tbuckley/cabot-search huggingface dataset:
Abstract Mo047: DNA METHYLATION AND TRANSCRIPTIONAL MARKERS OF MYOCARDIAL REVERSE REMODELING IN HF PATIENTS ON LVAD SUPPORT
Authors: Thirupura S Shankar, Marco Marchetti, Harini Srinivasan, J Ty Lunde, Craig Selzman, Edgar Javier Hernandez, Martin Tristani-Firouzi, Stavros Drakos, Omar Wever-Pinzon
Journal: Circulation Research (2024)

Heart failure (HF) involves global genome modifications. Adverse structural and function remodeling that occurs in HF was initially considered to be irreversible. However, ~15% of HF patients on left ventricular assist device (LVAD) support show significant structural and functional reverse remodeling, distinguishing them as responders (R), while ~85% are non-responders (NR). DNA methylation and transcriptional changes that exert myocardial recovery is not well understood. To address this, we performed whole-genome bisulphite sequencing and RNA sequencing on paired myocardial biopsies from HF patients (n=84) before (pre-LVAD) and after LVAD unloading (post-LVAD). Post-LVAD samples showed activation of pathways including TGFβ signaling, PTEN and Fatty acid oxidation compared to pre. Integrating DNA methylation and transcriptomics data revealed hypomethylation (upregulation) of genes including Scaper, Frat1 and Magi1 and hypermethylation (downregulation) of Tpp3 and Icam. Overall, suggesting a unique ECM-specific stabilization pathway post-LVAD unloading. Segregating samples based on their response to LVAD unloading, pathways such as inflammation and fibrotic signaling were activated in postNR compared to preNR. Hypomethylation of Magi1 and hypermethylation of Tppp3 was observed in postNR suggesting cross-linking proteins and destabilization of microtubule formation as potential mechanisms of functional regression in NR. Conversly, postR showed activation of deubiquitination, oxidative phosphorylation and NLR signaling compared to preR. Finally, we observed integrin signaling, syndecan interaction, collagen degradation and wound healing pathways were activated in postR compared to postNR. Hypomethylation of actin cytoskeleton ( Myl12a ), microtubule/motor proteins ( Kif5b ) and tropomyosin ( Tpm3 ) were observed in postR, suggesting a plausible reverse remodeling mechanism mediated by ECM degradation and stabilization of myocardium in these patients. Western blot analysis also showed a trend towards an increase in collagen in postNR compared to postR (n=4, p=0.06). Finally, a mouse model of HF and recovery was used and pathways including ECM degradation and cardioprotective signaling were activated in mice that recovered from HF compared to mice that did not. Overall, our data suggests a unique ECM regulation pathway to contribute towards myocardial reverse remodeling in HF patients.
Abstract Tu108: Improved Skeletal Muscle Metabolism and Exercise Capacity Following Hydrogen Sulfide Therapy in Cardiometabolic HFpEF
Authors: Timothy Allerton, Heather Quiriarte, Zhen Li, James Stampley, Gregory Davis, Robert Noland, Jake Doiron, Thomas Sharp, Huijing Xia, Sanjiv Shah, Brian Irving, David Lefer
Journal: Circulation Research (2024)

Introduction: Exercise intolerance is the cardinal feature of cardiometabolic HFpEF that is largely driven by impairments in skeletal muscle metabolism and function. Hydrogen sulfide (H 2 S) is an essential regulator of mitochondrial function and metabolic homeostasis. We have previously demonstrated reductions in skeletal muscle enzymatic H 2 S production and H 2 S bioavailability in rodent models of HFpEF. The potential for H 2 S donor therapy to restore skeletal muscle metabolism and improve exercise capacity in HFpEF has not been explored. Hypothesis: We tested the hypothesis that oral H 2 S donor (SG-1002) therapy would restore critical aspects of skeletal muscle metabolism and exercise performance in cardiometabolic HFpEF. Methods: Five weeks after the onset of HFpEF (high-fat diet + L-NAME) male C57BL6/J mice (n=12 per group) were administered