tbuckley/cabot-search huggingface dataset:
Abstract We133: <i>In Silico</i> Model of Cardiomyopathy Caused by a MYBPC3-induced HCM Mutation
Authors: Pichayathida Luanpaisanon, Matthew Wolf, Jeffrey Saucerman
Journal: Circulation Research (2024)

Introduction: Hypertrophic cardiomyopathy (HCM) is the most frequent hereditary cardiomyopathy and a leading cause of sudden cardiac death, particularly in adolescent. Despite recent improved prognosis and FDA approved drugs, current cardiac therapeutics cannot efficiently prevent remodeling across all HCM patient populations due to genetic variability. Therefore, we require a systems biology approach to understand how the complex cardiac signaling networks regulate cardiomyocyte shape and size due to an HCM mutation. This study leverages a computational systems approach to explore MYBPC3-induced HCM mutation regulation of cardiomyocyte hypertrophy. Method: For the signaling network model, we employed a logic-based differential equations model (NETFLUX) to construct a signaling network validated using in vitro data from published studies. This model captured signaling dynamics and cross talks between pathways governing cardiomyocyte size and shape. Additionally, a MYBPC3 HCM mutation is integrated in the model as an input providing a comparison between a control and simulated HCM responses. Sensitivity analyses are performed to investigate the most influential signaling pathways that mediate changes in mass. Results: We created a MYBPC3-induced HCM model by integrating an additional cardiac myosin binding protein-C node to a previously validated inherited cardiomyopathy signaling network. This updated model predicted an increase in mass when the HCM mutation is simulated. The simulated inhibition of each node in the model revealed that inhibition of calcium pathway, growth factor receptor, and titin stiffness could mitigate the HCM phenotype by reducing mass. The sensitivity analysis results also suggested PI3K, Ras, and ERK1/2 pathways as potential mechanisms mediating the decrease in mass and served as potential therapeutic targets. Discussion and Future Work: Our systems approach gave insights on the mechanisms governing the cardiomyocyte response to a MYBPC3-induced HCM mutation. This will be further validated via a MYBPC3-HCM model using human iPSC-CMs with MYBPC3-induced mutations. This will create a cell line that will provide genetically precise comparisons for identifying new molecular mechanisms of disease, thereby offering a strategy for advancing precision medicine.
Abstract We014: Creating Cell-specific Computational Models of Stem Cell-derived Cardiomyocytes Using Optical Experiments
Authors: Janice Yang, Neil Daily, Taylor Pullinger, Tetsuro Wakatsuki, Eric Sobie
Journal: Circulation Research (2024)

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction as a powerful model in cardiac disease and therapeutics research, since iPSCs are self-renewing and can be derived from healthy and diseased patients without invasive surgery. However, current iPSC-CM differentiation methods produce cardiomyocytes with immature, fetal-like electrophysiological phenotypes, and the variety of maturation protocols in the literature results in phenotypic differences between labs. Heterogeneity of iPSC donor genetic backgrounds contributes to additional phenotypic variability. Several mathematical models of iPSC-CM electrophysiology have been developed to help understand the ionic underpinnings of, and to simulate, various cell responses, but these models individually do not capture the phenotypic variability observed in iPSC-CMs. Here, we tackle these limitations by developing a computational pipeline to calibrate cell preparation-specific iPSC-CM electrophysiological parameters. We used the genetic algorithm (GA), a heuristic parameter calibration method, to tune ion channel parameters in a mathematical model of iPSC-CM physiology. To systematically optimize an experimental protocol that generates sufficient data for parameter calibration, we created simulated datasets by applying various protocols to a population of in silico cells with known conductance variations, and we fitted to those datasets. We found that calibrating models to voltage and calcium transient data under 3 varied experimental conditions, including electrical pacing combined with ion channel blockade and changing buffer ion concentrations, improved model parameter estimates and model predictions of unseen channel block responses. This observation held regardless of whether the fitted data were normalized, suggesting that normalized fluorescence recordings, which are more accessible and higher throughput than patch clamp recordings, could sufficiently inform conductance parameters. Therefore, this computational pipeline can be applied to different iPSC-CM preparations to determine cell line-specific ion channel properties and understand the mechanisms behind variability in perturbation responses.
Abstract Mo086: Functional impact of phosphorylation-site mutations on PKA response of Kir2.1
Authors: Saba Munawar, Corey Anderson, Louise Reilly, Molly Melnick, Kyle Brown, Ying Ge, Lee Eckhardt
Journal: Circulation Research (2024)

