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Sobie, E. A.

Publications and source records attributed to Sobie, E. A..

9 recordsLinked to original sources

Creating cell-specific computational models of stem cell-derived cardiomyocytes using optical experiments

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. Author SummaryMany drug treatments or environmental factors can trigger cardiac arrhythmias, which are dangerous and often unpredictable. Human cardiomyocytes derived from donor stem cells have proven to be a promising model for studying these events, but variability in donor genetic background and cell maturation methods, as well as overall immaturity of stem cell-derived cardiomyocytes relative to the adult heart, have hindered reproducibility and reliability of these studies. Mathematical models of these cells can aid in understanding the underlying electrophysiological contributors to this variability, but determining these models parameters for multiple cell preparations is challenging. In this study, we tackle these limitations by developing a computational method to simultaneously estimate multiple model parameters using data from imaging-based experiments, which can be easily scaled to rapidly characterize multiple cell lines. This method can generate many personalized models of individual cell preparations, improving drug response predictions and revealing specific differences in electrophysiological properties that contribute to variability in cardiac maturity and arrhythmia susceptibility. GLOSSARYO_LIModel/parameter calibration: tuning one or more parameters in the computational model so that the model output more closely matches experimental data C_LIO_LIExperiment/protocol optimization: the process of determining what type and amount of data is sufficient but also feasible for our model calibration goals O_LIProtocol conditions - buffer calcium, potassium, or sodium concentrations; addition or removal of stimulus; pacing rates; channel block; etc. C_LIO_LIProtocol length(?) - number of protocol conditions C_LIO_LIProtocol data type(?) -AP, CaT, or both; normalized or non-normalized data C_LIO_LIModel prediction: using the calibrated computational model to simulate response to new (unseen) conditions, drugs, or perturbations (in our case, IKr block) C_LIO_LIPapers on independent validation/prediction C_LI C_LI Computational pipeline: the full process of iPSC-CM computational model calibration; Includes iPSC-CM data acquisition/simulation -> data processing -> parameter calibration using genetic algorithm -> validation of calibrated models on an unseen condition (i.e. evaluating model predictions)

pharmacology and toxicology↗

Coupling and Heterogeneity Modulate Pacemaking Capability in Healthy and Diseased Two-Dimensional Sinoatrial Node Tissue Models

Both experimental and modeling studies have attempted to determine mechanisms by which a small anatomical region, such as the sinoatrial node (SAN), can robustly drive electrical conduction in the human heart. However, despite important advances from prior research, important questions remain unanswered. This study aimed to investigate, through mathematical modeling, the role of intercellular coupling and cellular heterogeneity in synchronization and pacemaking within the healthy and diseased SAN. In a multicellular computational model of a monolayer of either human or rabbit SAN cells, simulations revealed that heterogenous cells synchronize their discharge frequency into a unique beating rhythm across a wide range of cellular heterogeneity and intercellular coupling. However, an insightful and unanticipated behavior appears in pathological conditions precipitated by perturbations of certain ionic currents (gCaL = 0.5): an intermediate range of intercellular coupling (900-4000 M{Omega}) is beneficial to the human SAN automaticity, enabling a very small portion of tissue (3.4%) to drive propagation, which fails with both lower and higher resistances. This protective effect of intercellular coupling and heterogeneity, seen in both human and rabbit tissues, highlights the remarkable resilience of the SAN. Overall, the model here developed allowed insight into the mechanisms of automaticity of the human sinoatrial node. The simulations suggest that certain degrees of gap junctional expressions protect the SAN from ionic perturbations that might be caused by drugs or mutations.

physiology↗

PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

cell biology↗

A Library Of Induced Pluripotent Stem Cells From Clinically Well-Characterized, Diverse Healthy Human Individuals

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22-61. The hiPSCs match the karyotype and short tandem repeat identity of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of Mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-seq derived cell clusters and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.

cell biology↗

A tissue- and organ-based cell biological atlas of obesity-related human genes and cellular pathways

Over the last decades, several features of obesity have been identified at behavioral, physiological, endocrine and genomic levels, and they have revealed the complexity of the disease; obesity results from a combination of genetic predisposition, endocrine disorders, and dysregulation of both food intake and energy expenditure. This complexity makes the development of new therapeutic regimens challenging and bariatric surgery is still the treatment of choice for many obese patients. Given the need for noninvasive therapeutic intervention strategies, we sought to systematically study the biological manifestations of obesity in peripheral organs. We analyzed publicly available datasets of genes, genomic determinants, and levels of obesity-related hormones in the blood, using a combination of methodologies, including graph theory and dynamical modeling, that allow for the integration of different types of datasets. The analysis revealed tissue- and organ-specific metabolic impairments and potential new drug targets. All the data are organized into a tissue/organ-based subcellular-function atlas for human obesity. The data show that the complexity of the obesity arises due to the multiplicity of subcellular processes in different peripheral organs.