dietary SG-1002 (95 mg/kg/day) for 5 weeks and compared to HFD + L-NAME alone mice (i.e., control). Exercise performance was assessed using forced treadmill and voluntary wheel running (VWR) with continuous monitoring of indirect calorimetry. Skeletal muscle (soleus and gastroc) mitochondrial function was assessed by high-resolution respirometry. Palmitate (fatty acid oxidation, FAO) and leucine (BCAA) oxidation were determined by radiolabeled isotope ( 14 C) assays. A force grid meter was used to determine muscle grip force production. GC-chemiluminescence was used to measure H 2 S bioavailability. Results: SG-1002 therapy significantly (p &lt; 0.001) increased circulating H 2 S bioavailability (0.57 ± 0.15 vs. 0.22 ± 0.08 µM) when compared to HFpEF control mice. Forced treadmill running (125 ± 87 vs 64 ± 33 meters), grip force production (9.6± 1.1 vs. 7.5 ± 1.3 g/g lean mass) were increased (p &lt; 0.05 vs. control for both) following SG-1002 treatment. During VWR, whole-body fat oxidation was increased (p=0.0008, 0.3 ± .01 lower RER) in SG-1002-treated mice. At the skeletal muscle level, SG-1002 treatment reduced LEAK state respiration by 52% (p &lt; 0.0001 vs. control) and increased complete FAO by 30% (p &lt; 0.05) while reducing incomplete FAO by 33% (p &lt; 0.05 between groups). Conclusion: We demonstrate that H 2 S donor therapy significantly improves exercise capacity and underlying skeletal muscle dysfunction in a mouse “two-hit” model of cardiometabolic HFpEF.
Abstract Mo120: Overexpression of Prostaglandin E2 EP3 Receptor Subtype Alters Calcium-Handling Proteins in Mouse Hearts
Authors: Shaheen Bhat, Timothy Bryson, David Taube, Pamela Harding
Journal: Circulation Research (2024)

Prostaglandin E2 (PGE2) is an autacoid that acts through G-protein coupled receptors (EP1-EP4) in various cell types within the heart, including cardiomyocytes in which EP3 and EP4 predominate. We previously reported that mice with cardiomyocyte-specific overexpression of the EP3 receptor (EP3 TG) demonstrate reduced cardiac function and diminished cardiomyocyte contractility in isolated cardiomyocytes. We therefore hypothesized that PGE2 via the EP3 receptor regulates calcium handling proteins to reduce contractility. To test our hypothesis, we performed RNA sequencing (RNA seq) on the left ventricle of fifteen-week-old male EP3 TG mice along with their WT controls. Western blots were used to confirm changes in the expression of calcium-handling proteins such as Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Ryanodine receptor 2 (RYR2), Phospholamban (PLB), Troponins (C, I, T) and voltage-gated calcium channel (CACNB2). For RNA seq, all data is reported as Log2Fold change, adjusted P value, and western blot data is reported as mean ± standard error (SE) of the mean. Additionally, isolated cardiomyocyte contractility was assessed using the IonOptix system, and data is reported as the mean ± SE. RNA seq data shows that SERCA2a and RYR2 expression were significantly downregulated in TG mice (SERCA2a -0.44, p=4.72 x 10 -6 ; RYR2: -0.86, p=4.08 x 10 -5 ) in addition to significant reductions in Troponin I (-0.43, p=0.038) and the Voltage-gated calcium channel (CACNB2: -0.71,p=0.0055). Western blot analyses confirmed the reductions in SERCA2a, RYR2, and CACNB2 although these changes were only significant for CACNB2 (1.0 ± 0.03 for WT vs 0.59 ± 0.04 for TG, p = 0.016). Expression of Troponins did not change in TG mice. Consistent with our previous data, IonOptix analysis shows reduced contractility of myocytes from EP3 TG mice (bl% peak h is 3.47 ± 0.20 for WT vs 1.0 ± 0.14 for TG, p&lt;0.0001) despite a similar increase in the Ca 2+ transients (Ca 2+ peak h for WT= 0.86 ± 0.04 and TG=0.86 ± 0.07) suggesting potential changes in the sensitivity of the myofilament to Ca 2+ . In conclusion, the reduced contractility observed in EP3 TG mice may be due to alterations in calcium handling proteins and/or changes in calcium sensitivity at the level of the myofilament.