Background: Kir2.1 channels maintain the cardiac resting membrane potential and assist in phase 3 repolarization. PKA-mediated phosphorylation modulates Kir2.1 function, impacting heart excitability and rhythm. Using as mass-spetometry based integrated top-down and bottom up proteomic approach, we identified five novel and confirmed a 6 th phosphorylation site. However, which of these residues impact channel function has not been previously identified. Methods: HEK293 cells were engineered to stably express either WT-Kir2.1. Ablation of the phosphorylation site was achieved with an alanine substitution of the PKA phosphorylation residue (S13A, S14A, S313A,Y326A, T347A, and S425A). Whole-cell patch clamp experiments of WT-Kir2.1 or the phosphorylation mutants were performed using a standard protocol. Following the initial recording, cells were perfused with PKA-stimulating cocktail (20µM Forskolin and 10µ IBMX) for 4 minutes and the protocol was repeated. Clampfit 10.7, GraphPad Prism, and Origin 20 were used for Data Analysis. Results: HEK293 cells stabling expressing Kir2.1 demonstrate typical inward rectification with a maximum outward current at -50mV and responds to PKA-cocktail with 2.0 pA/pF increase in maximum outward current. Both S13A and S313A had a normal response PKA stimulation. In contrast, ablation of S425 and S14 resulted in no PKA response. T347A showed little response to PKA; however, the current density is very low at baseline, indicating that this residue is necessary for normal Kir2.1 current density. The Y326 is known to be critical for normal protein folding, and ablation of the site resulted in a complete loss of function. Discussion: Of the 6 PKA sites identified by a proteomic approach, only S425 and S14 functionally influence Kir2.1 response to PKA stimulation. Further molecular dynamics simulation investigation will provide deeper insight into the conformational changes of Kir2.1 in response to phosphorylation at these sites and the relationship of channel phosphorylation with clinical channel mutation residues.
Abstract We139: 3D Chromatin Architectures and Transcription Regulation in Diabetic Endothelial Dysfunction
Authors: Yuliang Feng, Liuyang Cai, Yigang Wang, wei huang, Lei Jiang
Journal: Circulation Research (2024)

Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC high-glu ). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC high-glu and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC high-glu and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC high-glu . We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC high-glu significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.
Abstract We017: Tnni3k mediates postnatal reactive oxygen species through Prdx2 signaling to induce polyploidization in cardiomyocytes
Authors: Baylee Westbury, Henry Sucov
Journal: Circulation Research (2024)

Fetal CMs are characteristically diploid and regenerative, while adult CMs are polyploid and nonregenerative. Increased polyploidy in postmitotic CMs remains a significant barrier to regeneration in the adult heart. Previously, through the analysis of multiple mouse strains, our lab has shown that Tnni3k, a cardiac-specific kinase, increases polyploidy in the adult heart. The mechanisms that induce CM polyploidization are unknown but could be due to a multitude of changes including the postnatal increase in reactive oxygen species (ROS). ROS is essential in cardiac development and works to promote maturation and facilitate cell signaling. Using HEK293 cells, I have found that Tnni3k physically interacts with Prdx2, a prominent cytoplasmic ROS sensor. This interaction results in hyperactivated Tnni3k kinase activity. Comparing Tnni3k kinase activity with interaction of WT or catalytically dead Prdx2 showed hyperactivity dependent on Prdx2 catalysis. Polyploidization increases along with the increase in ROS after birth, and Tnni3k contributes to increased ploidy in mice. I hypothesize that Tnni3k mediates postnatal ROS through Prdx2 signaling to induce polyploidization in postnatal CMs. Discovering the consequences of this interaction could lead to advanced knowledge of CM cell cycle and provide new therapeutic targets.
Abstract We132: Genetic Blockade of PKA-mediated Phosphoregulation of 26S Proteasomes Exacerbates Pathology in Both the Brain and Heart of an Alzheimer’s Disease Model
Authors: Saima Ejaz, Jack Sternburg, Hongmin Wang, Rezvani Khosrow, Xuejun Wang
Journal: Circulation Research (2024)