systems biology↗

Proteomic cellular signatures of kinase inhibitor-induced cardiotoxicity: Mount Sinai DToxS LINCS Center Dataset

The Drug Toxicity Signature Generation Center (DToxS) at the Icahn School of Medicine at Mount Sinai is one of the centers of the NIH Library of Integrated Network-Based Cellular Signatures (LINCS) program. A key aim of DToxS is to generate both proteomic and transcriptomic signatures that can predict adverse effects, especially cardiotoxicity, of kinase inhibitors approved by the Food and Drug Administration. Towards this goal, high throughput shot-gun proteomics experiments (317 cell line/drug combinations + 64 control lysates) have been conducted at the Center for Advanced Proteomics Research at Rutgers University - New Jersey Medical School. Using computational network analyses, these proteomic data can be integrated with transcriptomic signatures generated in tandem to identify cellular signatures of cardiotoxicity that may predict kinase inhibitor-induced toxicity and possible mitigation. Both raw and processed proteomics data have been carefully screened for quality and made publicly available via the PRIDE database. As such, this broad protein kinase inhibitor-stimulated cardiomyocyte proteomic data and signature set is valuable for the prediction of drug toxicities. Links to: Metadata Tables O_TBL View this table: org.highwire.dtl.DTLVardef@1cda9f8org.highwire.dtl.DTLVardef@1520e05org.highwire.dtl.DTLVardef@16a35borg.highwire.dtl.DTLVardef@3ee6e7org.highwire.dtl.DTLVardef@1a98664_HPS_FORMAT_FIGEXP M_TBL C_TBL

pharmacology and toxicology↗

Population-based mechanistic modeling allows for quantitative predictions of drug responses across cell types

Quantitative mismatches between human physiology and experimental models can present serious limitations for the development of effective therapeutics. We addressed this issue, in the context of cardiac electrophysiology, through mechanistic mathematical modeling combined with statistical analyses. Physiological metrics were simulated in heterogeneous populations describing cardiac myocytes from adult ventricles and those derived from induced pluripotent stem cells (iPSC-CMs). These simulated measures were used to construct a cross-cell type regression model that predicts adult myocyte drug responses from iPSC-CM behaviors. We found that quantitatively accurate predictions of responses to selective or non-selective drugs could be generated based on iPSC-CM responses and that the method can be extended to predict drug responses in diseased as well as healthy cells. This cross-cell type model can be of great value in drug development, and the approach, which can be applied to other fields, represents an important strategy for overcoming experimental model limitations.

systems biology↗

Fragile dynamics enable diverse genomic determinants to influence arrhythmia propensity

Genotype-phenotype relationships are determinants of human diseases. Often, we know little about why so many genes are involved in complex common diseases. We hypothesized that this multigene effect arises from the relationship between genes and physiological dynamics. We tested this hypothesis for arrhythmias as physiological dynamics define this disease. We integrated graph theory analysis of genomic and protein-protein interaction networks with dynamical models of ion channel function to identify the physiological dynamics of genome wide variation for five different arrhythmias. Regulatory networks for the cardiac conduction system and arrhythmias were constructed from GWAS and known disease genes. Electrophysiological models of myocyte action potentials were used to conduct extensive parameter variations to identify robust and fragile kinetic parameters that were then, using regulatory networks, associated with genomic determinants. We find that genome-wide determinants of arrhythmias that represent many cellular processes are selectively associated with fragile physiological dynamics of ion channel kinetics. This association predicts disease propensity. Deep RNA sequencing from human left ventricular tissue of arrhythmia and control subjects confirmed the predictive relationship. Taken together these studies show that the varied multigene effects of arrhythmias arises because of associations with fragile kinetic parameters of cardiac electrophysiology.\n\nSignificance StatementOur understanding of the genetics of common diseases has advanced exponentially over the past decade. We now know that differences and variation in multiple genes contribute to disease susceptibility with significant heterogeneity in the phenotype. However, how genetic variation contributes to disease phenotypes remains unknown. We hypothesized that the relationships between physiological dynamics and genetic architecture is a fundamental determinant of disease susceptibility and genetic heterogeneity. To test our hypothesis, we integrated mathematical models of cardiac electrophysiology with genetic network models of cardiac arrhythmias. We found that disease related genome variants were selectively associated with fragile kinetic parameters that predict disease propensity and identified several novel cellular processes associated with arrhythmogenesis.

systems biology↗

A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries

Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 3 end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 3-digital gene expression (3-DGE) approach with \"conventional\" random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 3-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

genomics↗