bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu037: Unraveling the Cellular Mechanisms of Cardiogenic Liver Disease
Authors: Park Arick , Parvathaneni Adeesh , Fu Christina , Byrnes Kathleen , Schilling Joel
Published: October 2024

Background:
Right heart failure (RHF) is associated with chronically elevated central venous pressures which can lead to hepatic dysfunction, fibrosis, and ultimately cirrhosis. Despite the prognostic importance of liver disease in RHF little is understood about the cellular and molecular changes that define cardiogenic liver disease. Filling this knowledge gap has the potential to lead to better diagnostic and therapeutic approaches for patients with heart failure and liver dysfunction.

Aims:
1) To evaluate the key clinical and pathologic features of cardiogenic liver disease in humans 2) To explore shifts in the cellular landscape in the congested liver using human samples and a mouse model congestive hepatopathy.

Methods:
We identified 34 patients with heart failure who underwent liver biopsy due to concern for cardiogenic liver disease. These biopsy samples were stained for numerous cellular markers (macrophage subsets, activated stellate cells, endothelial cells, biliary cells) and compared to normal controls. Clinical data was also collected for the included patients. To model cardiogenic liver disease in a mouse we performed partial IVC ligation (pIVCL) to induce liver congestion and then assessed the liver response using flow cytometry, tissue imaging and mRNA analysis.

Results:
Liver biopsies from patients with cardiogenic liver disease demonstrated increased liver fibrosis, macrophage aggregates, and biliary metaplasia. The severity of biliary metaplasia was closely associated with the extent of liver congestion. Using multi-marker staining the metaplastic cells were found to be of hepatocyte origin. Interestingly, the liver niche containing metaplastic biliary cells was also enriched for recruited macrophages and activated stellate cells. Similar pathologic features were observed in the mouse model of liver congestion.

Conclusions:
In this study we identified biliary metaplasia and macrophage recruitment as key pathologic features of cardiogenic liver disease. Ongoing investigation of the interplay between macrophages and metaplastic biliary in humans and animal models of congestive hepatopathy may lead to the discovery of novel disease mechanisms and biomarkers for liver dysfunction in heart failure.
Abstract Tu039: Intestinal atrophy contributes to low-grade inflammation, cardiac dysfunction and metabolic syndrome-like phenotype in a model of heart failure with preserved ejection fraction.
Authors: Tran Duyen , Neves Liomar , Bonilla Karina , Chowdhury Shamim , Malczewski Natalie , Rosas Paola
Published: October 2024

Introduction:
Heart failure with preserved ejection fraction (HFpEF) comprises half of all HF cases and lacks effective treatment. HFpEF is now seen as a systemic syndrome often linked to comorbidities like hypertension, obesity, and diabetes. These conditions are commonly associated with unhealthy diets rich in fats but lacking in fiber. We explored the effects of a 3-week exposure to excess mineralocorticoids, salt, and high fat diet (HFD) deficient in fiber. We hypothesize that these challenges lead to intestinal atrophy, contributing to low-grade inflammation that promotes the progression of heart failure and metabolic disease.

Methods:
We used a mix of 1% saline in drinking water and subcutaneous implantation of controlled-release DOCA pellets (0.83 mg/day) alongside a high-fat diet (60% fat) in 8-week-old female and male C57BL6/J mice. Control mice received a standard chow diet and regular water.

Results:
The challenge resulted in significant reductions in colon mass, colon length, crypt length, and mucus layer thickness compared to the control group. Additionally, elevated levels of C-reactive protein and IL-6 were detected in the plasma. These changes were accompanied by alterations in diastolic function, including decreased peak myocardial relaxation velocity (e'), elevated E/e' ratio, increased E/A ratio, and prolonged isovolumetric relaxation time (IVRT), while the ejection fraction (EF) remained unchanged. Contractility parameters, such as peak myocardial contraction velocity (s'), declined in challenged mice, along with diminished stroke volume (SV). We also found increased organ weight in the liver, spleen, and lungs indicative of organ congestion. Furthermore, signs of metabolic syndrome were identified, including increased body weight, increased adiposity characterized by gonadal and subcutaneous fat accumulation, and dysglycemia (see Data Table).

Conclusions:
Our data suggests that in this model, heart failure triggers intestinal hypoperfusion, resulting in intestinal atrophy and compromising the integrity of the intestinal lining. Additionally, a diet rich in fat and lacking in fiber alters the gut environment by diminishing mucus production and promoting a closer interaction between the microbiome and host cells. This compromised mucus layer and intestinal barrier facilitates bacterial wall products to move into the circulation, resulting in a pro-inflammatory state that subsequently affects both cardiac function and metabolism.
Abstract Tu055: Prominent effects of p38 MAPK inhibition on the phosphoproteome of a guinea pig model of heart failure and sudden cardiac death
Authors: Sedighi Sogol , Liu Ting , O'Rourke Brian , Foster D
Published: October 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 <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 Tu060: Unveiling Arrhythmogenic Right Ventricular Dysplasia In Pregnancy
Authors: Jyothi Ramachandran Nair Devi Parvathy , SHAH SMIT , Scheffler Morgan , Basnet Arjun , Shaikh Atif
Published: October 2024

Introduction:
Arrhythmogenic right ventricular dysplasia (ARVD) is a rare autosomal dominant disease, caused by desmosomal gene mutations, and characterized by fibrofatty replacement of right ventricular (RV) myocardium with left ventricular (LV) involvement in advanced stages. It is mostly diagnosed between the second and forth decades of life and can present with ventricular tachycardia and arrhythmic sudden death. Here we present a case of ARVD diagnosis in a female in her forth pregnancy.