Abstract Mo122: Investigating the Role of the Lysine Methyltransferase SMYD1 in Striated Muscle Motor Domain Folding
Authors: Dakota Hunt, Thomas Martin, Leslie Leinwand
Journal: Circulation Research (2024)

Properly coordinated folding and assembly of myosin into the sarcomere thick filament is critical for cardiac and skeletal muscle development, function, growth, and maintenance. The motor domain of myosin heavy chain (MHC) requires muscle-specific chaperones for proper folding, making it currently impossible to fold in non-muscle cells. Previous studies found that HSP90 and UNC45B are necessary, but not sufficient, for folding the motor domain, indicating other factors are involved. Defining the minimal set of factors for motor domain folding would allow for development of an improved expression system and accelerate the study of myosin mutations and myosin-targeted drug development. We hypothesize that the muscle-specific lysine methyltransferase SMYD1 contributes to MHC folding and stability. We found SMYD1 associates with UNC45B, HSP90, and MHC in the adult human heart. To study motor domain folding we generated a human MYH7 motor domain fused to GFP (MD::GFP). Transfection into COS-7 cells resulted in unfolded aggregates while adenoviral infection of C 2 C 12 myotubes leads to folded and functional MD::GFP. Interestingly, the folded MD::GFP contains lysine trimethylation, which may arise from the methyltransferase activity of SMYD1. When COS-7 cells were co-transfected with UNC45B and SMYD1, we observed increased total protein levels of non-functional MD::GFP without affecting RNA levels as assessed by RT-qPCR. This effect was not observed when MD::GFP was co-transfected with UNC45B or SMYD1 alone and is dependent on HSP90. We found that UNC45B and SMYD1 stabilized MD::GFP as determined by cycloheximide chase assays. We also found SMYD1 associates with MD::GFP, suggesting this stabilizing effect is due to a direct interaction. Interestingly, a catalytically inactive SMYD1 also increased MD::GFP protein levels in COS-7 cells, consistent with our observation that no significant changes in global protein methylation were observed in the presence of WT SMYD1. Our data suggests that SMYD1 stabilizes the motor domain of striated muscle myosin in conjunction with UNC45B and HSP90, in a manner that is independent of its methyltransferase activity. However, UNC45B and SMYD1 are insufficient for motor domain folding in a non-muscle cell line, suggesting that additional folding factors or proper stoichiometric ratios of chaperones/co-chaperones may be required.
