mitochondria, which we call “mitochondrial actin cages” which appear to limit the ability of mitochondria to swell under stress conditions. Conclusions: GJA1-20k induced actin cages could be critical to GJA1-20k mediated protection against IR injury. The observed actin around mitochondria could prevent pathological mitochondrial swelling, preserving mitochondrial integrity and function in oxidative stress conditions.
Abstract Tu056: A Two-Hit HFpEF-like Mouse Model with Accelerated Disease Onset
Authors: Sarita Nehra, Manoj Dokania, Ankit Gupta, Ricardo Garcia, Shailesh Dudhgaonkar, Debarati Mazumder Tagore, Neethu CK, Somnath Wagh, Prajakta Kale, Sabariya Selvam, Amit Anand, Joseph Luettgen, Nikki Bennett, Jyoti Gulia
Journal: Circulation Research (2024)

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a heterogenous syndrome with multiple co-morbidities and has limited therapeutic options. To better understand the underlying mechanisms and develop novel therapies, it is important to focus on development of pre-clinical models that mimic the multi-factorial nature of HFpEF. Goal &amp; Methodology: We aimed to develop a mouse HFpEF model by incorporating unfavourable metabolic axis, in db/db mice carrying mutation in lectin receptor, with Ang-II induced impaired hemodynamic conditions. Briefly, we infused Angiotensin II (Ang II, 1500 ng/kg/min) subcutaneously in 12-week-old wild type (WT) and db/db mice for 4 weeks (Fig 1). SGLT2 inhibitor (SGLT2i) was used as a pharmacological intervention. Results: The db/db + Ang II mice showed impaired diastolic function reflected by increase in E/A, E/e’ and global longitudinal strain. This increase was not observed in the db/db + Ang II mice upon treatment with SGLT2i. The ejection fraction was preserved across all groups. Interestingly, db/db + Ang II group did not develop cardiac hypertrophy &amp; fibrosis as indicated by unchanged LV mass, picosirius red staining and unaltered expression of Nppa, Nppb &amp; Acta2 transcripts. Investigation of lipid metabolism showed reduction of fatty acid synthase in db/db (+/-Ang II) groups in comparison to WT animals. Also, phosphorylation of ATP citrate lyase in db/db + Ang II group was diminished compared to db/db animals. Among the glucose metabolism markers, lactate dehydrogenase and pyruvate dehydrogenase showed decreasing trend in all groups compared to WT group. Conclusion: The present mouse model recapitulates metabolic and hemodynamic aspects of HFpEF pathophysiology. Our model is useful for basic research as well as quick evaluation of targets and assets focused on cardiac metabolism with the advantage of faster disease onset and clinically relevant echocardiographic endpoints.
Abstract Tu055: Prominent effects of p38 MAPK inhibition on the phosphoproteome of a guinea pig model of heart failure and sudden cardiac death
Authors: Sogol Sedighi, Ting Liu, Brian O'Rourke, D Foster
Journal: Circulation Research (2024)