Case description:
A 28-year-old female, on her 27
th
week of gestation (G4P3) was admitted to the hospital with preterm premature rupture of membranes. She also endorsed intermittent chest pressure, palpitations and shortness of breath. Her vital signs were stable, and electrocardiogram (ECG) revealed normal sinus rhythm with frequent premature ventricular contractions (PVCs) and T wave inversions in leads V1- V3. Laboratory studies showed normal troponin and D-dimer levels, and slightly elevated brain natriuretic peptide 240 pg/mL. Patient notably had frequent runs of monomorphic non-sustained ventricular tachycardia (NSVT) arising from RV apical territory, with left bundle branch morphology on telemetry. An echocardiogram revealed a dilated right atrium and RV with hypokinesis of RV; LV size and function were normal. Overall, patient met the revised task force diagnostic criteria for ARVD based on PVC morphology, abnormal ECG and dilated and hypokinetic RV. She had no sustained or hemodynamically unstable VT, no history of syncope or family history of sudden cardiac death and did not meet class I indication for implantable cardioverter defibrillator. She was started on oral metoprolol succinate 25 mg daily with which her arrhythmias significantly improved. She was recommended a cardiac magnetic resonance imaging and genetic testing for confirmation and to assess familial implications.

Conclusion:
Recognition of ARVD during pregnancy is challenging given the overlapping symptoms of normal pregnancy-related changes. Hormonal surge and sympathetic drive may have driven the increased frequency of NSVT in our patient and beta blocker was effective in managing these. Early recognition and tailored management are crucial to ensure the well-being of both the mother and the fetus. A close follow-up is necessary to understand the long-term implications for the patient and potential genetic predisposition in the family.
Abstract Tu065: The role of heavy metals in pulmonary arterial hypertension pathogenesis
Authors: Cathey Dakotah , Cai Lu , Cai Jun , TAN YI , Huang Jiapeng
Published: October 2024

Pulmonary arterial hypertension (PAH) is a disease where chronic high pulmonary arterial pressure causes inflammation, remodeling, and dysfunctions of lung endothelial/smooth muscle cells, and right ventricle (RV) cardiomyocytes. Pathogenesis is caused by a mixture of genetic, epigenetic, and environmental factors. Our clinical studies found PAH patients had elevated cadmium (Cd) and antimony (Sb) levels in the blood and/or urine, which correlated with increased severity of PAH parameters. Thus, we hypothesize that environmental factors, specifically chronic heavy metal exposure, could contribute to PAH pathogenesis either directly or indirectly.

We tested if Cd could induce PAH or worsen PAH parameters in the mouse SU5416 and hypoxia (SuHx) PAH model. Forty male C57/6J mice were initially split in 2 groups: control and 5ppm Cd in drinking water (n=20/each) for 8 weeks (wks). Diastolic and systolic functions were evaluated with echocardiography (echo) at baseline, and 8ks. Then, half of these mice were subject to PAH induction, netting 4 subgroups (n=10/group): CTRL, Cd, PAH, and Cd+PAH. Echo was performed again 4 wks post-PAH induction, followed by RV systolic pressure (RVSP) measurement (n=3/group). The remaining 7 mice/group were euthanized for heart/lung histopathology focusing on fibrosis and hypertrophy, and protein western blot analyses. GraphPad Prism 8 software was used for statistical analysis with normality checked. When data did not follow normality, the non-parametric equivalence was used. Two-way and one-way ANOVA with Tukey were performed for echo/RVSP and histological/biochemical analyses respectively, with a p-value<0.05 being significant.

Results showed 12-wks Cd exposure alone caused: (1) significantly increased RV systolic function indicating systolic stimulation, and (2) significantly increased collagen contents in both ventricles and LV cardiomyocyte area when compared to CTRL. Twelve-wks Cd+PAH group demonstrated (1) significantly decreased RV cardiac output and RVSP measurements indicating RV failure; (2) no further RV changes in collagen contents or hypertrophy; (3) LV western blot analysis revealed significant upregulation of antioxidant proteins, including catalase and superoxide dismutase 2, when compared to PAH group. Further mechanistic experiments are being conducted, including whether Sb has similar results as Cd.
Abstract Tu068: Calmodulin Kinase II is a Mutation-Specific Driver of Disease in Hypertrophic Cardiomyopathy
Authors: Hauck Garrett , Vasquez Catherine , Lynn Melissa , Klass Matthew , Langlais Paul , Hamilton Shanna , Tardiff Jil
Published: October 2024

