Abstract Background Mutations in LMNA , encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. Results Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA -related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. Conclusions Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.
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A Study of Gene Expression, Structure, and Contractility of iPSC-Derived Cardiac Myocytes from a Family with Heart Disease due to LMNA Mutation
Genetic mutations to the Lamin A/C gene (
- Award ID(s):
- 1763272
- NSF-PAR ID:
- 10308259
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Annals of Biomedical Engineering
- ISSN:
- 0090-6964
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Key points Induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) capture patient‐specific genotype–phenotype relationships, as well as cell‐to‐cell variability of cardiac electrical activity
Computational modelling and simulation provide a high throughput approach to reconcile multiple datasets describing physiological variability, and also identify vulnerable parameter regimes
We have developed a whole‐cell model of iPSC‐CMs, composed of single exponential voltage‐dependent gating variable rate constants, parameterized to fit experimental iPSC‐CM outputs
We have utilized experimental data across multiple laboratories to model experimental variability and investigate subcellular phenotypic mechanisms in iPSC‐CMs
This framework links molecular mechanisms to cellular‐level outputs by revealing unique subsets of model parameters linked to known iPSC‐CM phenotypes
Abstract There is a profound need to develop a strategy for predicting patient‐to‐patient vulnerability in the emergence of cardiac arrhythmia. A promising
in vitro method to address patient‐specific proclivity to cardiac disease utilizes induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). A major strength of this approach is that iPSC‐CMs contain donor genetic information and therefore capture patient‐specific genotype–phenotype relationships. A cited detriment of iPSC‐CMs is the cell‐to‐cell variability observed in electrical activity. We postulated, however, that cell‐to‐cell variability may constitute a strength when appropriately utilized in a computational framework to build cell populations that can be employed to identify phenotypic mechanisms and pinpoint key sensitive parameters. Thus, we have exploited variation in experimental data across multiple laboratories to develop a computational framework for investigating subcellular phenotypic mechanisms. We have developed a whole‐cell model of iPSC‐CMs composed of simple model components comprising ion channel models with single exponential voltage‐dependent gating variable rate constants, parameterized to fit experimental iPSC‐CM data for all major ionic currents. By optimizing ionic current model parameters to multiple experimental datasets, we incorporate experimentally‐observed variability in the ionic currents. The resulting population of cellular models predicts robust inter‐subject variability in iPSC‐CMs. This approach links molecular mechanisms to known cellular‐level iPSC‐CM phenotypes, as shown by comparing immature and mature subpopulations of models to analyse the contributing factors underlying each phenotype. In the future, the presented models can be readily expanded to include genetic mutations and pharmacological interventions for studying the mechanisms of rare events, such as arrhythmia triggers. -
null (Ed.)Genetic mutations in genes encoding for potassium channel protein structures have been recently associated with episodes of atrial fibrillation in asymptomatic patients. The aim of this study is to investigate the potential arrhythmogenicity of three gain-of-function mutations related to atrial fibrillation—namely, KCNH2 T895M, KCNH2 T436M, and KCNE3-V17M—using modeling and simulation of the electrophysiological activity of the heart. A genetic algorithm was used to tune the parameters’ value of the original ionic currents to reproduce the alterations experimentally observed caused by the mutations. The effects on action potentials, ionic currents, and restitution properties were analyzed using versions of the Courtemanche human atrial myocyte model in different tissues: pulmonary vein, right, and left atrium. Atrial susceptibility of the tissues to spiral wave generation was also investigated studying the temporal vulnerability. The presence of the three mutations resulted in an overall more arrhythmogenic substrate. Higher current density, action potential duration shortening, and flattening of the restitution curves were the major effects of the three mutations at the single-cell level. The genetic mutations at the tissue level induced a higher temporal vulnerability to the rotor’s initiation and progression, by sustaining spiral waves that perpetuate until the end of the simulation. The mutation with the highest pro-arrhythmic effects, exhibiting the widest sustained VW and the smallest meandering rotor’s tip areas, was KCNE3-V17M. Moreover, the increased susceptibility to arrhythmias and rotor’s stability was tissue-dependent. Pulmonary vein tissues were more prone to rotor’s initiation, while in left atrium tissues rotors were more easily sustained. Re-entries were also progressively more stable in pulmonary vein tissue, followed by the left atrium, and finally the right atrium. The presence of the genetic mutations increased the susceptibility to arrhythmias by promoting the rotor’s initiation and maintenance. The study provides useful insights into the mechanisms underlying fibrillatory events caused by KCNH2 T895M, KCNH2 T436M, and KCNE3-V17M and might aid the planning of patient-specific targeted therapies.more » « less
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During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation-stage human embryos. Human-induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single-cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single-cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.more » « less
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Abstract Aims Dissecting complex interactions among transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are central for understanding heart development and function. Although computational approaches and platforms have been described to infer relationships among regulatory factors and genes, current approaches do not adequately account for how highly diverse, interacting regulators that include noncoding RNAs (ncRNAs) control cardiac gene expression dynamics over time.
Methods To overcome this limitation, we devised an integrated framework, cardiac gene regulatory modeling (CGRM) that integrates LogicTRN and regulatory component analysis bioinformatics modeling platforms to infer complex regulatory mechanisms. We then used CGRM to identify and compare the TF-ncRNA gene regulatory networks that govern early- and late-stage cardiomyocytes (CMs) generated by in vitro differentiation of human pluripotent stem cells (hPSC) and ventricular and atrial CMs isolated during in vivo human cardiac development.
Results Comparisons of in vitro versus in vivo derived CMs revealed conserved regulatory networks among TFs and ncRNAs in early cells that significantly diverged in late staged cells. We report that cardiac genes (“
heart targets ”) expressed in early-stage hPSC-CMs are primarily regulated by MESP1, miR-1, miR-23, lncRNAs NEAT1 and MALAT1, while GATA6, HAND2, miR-200c, NEAT1 and MALAT1 are critical for late hPSC-CMs. The inferred TF-miRNA-lncRNA networks regulating heart development and contraction were similar among early-stage CMs, among individual hPSC-CM datasets and between in vitro and in vivo samples. However, genes related to apoptosis, cell cycle and proliferation, and transmembrane transport showed a high degree of divergence between in vitro and in vivo derived late-stage CMs. Overall, late-, but not early-stage CMs diverged greatly in the expression of “heart target” transcripts and their regulatory mechanisms.Conclusions In conclusion, we find that hPSC-CMs are regulated in a cell autonomous manner during early development that diverges significantly as a function of time when compared to in vivo derived CMs. These findings demonstrate the feasibility of using CGRM to reveal dynamic and complex transcriptional and posttranscriptional regulatory interactions that underlie cell directed versus environment-dependent CM development. These results with in vitro versus in vivo derived CMs thus establish this approach for detailed analyses of heart disease and for the analysis of cell regulatory systems in other biomedical fields.