Introduction: The p38 mitogen-activated protein kinase (p38 MAPK), activated by stress, plays a role in cardiac hypertrophy and heart failure (HF). The precise mechanism of action of p38 MAPK remains unclear, which could explain the mixed outcomes in P38 inhibitor trials. Here, we examine the impact of p38 MAPK inhibition on cardiac function and the proteome/phosphoproteome of the failing heart. Objective: To ascertain p38 MAPK’s impact on the progression of HF by inhibiting it with SB203580 (SB) in a guinea pig model of HF and sudden cardiac death. Methods: We assessed the proteomic changes in a previously described guinea pig HF model of ascending aortic constriction and once-daily isoproterenol administration (ACi). Three groups were included: sham-operated controls (Control), ACi, and ACi plus SB treatment (ACiSB). Cardiac function was evaluated by M-Mode echocardiography and proteome/phosphoproteome analysis was performed by multiplexed TMT-labeling and LC-MS/MS. Results: Inhibition of p38 MAPK with SB attenuated but did not fully block HF progression. At 5 weeks, fractional shortening was significantly improved in the ACiSB group compared to ACi (p=0.0037). Furthermore, it significantly decreased cardiac hypertrophy and lung edema (p-values &lt;0.0001 and 0.0003 respectively). Compared to our previous study on the impact of a mitochondrially-targeted antioxidant (MitoTEMPO), SB protected a smaller subset of protein expression changes in HF; However, its impact on the phosphoproteome was substantial, extending beyond known p38 substrates. SB suppressed HF-induced inflammatory pathway proteins, modestly protected against downregulation of some mitochondrial respiratory chain proteins, and prevented maladaptive phosphorylation of several proteins involved in excitation-contraction coupling, including the ryanodine receptor, phospholamban, and cytoskeletal protein phosphorylation. Conclusion: p38 MAPK inhibition offered significant protection against HF, primarily affecting the phosphoproteome with modest effects on protein expression. The data suggests that narrowly targeted p38 MAPK-driven phosphoproteome modifications contribute to cardiac decompensation, despite broad protein expression changes in HF. Differences between MitoTEMPO and SB effects on the HF proteome highlight the plethora of remodeling pathways triggered by mitochondrial ROS including p38 MAPK activation. Our findings emphasize the importance of preventing p38 MAPK to preserve cardiac function.
Abstract Or103: Troponin I Serine 150 Phosphorylation as a Novel Cardiac Inotrope Without Detrimental Effects
Authors: Lorien Salyer, Sarah Sturgill, Helena Zanella, Elizabeth Brundage, Cemantha Lane, Thomas Hund, Mark Ziolo, Brandon Biesiadecki
Journal: Circulation Research (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 Mo116: Evaluating <i>MYBPC3</i> and <i>MYBPHL</i> missense mutations on sarcomere incorporation
Authors: Kelly Araujo, Alexandra Pena, Alejandro Alvarez-Arce, Lucas Wittenkeller, David Barefield
Journal: Circulation Research (2024)

Hypertrophic cardiomyopathy (HCM) is one of the common genetic heart diseases and causes hypercontractility and pathological thickening of the myocardium. Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of cardiac contractility and MYBPC3 mutations accounts for 40% of HCM cases, resulting in the truncation and loss of cMyBP-C. While cMyBP-C is expressed in both atria and ventricle, its recently identified homologous partner, myosin binding protein-HL (MyBP-HL) is solely expressed in the atria. Loss of function mutations in MYBPHL cause dilated cardiomyopathy in humans and mice. MyBP-HL and cMyBP-C bind to specific positions on the thick filament within the C-zone of sarcomere. In the atria, there is a 1:1 ratio between MyBP-HL and cMyBP-C, whereas cMyBP-C is solely expressed in the ventricle at double the cMyBP-C content of the the atria, establishing a stoichiometric relationship between these two proteins in their ability to bind to the thick filament within the sarcomere. Within the ventricle, heterozygous missense mutations within the necessary myosin binding domains of cMyBP-C may prevent proper myosin binding and sarcomere incorporation, but function may still be preserved due to the other unaffected allele. Since the atria expresses MyBP-HL and cMyBP-C, we hypothesize that MYBPC3 and MYBPHL missense mutations are more severe due to the competition for binding sites within the sarcomere between these proteins. We have identified MYBPC3 and MYBPHL missense mutations and variants to evaluate their pathogenicity and sarcomere incorporation via immunofluorescence microscopy within neonatal rat ventricular cardiomyocytes. To test these mutations, we created a “Mini-C” construct consisting of the thick filament binding domains of cMyBP-C. We validated our Mini-C construct via transfection and co-localization with endogenous cMyBP-C in neonatal rat ventricular cardiomyocytes (NRVMs). Our data demonstrate that known pathogenic MYBPC3 missense mutations have improper sarcomere incorporation within NRVMs. Disease associated MYBPHL missense mutations also show improper sarcomere incorporation. To test the effects of missense mutations on myosin binding protein stoichiometry within the sarcomere, we have created and validated a T2A/P2A construct, expressing our Mini-C construct, a fluorescent reporter, Td-Tomato, and our hMyBP-HL construct at consistent and reproducible expression levels via western blot and mass spectrometry.
Abstract Tu145: Exploring Naltrindole’s hypercontracture attenuating effects during myocardial ischemia via a novel mechanism that produces cardioprotective effects in myocardial ischemia/reperfusion injury
Authors: Cameron Stinson, Mai An Le, Logan Clair, James Ramsarran, Desmond Boakye Tanoh, Juliet Melnik, Tameka Dean, Qian CHEN, Robert Barsotti, Lindon Young
Journal: Circulation Research (2024)