In the heart, Ca/Calmodulin Kinase IIδ (CaMKIIδ) functions to maintain electromechanical and calcium homeostasis. Post-translational modifications (PTMs) of CaMKIIδ render its activity autonomous of Ca/CaM. In hypertrophic cardiomyopathy (HCM), increased levels of autonomous CaMKIIδ are linked to disease progression. We have shown that the pathogenic role of CaMKIIδ in HCM is mutation-specific: autonomous CaMKIIδ is elevated in cardiac troponin T (cTnT)-R92W mice, but not in cTnT-R92L mice. These results pose a clinically relevant and critical question: what is the trigger for CaMKIIδ dysregulation in HCM, such that it is only found with certain mutations? We hypothesize that CaMKIIδ dysregulation results from distinct modifications in thin filament (TF) biophysical properties altered by point mutations. cTnT-R92W TFs display faster calcium dissociation kinetics compared to wild type TFs, whereas cTnT-R92L TFs have slower dissociation kinetics, thus we propose accelerated calcium dissociation kinetics as a mutation-specific trigger of CaMKIIδ dysregulation. To test this, we will use mass spectrometry following CaMKIIδ immunoprecipitation to assess the levels of phosphorylation at Thr-287 and oxidation at Met-281/282 in mice expressing the R94H, I79N, or R92W mutations in cTnT, which exhibit differing calcium dissociation kinetics. Preliminary data shows that CaMKIIδ PTMs can be readily detected. We will also use the CaMKIIδ biosensor CaMKAR in cardiomyocytes isolated from these mice to evaluate CaMKIIδ activity. We have shown that genetic inhibition of CaMKIIδ in cTnT-R92W mice improves diastolic function and atrial remodeling. We also aim to test the efficacy of the small molecule CaMKIIδ inhibitor ruxolitinib (rux) in cTnT-R92W mice. Two-week treatment of these mice with rux improves systolic and diastolic function, and has no effect in nontransgenic controls, implicating small molecule inhibition of CaMKIIδ as a potential therapeutic option in HCM. To continue these studies, we will explore changes in calcium handling in the three mouse models to further establish potential mechanistic pathways for CaMKIIδ dysregulation in HCM and will extend rux treatment to longer timepoints to further evaluate its efficacy as an HCM therapeutic.
Abstract Tu071: Computational Modeling to Predict Mechanisms of DYRK1A-mediated Inhibition of Cardiomyocyte Proliferation
Authors: Murillo Bryce , Zhao Catherine , Wu Michelle , Harris Bryana , Wintruba Kaitlyn , Saucerman Jeffrey , Wolf Matthew
Published: October 2024

Introduction and Background:
Adult cardiomyocytes lack strong proliferative capability with only a small subset being able to undergo mitosis. This can lead to poor functional recovery after heart injuries such as myocardial infarctions (MI). We have previously demonstrated that inhibition of DYRK1A, a regulator of the DREAM complex and cell quiescence, increases cardiomyocyte cycling. However, the mechanism of DYRK1A inhibition that induces this increased cycling has yet to be elucidated. Therefore, we built a network model that encapsulates potential components that can lead to cardiomyocyte proliferation.

Methods:
We developed a logic-based differential equations model to simulate DYRK1A-mediated contributors of cardiomyocyte proliferation and G1/S phase transition. Various knock-down/knock-out and overexpression simulations were performed to predict targets of cell cycle regulation. Literature validation was performed to compare experiments across cancer, cardiomyocytes, and broader cell types to independent model simulations. A sensitivity analysis was used to identify the influence of particular proteins/genes on cardiomyocyte proliferation.

Results:
Our model simulations validated against (n = # of primary literature articles) 84.6% of literature experiments in cancer cell lines (n = 13), 88.8% in broader cell types (n = 9), and 100% in cardiomyocytes (n = 5). Simulated knockouts of DYRK1A and overexpression of Cyclin-D, Cyclin-E, and B-MyB increased DNA replication, indicating exit from G
0
phase. Sensitivity analysis showed that a DYRK1A knockout was the most influential in terms of increasing DNA replication.

Conclusion:
Our model’s predictions were successfully validated against literature in several different types of cell lines. Furthermore, it gives valuable insight on possible targets that can induce cardiomyocyte proliferation in addition to DYRK1A. Further in vitro and in vivo experiments are necessary to fully evaluate our simulations in cardiomyocytes.
Abstract Tu076: N4-acetylcytidine-regulated Cardiomyocyte Ferroptosis Mediates Cardiac Benefits of Exercise in Aging
Authors: Kang Jiayi , wang Chunyan , Sugai-Munson Aya , Liu Zipeng , Rhee James , LI Haobo
Published: October 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<0.05 vs Sed) and senescence (increased gene expression of p16, p21, and PAI, all p<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<0.05 vs Sed), and decreased senescence gene expression. Cardiac NAT10 was downregulated in aged mice and was upregulated by exercise (all p<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<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 Tu077: Targeting β-adrenergic receptor resensitization attenuates cardiac dysfunction
Authors: Sun Yu , Mohan Maradumane , Stenson Kate , Naga Prasad Sathyamangla
Published: October 2024