Background: Alzheimer’s disease (AD) patients often display not only cognitive impairment but also cardiac malfunction, but the underlying factors remain elusive. Phosphorylation of Ser14 of RPN6 (p-S14-RPN6) is required for cAMP/protein kinase A (PKA) to activate 26S proteasomes. Proteasome priming is implicated in the protection by cAMP-PKA augmentation against AD, but this remains to be established. Hence, we conducted this study to address these critical gaps. Methods: The Rpn6 S14A knock-in mice recently created for genetic blockade of p-S14-Rpn6 were crossbred with the PS19 tauopathy model (Jackson Lab Stock # 008169). Littermate wild type (WT), PS19, and PS19::S14A mice were compared. Cardiac function was evaluated with echocardiography (Echo). Hippocampal and myocardial tissues were used for western blot analyses (WB) and histopathology. Results: Increases in hippocampal insoluble tau proteins and in total and K48-linked ubiquitin conjugates were markedly greater in PS19::S14A mice than PS19 and WT mice. WBs for PSD95, synaptotagmin, Neu N, GFAP, and IBA1 revealed exacerbated synaptic loss, neuronal loss, gliosis, and microglial activation in the hippocampus of PS19::S14A mice in comparison to PS19 and WT mice. A more significant decline in cognitive function indicated by object recognition index was found in PS19::S14A mice vs. PS19 mice at 6 months. More interestingly, as compared to PS19 and WT littermates, PS19::S14A mice showed greater increases in myocardial tau proteins, ubiquitin conjugates, and cleaved caspase 3 and displayed more cardiac fibrosis, as reflected by increased mRNA expression of collagen 1 and picrosirius red staining. Echo revealed that PS19::S14A had significantly reduced fractional shortening, ejection fraction, stroke volume, and cardiac output at 9 months. Conclusions: The findings of this study demonstrate that genetic blockade of p-S14-RPN6 exacerbates tauopathy pathology in both the brain and heart. This is the first to have established the role of p-S14-RPN6 and, by extension, the significance of PKA-mediated proteasome activation in AD/tauopathy. Proteasome activation via augmentation of cAMP/PKA signaling should be explored for the alleviation of AD-associated brain and cardiac pathologies.
Abstract We018: The Molecular Mechanism of Coronary Collateral Growth Induced by Repetitive Ischemia by Single Cell-RNA Sequencing
Authors: Jian Shi, Yang Wang, Cody Juguilon, James Gadd, Molly Enrick, Anurag Jamaiyar, Laura Zhang, Feng Dong, Yanqiao Zhang, Liya Yin
Journal: Circulation Research (2024)