Introduction: Previously, naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5µM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. Additionally, we recently showed that NTI 100-200µM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. Hypothesis: We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca 2+ ). Testing this hypothesis, we conducted isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca 2+ via inhibition of reverse mode Na + /Ca 2+ exchanger and, therefore, should attenuate myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). We predict that both NTI and KB will improve LVEDP and decrease infarct size compared to vehicle (dH 2 O) control and naloxone (NX) used as a negative control. Methods: Hearts were isolated from anesthetized male Sprague Dawley (SD) rats (~300g). We then infused NTI 1.25-5µM, KB 10-20µM, or NX 10µM for 5 min into perfused hearts, just prior to global I(30min)/R(45min). Cardiac left ventricular function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in the MIR assay. Data were analyzed using ANOVA and Fishers post-hoc analysis, with p&lt;0.05 considered statistically significant. Results: MIR assay: NTI 5µM, 2.5µM, 1.25µM and KB 20 µM reduced infarct size (4.8±3%, n=5, p&lt;0.05), (6.8±0.8%, n=6, p&lt;0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p&lt;0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10µM controls (18±3%, n=3). NTI 5 µM and KB 20 µM had significantly improved LVEDP to (32.4±11%, n=5, p&lt;0.05) and (22.8±4%, n=5, p&lt;0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10µM controls (54.2±8%, n=3) Conclusion: Both NTI (2.5µM, 5µM) and KB (20µM) reduced infarct size and improved LVEDP. The novel MOA of NTI may be mediated via a reduction in intracellular Ca 2+ during myocardial ischemia. The infarct reducing effect of NTI can benefit patients undergoing elective PCI, CABG, and transplant procedures.
Abstract Mo112: ARA290, a small peptide non-erythropoietic Erythropoietin derivative, ameliorates heart functional deterioration in a chronic cardiac stress model
Authors: Suri Gime, Nolan Winicki, Ismayil Ahmet, Christopher Morrell, Edward Lakatta
Journal: Circulation Research (2024)

Objective: To determine whether ARA290 can prevent or delay age-related declines in cardiac function and structure in the TG AC8 heart failure model. Methods: The study consists of 9 TG AC8 + mice that were randomly assigned to treatment group (n=5) or non-treatment saline group (n=4). Both groups received intraperitoneal injections of ARA290 (100 μg/kg) or saline tri-weekly. Monthly echocardiographic assessment of heart systolic function prior to, and monthly during 7 months of treatment was performed starting at 10 months of age. Two Way ANOVA was used to analyze the data. Results: At baseline, 10 months of age, prospective treatment and non-treatment group did not exhibit any statistically significant difference in their measure of ejection fraction (EF), fractional shortening (FS), and stroke volume (SV) ( P &gt; 0.05). ARA290 treatment preserved EF, FS, and SV, (compared to a progressive, time dependent deterioration of these systolic functions in the saline treated group). Significant interaction terms for EF ( P &lt; 0.04), FS ( P &lt; 0.03), and SV ( P &lt; 0.02), and time indicated that the ARA290 effect depended upon treatment time. Conclusion: The prevention of the decline in heart systolic functional by ARA290 suggests that this small peptide derivative of EPO has therapeutic potential as a novel intervention to retard declines in cardiac function that accompany chronic heart diseases.