β-adrenergic receptor (βAR) is a key regulator of cardiac function. Agonist activation of βAR leads to phosphorylation-mediated desensitization resulting in βAR trafficking to endosomes. Endosomal βARs undergo resensitization through protein phosphatase 2A (PP2A)-mediated dephosphorylation. We have shown that stimulation of βARs activates phosphoinositide 3-kinase γ (PI3Kγ) that phosphorylates endogenous inhibitor of PP2A (I2PP2A) promoting robust binding to PP2A resulting in PP2A inhibition. βAR dysfunction due to desensitization is considered as a classical hallmark of heart failure, while less is known about resensitization. Plasma membrane and endosomal fractions were isolated from non-failing and failing patient samples to determine whether βAR resensitization is altered in human heart failure. Endosomes from heart failure samples showed significant accumulation of phosphorylated β2ARs which was associated with reduced adenylyl cyclase and βAR-associated PP2A activity compared to non-failing showing that resensitization is inhibited in human heart failure. Since PP2A activity is inhibited by robust binding of I2PP2A to PP2A, we hypothesized that disrupting I2PP2A interaction with PP2A would unlock PP2A inhibition normalizing βAR resensitization. In silico modeling and biochemical studies mapped the PP2A interaction with I2PP2A to the C-terminal region of PP2A (PP2Act). Agonist isoproterenol (ISO) treatment of cells expressing PP2Act showed significant reduction in β2AR phosphorylation along with increased cAMP generation and normalized PP2A activity showing preserved βAR resensitization. While expression of PP2A mutant with deletion in the PP2Act (ΔPP2Act) did not preserve βAR resensitization. Challenging mice with cardiomyocyte-specific expression of PP2Act to ISO showed preserved cardiac function providing insights that targeting resensitization could potentially be a novel therapeutic strategy.

Key words:
Resensitization, I2PP2A, PP2A, Cardiac hypertrophy
Abstract Tu081: T-tubule microdomains promote protective mitophagy in failing hearts
Authors: Richmond Bradley , Li Jing , Funai Katsuhiko , Shaw Robin , Hong Tingting
Published: October 2024

Heart failure (HF) with preserved ejection fraction (HFpEF) represents about 50% of all HF incidence, yet with limited therapeutic options. Although poorly understood, a key pathophysiology of HFpEF includes mitochondrial dysfunction and increased oxidative stress. We recently identified that abnormal remodeling of the cardiomyocyte t-tubule microdomains organized by the scaffolding protein cardiac bridging integrator 1 (cBIN1) contributes to HFpEF progression in
db/db
mice with type 2 diabetes, which can be rescued by cBIN1 gene therapy. In
db/db
mice, cBIN1 gene therapy (2x10
12
vg/kg) rescues HFpEF by normalizing t-tubule microdomains. With transmission electron microscopy imaging, here we report that t-tubule cBIN1-microdomains are in close proximity to mitochondria and can correct abnormal mitochondrial accumulation in
db/db
cardiomyocytes (Figure 1A-B). Oroboros and seahorse analysis indicates that reduced mitochondrial calcium retention, elevated oxidative stress, and impaired oxygen consumption in diabetic cardiomyocytes can be normalized by cBIN1 therapy (Figure 1C-E). Furthermore, proteomics analysis indicates accumulation of mitochondrial proteins in
db/db
cardiomyocytes can be reversed by the therapy. cBIN1-normalized mitochondrial number, proteomics, and function in
db/db
cardiomyocytes are associated with activation of LC3 but not changes in PGC1a, indicating increased mitophagy by the therapy. Mechanistic exploration in rat myoblast H9C2 cells indicates that FCCP stress induces mitophagy as evidenced by elevated LC3II/I ratio, which is further accumulated with autophagic flux blockade with chloroquine. Knockdown of BIN1 results in further accumulation of LC3II/I in FCCP-stressed cells (Figure 1F with quantification in the bar graph below), which is abolished by chloroquine. These data indicate that cBIN1 promotes protective mitophagy in diabetic failing hearts, suggesting its therapeutic potential for HFpEF in general.
Abstract Tu082: Novel Structural and Biochemical Effects of Nexilin Associated Hypertrophic Cardiomyopathy
Authors: Chittora Harshil
Published: October 2024

