Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions.

Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electro-pathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide non-invasive and high- throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D- culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process.

Comprehensive detection of structural variation and transposable element differences between wild type laboratory lineages of C. elegans.

Genomic structural variations (SVs) and transposable elements (TEs) can be significant contributors to genome evolution, altered gene expression, and risk of genetic diseases. Recent advancements in long-read sequencing have greatly improved the quality of de novo genome assemblies and enhanced the detection of sequence variants at the scale of hundreds or thousands of bases. Comparisons between two diverged wild isolates of Caenorhabditis elegans, the Bristol and Hawaiian strains, have been widely utilized in the analysis of small genetic variations. Genetic drift, including SVs and rearrangements of repeated sequences such as TEs, can occur over time from long-term maintenance of wild type isolates within the laboratory. To comprehensively detect both large and small structural variations as well as TEs due to genetic drift, we generated de novo genome assemblies and annotations for each strain from our lab collection using both long- and short-read sequencing and compared our assemblies and annotations with that of other lab wild type strains. Within our lab assemblies, we annotate over 3.1Mb of sequence divergence between the Bristol and Hawaiian isolates: 337,584 SNPs, 94,503 small insertion-deletions (<50bp), and 4,334 structural variations (>50bp). Further, we define the location and movement of specific DNA TEs between N2 Bristol and CB4856 Hawaiian wild type isolates. Specifically, we find the N2 Bristol genome has 20.6% more TEs from the Tc1/mariner family than the CB4856 Hawaiian genome. Moreover, we identified Zator elements as the most abundant and mobile TE family in the genome. Using specific TE sequences with unique SNPs, we also identify 38 TEs that moved intrachromosomally and 9 TEs that moved interchromosomally between the N2 Bristol and CB4856 Hawaiian genomes. By comparing the de novo genome assembly of our lab collection Bristol isolate to the VC2010 Bristol assembly, we also reveal that lab lineages display over 2 Mb of total variation: 1,162 SNPs, 1,528 indels, and 897 SVs with 95% of the variation due to SVs. Overall, our work demonstrates the unique contribution of SVs and TEs to variation and genetic drift between wild type laboratory strains assumed to be isogenic despite growing evidence of genetic drift and phenotypic variation.