Abstract Mo059: Leucine-Rich Repeat-Containing Protein 10 Modulates Human Cardiac L-Type Ca <sup>2+</sup> Channels but not T-Type Ca <sup>2+</sup> Channels
Authors: Natthaphat Siri-Angkul, Marites Woon, Zachery Gregorich, OLUFUNKE OLORUNDARE, Youngsook Lee, Timothy Kamp
Journal: Circulation Research (2024)

Background: Leucine-rich repeat-containing protein 10 (LRRC10) is a novel regulator of the cardiac L-type Ca 2+ channel/current (LTCC/I Ca,L ). LRRC10 is crucial for cardiac regeneration in multiple species, and LRRC10 variants have been associated with dilated cardiomyopathy in humans. LRRC10 increases I Ca,L in HEK-293 cells expressing rabbit Ca V 1.2 subunit, and knockout of LRRC10 in zebrafish and mouse cardiomyocytes (CM) reduces I Ca,L . However, LRRC10-mediated regulation of human cardiac LTCC has not been demonstrated, and the mechanism by which LRRC10 modulates LTCC is not known. Moreover, the effect of LRRC10 on the T-type Ca 2+ channel/current (TTCC/I Ca,T ), which has been implicated in CM cell cycle regulation, has never been tested. Methods: LRRC10 knockout ( LRRC10 –/– ) human induced-pluripotent stem cells (hiPSC) were generated from wild-type (WT) DF19-9-11T hiPSCs using CRISPR-Cas9 techniques. LRRC10 –/– and WT hiPSCs were differentiated to CMs using the standard GiWi protocol. Whole-cell I Ca,L and I Ca,T were examined in LRRC10 –/– and WT hiPSC-CMs. Whole-cell I Ca,L was also assessed in HEK-293 cells expressing human cardiac LTCC subunits (Ca V 1.2, β 2 , and α 2 δ) with or without human LRRC10. To identify the LRRC10-interacting site of Ca V 1.2, GST-pulldown assays were performed using purified GST-fusion constructs of the intracellular domains of rabbit Ca V 1.2 and lysates from HEK-293 cells expressing human LRRC10. Ca V protein sequences were analyzed using the Clustal Omega program. Results: Genetic ablation of LRRC10 in hiPSC-CMs decreased peak I Ca,L (WT -29.4±2.2 pA/pF, LRRC10 –/– -15.0±1.8 pA/pF, p&lt;0.01) but did not significantly affect I Ca,T magnitude (WT -3.9±0.5 pA/pF, LRRC10 –/– -4.0±0.7 pA/pF, p&gt;0.05 [NS]). LRRC10 increased peak I Ca,L in HEK-293 cells expressing human LTCC (with LRRC10 -156.0±19.8 pA/pF, without LRRC10 -60.2±12.0 pA/pF, p&lt;0.05). LRRC10 was pulled down by the GST-Ca V 1.2-N-terminus but not by other GST-Ca V 1.2 constructs. Sequence analyses revealed an invariant exon 2-encoded segment of the N-terminus (NT) that was present in both human and rabbit LTCC Ca V 1.2 but not in TTCC Ca V 3.1 and Ca V 3.2. Conclusions: The Ca 2+ influx-potentiating effect of LRRC10 is conserved in human LTCC and is specific to regulate I Ca,L , not I Ca,T . The invariant Ca V 1.2 NT segment is a potential LRRC10-interacting site. The LRRC10 –/– hiPSC line is a promising tool for future investigations into the roles of LRRC10-LTCC interaction in human cardiac biology and disease.
Abstract We051: Chronic mavacamten treatment prevents development of contractile dysfunction in cMyBP-C null engineered heart tissue constructs, but not in constructs carrying the cMyBP-C W792R missense mutation
Authors: Willem De Lange, Angeela Gauchan, Emily Farrell, J Carter Ralphe
Journal: Circulation Research (2024)

Hypercontractility, caused by mutations that increase the number of myosin heads in the disordered relaxed (DRX) state, is a prevalent cause of hypertrophic cardiomyopathy (HCM). A subgroup of HCM mutations cause primary hypocontractility, by stabilizing super-relaxed crossbridges (SRX). Mavacamten (mava) directly targets hypercontractility by reducing the number of DRX heads and improves outcomes in ~70% patients with obstructive HCM. We here assessed the efficacy of chronic mava treatment at preventing phenotype development in human iPSC engineered heart tissue (EHT) in a cMyBP-C null model (-/-), that increases the number of DRX heads, and EHT carrying the W792R (RR) HCM mutation. While the primary contractile defect caused by the W792R mutation is unknown, we postulated that it differed from the cMyBP-C -/- model, as RR mice die prior to weaning while -/- mice do not. Twitch force (TF) measurements performed on day 7 on control (+/+), -/- and RR EHT show that -/- EHT were hypercontractile