Hypertrophic cardiomyopathy is primarily a genetic disease that results in abnormal cardiac muscle phenotypes characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Both sarcomere structural and signaling genes are associated with HCM. Among various genes, one of the implicated genes for HCM is Nexilin (NEXN). Nexilin is an F-actin binding protein widely expressed in muscle cells and localized at sarcomere and T-tubules. It is known to have a surprising role in insulin signaling. The role of Nexilin mutations is poorly studied. We hypothesized that Nexilin missense variants in genetically engineered human pluripotent stem cells (hPSCs) perturb essential cardiac metabolic signaling pathways leading to HCM. In our Indian HCM cohort, we identified seven Nexilin mutations through exome sequencing and categorized them as pathogenic per ACMG/AMP classification. A representative novel mutant in Nexilin p.Q131T was introduced in hPSC with the CRISPR-Cas9 knock-in method and was differentiated into cardiomyocytes, recapitulating patient-specific heterozygous mutation. Our data shows that Nexilin p.Q131T causes an increase in cell size and activation of fetal gene transcriptional programs in hPSC-derived cardiomyocytes. Global transcriptomics was performed to understand transcriptional changes and investigate further. We observe the significant activation of insulin-related signaling pathways, including AKT/MTOR and MAPK, confirmed by immunoblotting. Akt-mTOR and MAPK cascades are well-known to be regulated by IRS-1 signaling. Our studies show hyperactivation of these cascades, suggesting IRS-1 involvement in the pathogenesis. Nexilin and IRS-1 interaction were probed and found to be perturbed, hence causing higher activation of IRS-1 and its downstream targets. In addition, Nexilin wild type and mutant protein structure-functional analysis were performed with purified proteins. Using cryo-EM structural analysis, we identified unique F-actin binding sites for Nexilin for the first time, and the mutant p.Q131T showed decreased binding affinity to F-actin. We report Nexilin's high-resolution cryo-EM structure and a novel metabolic signaling mechanism where mutations in Nexilin cause dysregulation of IRS-1 signaling in the gene-edited cardiomyocytes, leading to HCM pathogenesis.
Abstract Tu083: Oxytocin improves cardiovascular outcomes in a rat model of pressure-overload heart failure
Authors: Alber Bridget , Cheung Emily , Russo Rebekah , Mendelowitz David , Kay Matthew
Published: October 2024

Oxytocin (OT) is a peptide made in the hypothalamus and released into circulation by the pituitary gland. Known for its role in childbirth and lactation, recent research suggests that OT has significant cardiovascular benefits, reducing the risk of cardiovascular diseases such as hypertension and atherosclerosis. We tested if OT has beneficial effects on cardiovascular function in a rat model of pressure overload heart failure (HF). Neonatal Sprague Dawley rats underwent transaortic constriction (TAC). Pressure overload and cardiac dysfunction progressed as animals matured. Echocardiography at 5 weeks of age confirmed reduced ejection fraction. Animals were then randomly assigned to receive daily IP injections of either OT (treatment) or saline (control) for two weeks. Echocardiography was performed again before hearts were excised for optical mapping studies to test for electrophysiological differences between groups. Hearts were electromechanically uncoupled using blebbistatin and transmembrane potential (RH237) and intracellular Ca2+ (Rhod2am) were simultaneously mapped during a dynamic restitution protocol. Preliminary results indicate that rats in the treatment (OT, n=3) group had improved cardiac function compared to controls (saline, n=3), with higher ejection fraction (66.71 ± 9.44 vs. 25.83 ± 17.93, p=0.0125), higher cardiac output (62.4 ± 11.45 vs. 20.35 ± 6.12, p=0.0025), and greater stroke volume (180.2 ± 18.19 vs. 73.96 ± 37.29, p=0.0057). Preliminary optical mapping results indicate that the hearts of OT-treated rats (n=3) have lower steady state action potential duration (APD) compared to controls (n=2). APD60 decreased from 109.5 ± 10.09 to 75.79 ± 21.94 (p=.0725) and APD80 decreased from 133.1 ± 10.22 to 101.1 ± 24.5 (p=.09595), indicating preservation of repolarizing currents corresponding to healthier electrophysiological function. In summary, our preliminary results suggest exogenous IP OT administration could have a potent cardioprotective effect on chronic and severe pressure overload hearts. Future work will investigate potential mechanisms of OT mediated cardio-protection, including sites of action.
Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.
Authors: BEN DRISS Laura , Fandl Hannah , Wifak Imane , Wagner Julian , Kleefeldt Florian , van den Berg Daphne , Xu Lucy , Lee Richard
Published: October 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>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.
Abstract Tu100: Multiscale Drug Screening for Cardiac Fibrosis Identifies MD2 as a Therapeutic Target
Authors: Zhang Hao , Thai Phung , Shivnaraine Rabindra , Ren Lu , Wu Xuekun , Siepe Dirk , Liu Yu , Tu Chengyi , Caudal Arianne , Mukherjee Souhrid , Leitz Jeremy , Wen Wilson , Shin Hye , Liu Wenqiang , Chiamvimonvat Nipavan , Wu Joseph
Published: October 2024

