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CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes.

ABSTRACT: Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all-optical tools. More potent CRISPRi effectors, e.g., Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

SUBMITTER: Han JL

PROVIDER: S-EPMC10703822 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes.

Han Julie L JL Heinson Yuli W YW Chua Christianne J CJ Liu Wei W Entcheva Emilia E

Communications biology 20231207 1

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardia ...[more]

PMID: 38062109

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Project description:High-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology and by contact-free all-optical systems simultaneously measuring action potentials (APs) and Ca2+ transients (CaTrs). Parallel computational advances have shown that in silico simulations can predict drug effects with high accuracy. We combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPSC-CM populations and to predict and explain drug action mechanisms. Optically obtained hiPSC-CM APs and CaTrs were used from spontaneous activity and under optical pacing in control and drug conditions at multiple doses. An updated version of the Paci2018 model was developed to refine the description of hiPSC-CM spontaneous electrical activity; a population of in silico hiPSC-CMs was constructed and calibrated using simultaneously recorded APs and CaTrs. We tested in silico five drugs (astemizole, dofetilide, ibutilide, bepridil, and diltiazem) and compared the outcomes to in vitro optical recordings. Our simulations showed that physiologically accurate population of models can be obtained by integrating AP and CaTr control records. Thus, constructed population of models correctly predicted the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights; e.g., through simulations, bepridil's more proarrhythmic action in adult cardiomyocytes compared to hiPSC-CMs could be traced to the different expression of ion currents in the two. Therefore, our work 1) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally calibrated populations of in silico hiPSC-CMs, 2) offers insights into certain limitations when translating results obtained in hiPSC-CMs to humans, and 3) shows the strength of combining high-throughput in vitro and population in silico approaches.

Project description:The ability to accurately phenotype cells differentiated from human induced pluripotent stem cells (hiPSCs) is essential for their application in modeling developmental and disease processes, yet also poses a particular challenge without the context of anatomical location. Our specific objective was to determine if single-cell gene expression was sufficient to predict the electrophysiology of iPSC-derived cardiac lineages, to evaluate the concordance between molecular and functional surrogate markers. To this end, we used the genetically encoded voltage indicator ArcLight to profile hundreds of hiPSC-derived cardiomyocytes (hiPSC-CMs), thus identifying patterns of electrophysiological maturation and increased prevalence of cells with atrial-like action potentials (APs) between days 11 and 42 of differentiation. To profile expression patterns of cardiomyocyte subtype-associated genes, single-cell RNA-seq was performed at days 12 and 40 after the populations were fully characterized with the high-throughput ArcLight platform. Although we could detect global gene expression changes supporting progressive differentiation, individual cellular expression patterns alone were not able to delineate the individual cardiomyocytes into atrial, ventricular, or nodal subtypes as functionally documented by electrophysiology measurements. Furthermore, our efforts to understand the distinct electrophysiological properties associated with day 12 versus day 40 hiPSC-CMs revealed that ion channel regulators SLMAP, FGF12, and FHL1 were the most significantly increased genes at day 40, categorized by electrophysiology-related gene functions. Notably, FHL1 knockdown during differentiation was sufficient to significantly modulate APs toward ventricular-like electrophysiology. Thus, our results establish the inability of subtype-associated gene expression patterns to specifically categorize hiPSC-derived cells according to their functional electrophysiology, and yet, altered FHL1 expression is able to redirect electrophysiological maturation of these developing cells. Therefore, noncanonical gene expression patterns of cardiac maturation may be sufficient to direct functional maturation of cardiomyocytes, with canonical gene expression patterns being insufficient to temporally define cardiac subtypes of in vitro differentiation.

Project description:Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2 Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the IKs potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.

Dataset Information

CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes.

Publications

CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes.

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