while RR EHT were hypocontractile (Fig. 1). Following measurement, EHT from each genotype were either treated with vehicle or 100nM mava and cultured for 21 days. By day 28, TF production was similar between +/+ and -/- vehicle treated EHT while RR EHT remained hypocontractile (Fig. 2). Chronic mava treatment significantly reduced TF production in +/+ EHT but not in -/- or RR EHT (Fig. 2). Consistent with diastolic dysfunction, a hallmark of HCM in humans, day 28 -/- and RR EHT displayed slow relaxation, though the effect was more pronounced in RR EHT (Fig. 3). While chronic mava treatment accelerated relaxation in -/- EHT to levels that were similar to that observed in vehicle treated +/+ EHT, it failed to do so in RR EHT (Fig. 3). cMyBP-C -/- EHTs initially present with a hypercontractile phenotype, consistent with an increase in DRX crossbridges, while W792R RR EHT present with a hypocontractile phenotype. By 4 weeks in EHT culture, both genotypes present with impaired relaxation, that is more severe in RR EHT. While chronic mava treatment prevents development of slow relaxation in -/- EHT, it failed to do so in RR EHT. These findings suggest that the primary disease mechanism of the W792R mutation is not an increase in DRX crossbridges and that mavacamten may not be an effective treatment for patients carrying this mutation, further highlighting the need for studying the pathomechansims of HCM mutations and the development of novel therapies.
Abstract We097: Dual Targeting CXCR4 and CXCR7 Prevents Doxorubicin-Induced Cardiotoxicity by Eliminating Cardiac Inflammation and Preserving Cardiomyocyte Function
Authors: Xiaodan Hui, Chunjie Gu, Ahmed Abdelbaset-Ismail, Jianxiang Xu, William Tse, Ashok Raj, Zhongbin Deng, Wuqiang Zhu, Lu Cai, Yi Tan
Journal: Circulation Research (2024)

Background: Doxorubicin (DOX)-induced cardiotoxicity (DICT) has become a serious health burden for cancer survivors. The detrimental effects of CXCR4-mediated excessive inflammation in myocardium and the protective effects of CXCR7 in cardiomyocytes have been demonstrated. Aims: We tested the protective effects against DICT of a dual-targeting compound TC14012, which has specific antagonistic activity against CXCR4 and potent agonistic activity on CXCR7. Methods: Eight-week-old male C57BL/6J mice were injected with DOX for consecutive 5 weeks, during which mice were treated with TC14012 twice a week. One week after the last dose injection, mice were euthanized after heart function examination. Markers of cardiac inflammation and fibrosis were evaluated. Cardiac RNA-seq was performed to explore the underlying molecular pathways. Key findings: TC14012 supplement significantly prevented DOX-induced cardiac systolic dysfunction, reflected by improved ejection fraction and fraction shortening, along with remarkable attenuation of DOX-induced cardiac perivascular and interstitial collagen deposition and neutrophils and macrophage infiltration. RNA-seq revealed that TC14012 prevention of DICT was accompanied with a remarkable stimulation of signals involving in cardiac contraction and energy reserve metabolic process, particularly glycogen and glucan metabolism that are crucial for meeting the cardiac energy demand in response to DOX induction. These signals could be further enriched into AMPK signaling pathway and validated by upregulation of AMPK phosphorylation. Besides, TC14012 prevention of DICT was accompanied with a significant reduction in cardiac mast cell activation and degranulation. These signals could be further enriched into Rap1 signaling pathway, and TC14012 was validated to inhibit Rap1 activity and mast cell activation. Most importantly, TC14012 treatment did not affect the suppressive effects of DOX on tumor growth in a syngeneic EL4 lymphoma-bearing, immunocompetent mouse model. Conclusions: We preliminary conclude that TC14012 prevents DICT by activating cardiac CXCR7 to reprogram cardiomyocyte metabolism and prevent cardiac damage and dysfunction via activating AMPK, and by antagonizing CXCR4 to inhibit cardiac mast cell infiltration and activation via blunting Rap1 activity but does not affect the anti-tumor activity of DOX.