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. A well-developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct sizes following coronary occlusion. Stimulation of coronary collateral growth (CCG) is also an alternative therapeutic approach for patients with intractable angina pectoris. The importance of CCG is undisputable, but the process and mechanism underlying the CCG is unclear. We developed a mouse model of CCG by repetitive ischemia (RI) in adults, and the CCG was validated with contrast echo to measure the coronary blood flow 3D images of micro-CT to quantitate the CCG. In this study, we use single cell-RNA sequencing (scRNA-Seq) to analyze the molecular signaling of CCG at different time points during the RI process. We digested the hearts from RI days 5, 10, and 17 and control (naïve) mice, harvested non-cardiomyocytes, performed scRNA-Seq with 10x Genomics and sequenced with Next-Seq 500 (Illumina).The fastq files are converted to a gene count matrix using kallisto|bustools with refdata-gex-mm10-2020-A employed as the reference mouse genome. The Kallisto bustools output is then converted in R to files compatible for uploading to Cellenics, an open-source platform for single-cell RNA-Seq analysis. Dimensionality reduction is done with Principal Components Analysis (PCA) with 30 principal components. We will use UMAP to visualize cell clusters. Cellenics uses scType to annotate the cell populations automatically when building clusters of cell populations. Our preliminary data show dynamic kinetics of non-cardiomyocytes induced by RI over the timeline of CCG and different cell populations of non-cardiomyocytes and EC expression profiling varied in response to RI throughout CCG. We will validate the critical signaling in CCG and explore the potential targets to induce CCG.
Abstract We129: Endothelium-Targeted MiR-122 Inhibition Improves Vascular Endothelial Function In a High-Fat Diet Fed mice
Authors: Ravinder Gaddam, Karishma Dhuri, Mounika Pathuri, Veda Amalkar, YOUNG-RAE KIM, Julia Jacobs, Veeresh Kumar Sali, Krishna Nakuluri, Kaikobad Irani, Raman Bahal, Ajit Vikram
Journal: Circulation Research (2024)

Background: MicroRNA-122-5p (miR-122), a liver-specific miR, levels are increased in the blood during obesity and diabetes and is taken-up by the extra-hepatic tissues including vascular endothelium. Despite miR-122’s role in maintaining hepatic homeostasis, its uptake (from circulation) by other tissues disrupts their function and increases the risk of cardiovascular disorders. Hence, inhibiting extra-hepatic miR-122 is a viable approach to mitigate vascular disorders associated with diabetes. Hypothesis: Endothelium-targeted miR-122 inhibitor selectively targets miR-122 in endothelial cells and improves vascular endothelial function in high-fat-diet (HFD) fed pre-diabetic mice. Methods: To selectively inhibit miR-122 in the endothelial cells, we synthesized gamma-peptide nucleic acid miR-122 inhibitor (γP-122 I) tagged with vascular cell adhesion molecule 1 (VCAM1) targeted peptide (e-γP-122 I). The purity was assessed using HPLC. Pre-diabetic condition was induced by feeding mice with a HFD (for 8 weeks) and e-γP-122-I was injected to the HFD-fed mice (2 weeks after dietary intervention; 5 mg/kg/day, i.p., 6 weeks). MiR-122 expression was measured by real-time PCR. The vascular endothelial function was assessed by determining endothelium-dependent and independent relaxation of phenylephrine-induced precontracted aortic rings. Results were expressed as mean ± s.e.m. Results: The e-γP-122-I was &gt;95% pure. The HFD-fed mice show higher levels of serum and aortic miR-122 expression and impaired vascular endothelial function. e-γP-122-I treatment selectively inhibited aortic miR-122 expression while it has no effect on other organs (heart, kidney etc.) and improved vascular endothelial function and inflammation. Conclusions: e-γP-122-I selectively inhibits miR-122 in the endothelium and improves endothelial function in pre-diabetic mice.
Abstract We131: Systems-Enabled Drug Repurposing to Regulate Cardiomyocyte Proliferation
Authors: Kaitlyn Wintruba, Jeffrey Saucerman
Journal: Circulation Research (2024)

