Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging

Dempsey, Graham T.; Chaudhary, Khuram W.; Atwater, Nicholas; Nguyen, Cuong; Brown, Barry S.; McNeish, John D.; Cohen, Adam E.; Kralj, Joel M.

doi:10.1016/j.vascn.2016.05.003

Cited by 130 publications

(145 citation statements)

References 78 publications

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“…Recently described systems enabled simultaneous voltage and calcium imaging in free-running or electrically paced hiPSC-CM [21], optogenetic pacing and either voltage or calcium imaging [22], or optogenetic pacing with simultaneous voltage and calcium imaging [23]. …”

Section: Introductionmentioning

confidence: 99%

“…We applied simultaneous voltage and calcium imaging using a genetically encoded dual-function reporter, CaViar [23,24], to probe the functional maturation of hiPSC-CM grown on micro-fabricated islands of different sizes and shapes. HiPSC-CM grown on larger islands showed greater functional maturity by several measures of voltage and Ca 2+ dynamics.…”

Section: Introductionmentioning

confidence: 99%

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Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

et al. 2017

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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

et al. 2017

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“…voltage indicators based on rhodopsins have been used in numerous cell types, including neurons, cardiomyocytes, and bacteria (21)(22)(23). These improved sensors enable measurements in very small cells and avoid potential exclusion by the outer membrane and cell wall.…”

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confidence: 99%

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

Bruni

Weekley

Dodd

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.alcium is a universal and indispensable signaling ion used in all known eukaryotes (1). Calcium concentration gradients across the plasma membrane and intracellular organelles enable highly dynamic fluxes via orchestrated channel openings to generate tightly controlled spatial and temporal patterns. The dynamics in both space and time are encoded and decoded into varying and sometimes opposing cellular signals (2, 3). In electrically excitable cells, including neurons and muscle cells, voltage-gated calcium channels (VGCCs) couple membrane depolarization to calcium influx, which can then alter cellular physiology. The myriad uses of calcium by cells highlight its crucial role in cellular maintenance.Despite calcium's ubiquity and utility across biological domains, little is known about how calcium is regulated in bacteria, especially at the single cell level (4). Fluorescent calcium dyes used in eukaryotic cells are not able to pass through the cell wall without pretreatment with the chelating agent EDTA, prohibiting studies of calcium with high temporal and spatial resolution in bacteria. Isotope labeling and luminescent probes have contributed population-level measurements of calcium, but were unable to resolve any potential cellular heterogeneity. In population measurements, cytoplasmic ...

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“…In contrast, transmembrane voltage signals obtained using voltage-sensing optical techniques provides an efficient approach to assess the electrophysiological characteristics of hiPSC-CM at both baseline and following the addition of drug. Furthermore, voltage-sensitive fluorescent indicators have previously been used to accurately monitor electrical activity in cardiac cells (Dempsey et al , 2016; Klimas et al , 2016), and this capability offers significant potential when applied to monitoring electrical activity in hiPSC-CM. This combination of instrument plus indicator offers a robust platform on which to advance the use of hiPSC-CM within the proposed guidelines of CiPA.…”

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confidence: 99%

The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

et al. 2016

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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10–90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/normalTrise2 in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.

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Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging

Cited by 130 publications

References 78 publications

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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