Abstract Mo049: Loss of Y chromosome and cardiovascular events in chronic kidney disease
Authors: Andreas Zeiher
Journal: Circulation Research (2024)

Background: Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease. Methods: LOY was quantified in patients with stable CKD (CARE for HOMe study, derivation cohort, N=279) and dialysis patients (4D study, validation cohort, N=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific endpoints, and echocardiographic measures was assessed. Results: In CARE for HOMe, mortality was higher in men as compared to women across the entire range of impaired renal function. The frequency of LOY increased with age. LOY was associated with increased mortality (HR 2.58, 95%CI 1.33-5.03) and risk for cardiac decompensation or death (HR 2.30, 95%CI 1.23-4.27). Patients with LOY &gt;17 % showed a significant decline of ejection fraction and an increase of E/E’ within five years. Consistently, validation in the 4D study revealed that LOY was associated with increased mortality (HR 3.13, 95%CI 1.97-4.99), higher risk of death due to heart failure and sudden cardiac death (HR 4.14, 95%CI 2.00-8.57), but not atherosclerotic events (see figure). Patients with LOY &gt;17 % showed significantly higher plasma levels of soluble ST2, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY &gt;17 % showed significantly higher CCR2 expression and higher plasma levels of the CCR2 ligand CCL2, which may have contributed to increased heart failure events. Conclusions: LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.
Abstract We067: A meta-analysis of RNA sequencing data to characterize immune cell activation among patients with cardiac sarcoidosis
Authors: Natasha Banga, Yuhsin Huang, Harrison Chou, Maria Rosaria Vitale, Matthew Baker, Ronald Witteles, Han Zhu
Journal: Circulation Research (2024)

Introduction: Cardiac sarcoidosis (CS) is a life-threatening auto-immune disorder that causes the formation of non-caseating granulomas in cardiac tissue. Th17.1 cell have been found to be upregulated in patients with pulmonary sarcoidosis (PS) although data is lacking among CS patients. This meta-analysis integrates all publicly available granuloma single-cell and single-nuclei RNA sequencing data to identify patterns in immune cell activation across different granulomatous diseases while focusing on CD4+T cell activation among CS patients. Methods: Five studies were included, spanning cardiac tissue from healthy controls and DCM (Koenig 2022, PMID:35959412), cardiac tissue from ICM and cardiac sarcoidosis (Liu 2022, PMID:36111531), BAL from beryllium sensitized, chronic berylliosis, and PS (Liao 2021, PMID: 33602861), peripheral blood samples from sarcoidosis (Garman 2020, PMID: 33363531), and lesional and nonlesional skin tissue from sarcoidosis (Krausgruber 2023, PMID: 36750099) for a total of 122 patients. All integrative analysis was performed using the R package Seuratv5 on Stanford’s computing cluster, Sherlock. After quality control filtering, a total of 127,335 cells were integrated using Seurat’s reference-based integration. After labeling the UMAP with general immune cells (figure 1A), we subsetted T cells into CD4+ and CD8+ and examined the landscape of CD4+ T cells (figure 1B). Results: We found significantly more Tregs in CS than PS (P=0.013, figure 1C). Additionally, patients with CS had significantly more Th17.1 cells in CS in comparison to PS (P=0.0041). However, significantly less Th2 cells were found in CS than PS patients (P=0.0152). These differences remained statistically significant across various analyses comparing Tregs and Th17.1 cell as a proportion of CD4+, CD3+, and CD45+ cells. Conclusions: In comparison to patients with PS, pathways which differentiate naive CD4+ cells into TH17.1 and Tregs are upregulated among CS patients while the TH2 differentiation pathway is downregulated. Our findings indicate distinct CD4+ T cell patterns among CS and PS patients. Our ongoing work aims to further evaluate T cell proliferation and expression to elucidate mechanistic pathways as potential drug targets for CS.