Introduction: The inability of the adult heart to regenerate results in prevalent morbidity and mortality related to myocardial infarction and heart failure. This lack of cardiac regeneration is driven by the loss of cardiomyocytes (CMs), with limited supporting endogenous CM renewal. While many individual regulators of CM proliferation have been implicated, the field lacks strategies to identify druggable proliferative signaling pathways and prioritize candidate therapeutics. Our goal is to predict and experimentally validate FDA-approved drugs and their respective mechanisms that regulate CM proliferation. Methods: A logic-based differential equation model of CM proliferation signaling was integrated with drug-target interactions from a publicly available database (DrugBank) to simulate the effect of drugs on CM proliferation phenotypes (Binucleation, Cytokinesis, DNA Replication, Mitosis, and Polyploid). A virtual screen with knockdown combinations of regulators identified signaling mechanisms responsible for mediating the effect of drugs, and literature validation was performed against published experimental data investigating the drugs or the drug targets in CMs. Neonatal rat CMs were treated with the YAP-activating drug TT10 or drugs predicted to increase CM proliferation. Proliferation was measured by high-content microscopy and DNA replication and mitosis positive cells were identified from image analysis (CellProfiler) to discern CM proliferation from endoreplication. Results: We identified 16 unique drug-target interactions predicted to regulate CM proliferation phenotypes. Four drugs were predicted to increase CM phenotypic proliferation outputs. Experimental validation confirmed lithium, a GSK3beta-antagonist, induces cell cycle activity in neonatal rat CMs as evidenced by increased Ki67 expression in cardiac Troponin T positive cells. The model predicts cyclin D is critical in mediating this response and subsequent experiments measured changes in cyclin D expression in response to lithium treatment. Retrospective analysis of clinical data (TriNetX and AERSMine) indicates more adverse events of congenital cardiac disorders were reported in patients exposed to lithium. Conclusion: We identified lithium to promote DNA replication in CMs by upregulating cyclin D. This study advances the current knowledge of CM proliferation regulators by providing insight into new pathways targeted with existing drugs that can ultimately impact cardiac regeneration.
Abstract We006: Metabolic Reprogramming to Increase Mitochodnrial Mass, Fusion and Energetics Represents an Important Rate Limiting Step in Direct Cardiac Reprogramming
Authors: Brian Spurlock, Yifang Xie, Yiran Song, James Hua, Meenakshi Nishtala, Rustem Salmenov, Jiandong Liu, Li Qian
Journal: Circulation Research (2024)

Direct reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) shows promise for treating myocardial infarction (MI). However, low conversion efficiency and relative immaturity of iCMs have so far precluded its clinical translation. Native CM maturation and cell cycle exit is associated with a switch from glycolytic metabolism to mitochondrial fatty acid oxidation (FAO), accompanied by a shift in the dynamic balance of mitochondrial fission (dividing) and fusion (joining) toward fusion. Because CFs lack the energy requirements of CMs, we hypothesized that transdifferentiation of CFs to iCMs requires a similar switch. Combining confocal microscopy with metabolic flux assays, we directly compared native CF and CM mitochondrial morphology and energetics. CMs showed significantly higher mitochondrial mass, fusion and energetics, all metrics relevant for increased reliance on mitochondrial FAO. Applying these techniques to study reprogramming, we found that at later reprogramming time points key metrics move significantly toward the phenotypes of native CMs. We then hypothesized that facilitating metabolic reprogramming by inhibiting fission and/or promoting fusion could improve iCM conversion. A loss of function screen identified certain fission genes, particularly the little studied mitochondrial fission regulator 1-like protein (Mtfr1l), as potent barriers to iCM conversion and maturation. In contrast, the core fusion genes were necessary for successful generation of iCMs, particularly the key fusion effector optical atrophy 1 (Opa1). Mtfr1l has been shown to promote cleavage of Opa1, and indeed concurrent knockdown of Opa1 ablated improvements to reprogramming efficiency. In conclusion, we showed both that iCMs are associated with increased mitochondrial mass, fusion and energetics approaching those of native CMs and that inducing CM-like mitochondria by knocking down Mtfr1l dramatically improved reprogramming, potentially through increased fusion activity of Opa1. By incorporating metabolic reprogramming into traditional direct cardiac reprogramming strategies, we may be able to move the field closer to clinical application to improve outcomes after MI.