Cardiac fibrosis impairs cardiac function, but no clinical therapies exist. To address this unmet need, we employed a high-throughput drug screening for antifibrotic compounds using human iPSC-derived cardiac fibroblasts (CFs) (Figure 1). Counter-screening of the initial candidates using iPSC-derived cardiomyocytes and endothelial cells excluded hits with toxicity. This screening process identified artesunate as the lead compound. Following profibrotic stimuli, artesunate inhibited proliferation, migration, gel contraction in human primary CFs, reduced collagen deposition and improved contractile function in 3D-engineered heart tissues. Artesunate also attenuated cardiac fibrosis and improved cardiac function in a heart failure mouse model. Mechanistically, artesunate molecularly targeted myeloid differentiation factor 2 (MD2) and inhibited MD2/toll-like receptor 4 (TLR4) signaling pathway, alleviating fibrotic gene expression in CFs (Figure 2). Our study is the first to use multiscale drug screening, which combines human iPSC platform, tissue engineering, animal models,
in silico
simulation, and multiomics to identify MD2 as a therapeutic target for cardiac fibrosis.
Abstract Tu110: CD36-Mediated Transendothelial Fatty Acid Transport Determines Cardiomyocyte Uptake and is Critical to Limiting a Lipotoxic Ceramide Profile and Supporting Cardiac Function during Pathological Stress.
Authors: Challa Azariyas , Carley Andrew , Maurya Chandan Kumar , Wang Yang , Fasano Matthew , Goldberg Ira , Lewandowski E Douglas
Published: October 2024

The heart primarily relies on long chain fatty acid oxidation (LCFA) to meet ATP demand. CD36, is a transmembrane glycoprotein transferase for LCFA uptake widely expressed in several cell types including endothelial cells (ECs), cardiomyocytes (CMs), skeletal myocytes, adipocytes, and monocyte/macrophages. Notably, genetic deficiency of CD36 in humans is associated with hypertrophic cardiomyopathy. The role of CD36 in transendothelial LCFA delivery to CMs under physiologic vs pathologic conditions is unknown. We explored consequences of EC-specific CD36 deletion (EC-CD36 KO mice) on the cardiac response to pressure overload.

Lack of EC-CD36 increased severity of left ventricular hypertrophy (LVH), and systolic and diastolic dysfunction at 8 weeks of pressure overload via transverse aortic constriction (TAC), with increases in HW:TL ratio, LV mass by echo, reduced EF (p<0.05), and elevated E/E’ in EC-CD36-KO vs. flox/flox littermate controls (f/f) (p<0.5) (Fig 1). Dynamic mode
13
C NMR revealed delayed
13
C LCFA uptake kinetics (time constant, τ) in CMs of isolated, sham and TAC EC-CD36 KO hearts provided
13
C palmitate +
13
C oleate (0.02 mM each) + 10 mM glucose + 1 mM lactate (Fig 2).
13
C LCFA esterification into triglyceride (TG) was also reduced in EC-CD36-KO vs f/f controls, but without evidence for an additional effect of TAC.

The contribution of
13
C LCFA to acetyl CoA entry into oxidation via the TCA cycle, was reduced by lack of EC-CD36 in sham and TAC hearts (p<0.0001) (Fig 2). Consistent with diminished LCFA oxidation, phosphocreatine (PCr) to ATP ratio was also compromised in EC-CD36-KO-TAC hearts (p<0.05). Lack of EC-CD36 during TAC increased
13
C LCFA trafficking to lipotoxic C16 ceramide in EC-CD36 KO hearts (Fig 3).

CD36 in ECs is critical to LCFA delivery to CMs during pathological stress on the heart. The imbalance in intramyocellular LCFA dynamics due to restricted transendothelial delivery of LCFA to CMs increased lipotoxic ceramide formation. Thus, EC CD36 is critical to the adaptive cardiac response to pathological stress, and the absence of EC CD36-dependent delivery of LCFA to CMs exacerbates reduced energy potential, adverse cardiac remodeling, and dysfunction during afterload stress.
Abstract Tu114: Prostaglandin E2 Alters Mitochondrial Energy Metabolism in the Murine Heart
Authors: Bryson Timothy , Maxwell DruAnne , Taube David , Harding Pamela
Published: October 2024

