Cardiomyocytes (CMs) and fibroblast cells are two essential elements for cardiac tissue structure and function. The interactions between them can alter cardiac electrophysiology and thus contribute to cardiac diseases, such as arrhythmogenesis. One possible explanation is that fibroblasts can directly affect cardiac electrophysiology through electrical coupling with CMs. Therefore, detecting the electrical activities in the CM-fibroblast network is vital for understanding the coupling dynamics among them. Current commercialized platforms for studying cardiac electrophysiology utilize planar microelectrode arrays (MEAs) to record the extracellular field potential (FP) in real-time, but the prearranged electrode configuration highly limits the measurement capabilities at specific locations. Here, we report a custom-designed MEA device with a novel micropatterning method to construct a controlled network of neonatal rat CMs (rCMs) and fibroblast connections for monitoring the electrical activity of rCM-fibroblast co-cultures in a spatially controlled fashion. For the micropatterning of the co-culture, surface topographical features and mobile blockers were used to control the initial attachment locations of a mixture of rCMs and fibroblasts, to form separate beating rCM-fibroblast clusters while leaving empty space for fibroblast growth to connect these clusters. Once the blockers are removed, the proliferating fibroblasts connect and couple the separate beating clusters. Using this method, electrical activity of both rCMs and human-induced-pluripotent-stem-cell-derived cardiomyocytes (iCMs) was examined. The coupling dynamics were studied through the extracellular FP and impedance profile recorded from the MEA device, indicating that the fibroblast bridge provided an RC-type coupling of physically separate rCM-containing clusters and enabled synchronization of these clusters.
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OptoGap is an optogenetics-enabled assay for quantification of cell–cell coupling in multicellular cardiac tissue
Abstract Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and non-excitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g., cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells (e.g., cardiomyocytes) that are light-insensitive. Cell–cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modelling and proper calibration. Lastly, the sensitivity of OptoGap to intrinsic cell-scale excitability is robustly characterized via computational analysis.
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- NSF-PAR ID:
- 10224741
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning–based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.
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Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm 2 ) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.more » « less
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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. -
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Abstract Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC‐CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC‐CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two‐dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC‐CMs.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41598-021-88573-1