Prostaglandin E
2
(PGE2) is an autacoid that acts via 4 receptors (EP1-EP4), with EP3 and EP4 being the most prominent in the heart. We previously reported that cardiomyocyte specific knockout of EP4 (EP4 KO) results in dilated cardiomyopathy. Additionally, transgenic mice that overexpress EP3 in the cardiomyocyte (EP3 TG) develop heart failure. Recently published data from our laboratory suggests EP4 KO results in mitochondrial dysfunction, likely via EP3 receptor signaling. We therefore hypothesized that PGE2 alters energy metabolism in the heart via EP3. To test our hypothesis, we performed gene array analysis on left ventricles from adult EP4 KO and wild type (WT) mice. Significant reductions (2.1-fold) in carnitine palmitoyltransferase 2 (
Cpt2
) were observed in EP4 KO
vs.
EP4 WT. CPT2 is a key enzyme in fatty acid oxidation and deficiencies in humans have been shown to cause cardiomyopathy. Substantial reductions in CPT2 were also observed in C57 mice after 2 weeks of myocardial infarction (0.22 ± 0.04 a.u. in sham controls
vs.
0.039 ± 0.06 a.u. after MI. p<0.01) by western blot analysis. Moreover, treatment with the EP3 receptor agonist, sulprostone, reduced CPT2 protein expression significantly in cardiomyocytes isolated from C57 mice (1.0 ± 0.0 a.u. in vehicle control
vs.
0.67 ± 0.07 a.u. in sulprostone treated. p<0.005). These data suggest that PGE2 via its EP3 receptor reduces CPT2 expression. In fact, RNAseq analysis on left ventricles from EP3 TG and EP3 WT mice showed significant reductions in
Cpt
2 expression as early as 5 wks of age (log
2
FC = -1.17. p<0.01). RNAseq also showed reductions in other key fatty acid oxidation/transport genes in EP3 TG mice (
Acsl1
; log
2
FC= -0.73,
Acads
, log
2
FC= -0.59,
Cd36
, log
2
FC= -0.64. p<0.005). Lastly, to further investigate the role of the EP receptors on energy metabolism in the heart, we isolated cardiomyocytes from EP4 KO and EP4 WT mice and performed metabolomics. As anticipated, we observed significant reductions in L-carnitine (7.6 ± 1.13 fmol/cell in EP4 WT
vs.
2.1 ± 0.32 fmol/cell in EP4 KO. p<0.005) and several acyl carnitine products. Altogether our data supports our hypothesis that PGE2 signaling via its EP3 receptor impairs mitochondrial energy metabolism in the heart by reducing fatty acid oxidation.
Abstract Tu128: p22
phox
is a critical factor for the prevention of oxidation and stabilization of SERCA2a in the heart.
Authors: Titus Allen , Nakada Yasuki , Mizushima Wataru , Yang Yanfei , Zhai Peiyong , Oka Shinichi , Kashihara Toshihide , Fefelova Nadezhda , Liu Tong , Li Hong , Xie Lai-Hua , Sadoshima Junichi
Published: October 2024

Introduction:
Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22
phox
, a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22
phox
in oxidative stress and SERCA2a levels during pressure overload (PO).

Question:
Does loss of p22
phox
alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO?

Methods/approach:
Cardiac-specific

p22
phox

knockout (

p22
phox

-cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22
phox
interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling.

Results:
The

p22
phox

-cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H
2
O
2
levels compared to WT after 1-week TAC (p<0.01). Unexpectedly,

p22
phox

-cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22
phox
directly interacted with SERCA2a. Lack of p22
phox
led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and

p22
phox

-cKO mice.

Furthermore, in the absence of p22
phox
, SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22
phox
knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22
phox
knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice.

Conclusion:
The loss of p22
phox
exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.
Abstract Tu135: FYCO1 ameliorates cardiac remodeling in response to ischemia by amplifying autophagic flux
Authors: Senger Frauke , Remes Anca , Tannert Christin , Haas Emelie , Mueller Oliver , Frey Norbert
Published: October 2024

Background:
Dysregulation of autophagy is known to have detrimental effects on cardiac remodeling. FYCO1, a novel regulator of autophagy mediates the transport of autophagosomes, thereby amplifying autophagic flux. Here, we investigate the impact of FYCO1 on myocardial healing via autophagy modulation.

Methods:
FYCO1-Tg mice crossed with RFP-EGFP-LC3 reporter mice (FYCO1-TGxRGFP) were utilized to analyze autophagic flux via confocal microscopy following permanent ligation of the left anterior descending artery (LAD) to induce myocardial ischemia. Autophagy activity was evaluated via Western blot of p62 and LC3 II. Macrophage infiltration and apoptosis was assessed by immunofluorescence microscopy, while Masson's Trichrome staining detected fibrosis and infarct size. Cardiac function was determined by echocardiography.

Results:
FYCO1-TGxRGFP mice displayed significantly enhanced autophagic flux post MI, as evidenced by increased p62 (WT LAD: 2,9±0,4; TG LAD:4,0±0,5) and LC3 II (WT LAD: 1,6±0,2; TG LAD:14,0±1,4) protein levels. Confocal microscopy revealed an accumulation of autolysosomes in WT mice 30 days post MI (WT autophagosomes: 1±0,4; WT autolysosomes: 38,6±3,8), contrasting with the sustained ratio of autophagosomes to autolysosomes in FYCO1-TGxRGFP mice (TG autophagosomes:43,3±5,1; TG autolysosomes: 73,5±12,4), implying the preservation of adequate autophagic flux. FYCO1 overexpression reduced macrophage infiltration into the border zone post MI (WT LAD: 132,4 ±14,3; TG LAD: 18,91±3,7 and inhibited apoptosis induction shown by cleaved caspase7 activity (WT LAD: 64,6±10,1; TG LAD: 9,2±2,1). Moreover, FYCO1-TGxRGFP mice exhibited significantly reduced fibrosis (WT:19,6±1,8%; TG:10,6±1,6%) and infarct size (WT:34,5 ±3,9%; TG:5,5±2%) in the left ventricle (LV), accompanied by marked improvements in global heart function post MI (EF WT: 32,7±3,2%; TG: 56,4±3,9%) compared to WT mice.

Conclusion:
In conclusion, FYCO1 mitigates adverse consequences of ischemic injury via induction of autophagy and a previously unrecognized role in modulating inflammation in response to myocardial infarction.
Abstract Tu140: Evaluation of the effects of mitoquinone on doxorubicin-induced acute cardiac damage
Authors: Lyons Meagan , Melnik Juliet , Yan Lloyd , Mercado Kelly Annie , Ghofrani Sasha , Malik Nelma , Dawes Kimberly , Ghasb Gabby , Amora Jonathan , Young Lindon , Barsotti Robert , CHEN Qian
Published: October 2024

Background:
Doxorubicin (DOX)-induced cardiotoxicity remains a high priority to overcome due to irreversibility and lack of abundant preventions. DOX damages the heart tissue principally by overproduction of mitochondrial superoxide and intercalation with the topoisomerase II in the nucleus. Ultimately, cardiac cells become apoptotic, and heart function declines. Our lab has shown that Mitoquinone (MQ), a mitochondria-targeted antioxidant, improves H9c2 cell viability in the presence of DOX. We hypothesize that MQ would counteract DOX to preserve cardiac cells and function in isolated rat hearts, possibly by reducing mitochondrial superoxide production and intracellular accumulation.

Methods:
H9c2 cells (passages < 18) were incubated with DOX (40 µM) with or without MQ (0.005 μM - 5 μM) for 24 hours before measuring mitochondrial superoxide anion levels and DOX intracellular accumulation. Furthermore, heart rate (HR) and left ventricular end-systolic pressure (LVESP) were recorded before and after a 60-minute infusion of DOX or DOX with MQ in isolated rat hearts. TUNEL staining was performed on heart tissue slides to assess apoptosis. All the data was expressed as a ratio between treatment and DOX alone or between final to initial values. Statistical significance was evaluated using ANOVA with Student Newman Keuls post-hoc analysis.

Results:
Hearts perfused with DOX (25 µM, n = 5) displayed lower final LVESP with 39 ± 5% of the initial baseline. By contrast, hearts treated with 0.25 µM MQ (n = 4) or 1 µM MQ (n = 5) manifested exhibited a higher final LVESP to 44 ± 8% and 90 ± 6% of initial baselines, respectively. Additionally, MQ administration significantly reduced cardiac cell apoptosis to 8 ± 1% (0.25 µM, n = 2) and 6 ± 1% (1 µM, n = 5, p < 0.05) when compared to DOX (88 ± 2%, n = 4). In contrast to DOX, MQ (1 µM, n = 4) showed 27 ± 9% (n = 4) reduction in mitochondrial superoxide levels and 54 ± 6% (n = 6, p < 0.05) less intracellular DOX deposition in H9c2 cells.

Conclusion:
This study suggests acute DOX administration to the heart severely compromises cardiac systolic function with higher induction of cell apoptosis. MQ exerts cardio-protection with higher systolic function and cell viability by attenuating mitochondrial superoxide levels and intracellular DOX accumulation.
Abstract Tu141: Effect of Epicardial Application of Oxygenated Hydrogel on Scar Burden in a Rat Model of Myocardial Infarction
Authors: Sanadgol Ghazal , Guo Jie , van Kampen Antonia , Nahlawi Acile , Goudot Guillaume , Yaghoubian Koushiar , Ruskin Jeremy , Zhang Yu Shrike , Ptaszek Leon
Published: October 2024

Background:
Epicardial application of a bioadhesive at the time of experimental myocardial infarction (MI) has been shown to reduce ventricular scar burden in mice. This study assessed the impact of an oxygenated hydrogel (O
2
-hydrogel) on post-infarct cardiac function and scar burden in rats.

Method:
Experimental MI was performed in Lewis rats (age 12-15 weeks) by permanent ligation of the anterior interventricular artery. There were 3 cohorts in this study: 1. MI followed by epicardial application of hydrogel, 2. MI followed by epicardial application of O
2
-hydrogel, and 3. MI with no treatment. Left ventricular (LV) function was evaluated by transthoracic echocardiography (TTE) at baseline, immediately post-MI, and 3 weeks post-MI. LV scar burden was evaluated with histopathology performed 3 weeks post-MI.

Result:
Twenty-four rats were analyzed (hydrogel, n= 8; O
2
-hydrogel, n= 9; untreated, n=7). LV fractional shortening (FS) measured by TTE 3 weeks post-MI was significantly reduced compared with baseline in untreated rats (44.6±12.4 to 22.3±4.9, P=0.043; Figure 1) and hydrogel treated rats (32.4±6.0 to 14.9±5.2, P=0.018). No significant change in FS was observed in the O
2
-hydrogel treatment cohort (39.0±13.9 to 38.1±12.6, P=0.859). LV scar burden in the O
2
-hydrogel cohort was significantly lower than in the hydrogel alone and untreated cohorts (11.9% vs 39.9% and 36.2%, respectively; Figure 2).

Conclusion:
In this rat model, epicardial application of O
2
-hydrogel at the time of experimental MI is associated with greater preservation of LV function and significantly lower scar burden than application of hydrogel alone or no treatment.