An infrared optical pacing system for screening cardiac electrophysiology in human cardiomyocytes

McPheeters, Matthew T.; Wang, Yves T.; Werdich, Andreas A.; Jenkins, Michael W.; Laurita, Kenneth R.

doi:10.1371/journal.pone.0183761

Cited by 29 publications

(27 citation statements)

References 64 publications

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“…Still, as reported previously with voltage sensitive dyes, light intensity and duration can alter the APD 2,5,17 . This is believed to be the result of the generation of reactive oxidative species (ROS)…”

Section: Discussionsupporting

confidence: 67%

Optical Imaging of Isolated Murine Ventricular Myocytes

Han

Klos

Morgenstern

et al. 2020

JoVE

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The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it. Video Link The video component of this article can be found at https://www.jove.com/video/60196/ 9. Consequently, using a single workstation this protocol allows for a thorough examination of singly myocyte excitation-contraction coupling. Protocol All methods and procedures described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.

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Section: Discussionsupporting

confidence: 67%

Optical Imaging of Isolated Murine Ventricular Myocytes

Han

Klos

Morgenstern

et al. 2020

JoVE

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“…Further, it offers the possibility of combining differentiated cells from different wells or pooling data from individual wells of the 96-well format to balance out the inter-experimental variation and improve reproducibility. This protocol also demonstrates it is not necessary to first differentiate the cells in larger format before reseeding them in to 96 wells for experimentation such as high throughput analysis 41 , 42 .…”

Section: Discussionmentioning

confidence: 94%

A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates

Balafkan

Mostafavi

Schubert

et al. 2020

Sci Rep

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The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.

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“…Optical recordings of the membrane potential were accomplished with FluoVolt dye (Thermo Fisher Scientific, Waltham, MA), which was previously validated for the recording of neuronal APs in our group 25 and for cardiac APs by others. 36 The procedures for field stimulation of individually selected cells on a microscope stage were similar to those described previously. 25 In brief, a single selected cell was brought in the center of the microscope field of view, and a pair of tungsten rod electrodes (100 μm diameter, 170 μm interelectrode gap) was positioned above it with a help of a micromanipulator.…”

Section: Cell Imagingmentioning

confidence: 99%

Excitation of murine cardiac myocytes by nanosecond pulsed electric field

Azarov

Semenov

Casciola

et al. 2019

Cardiovasc electrophysiol

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Introduction Opening of voltage‐gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Methods and Results Fluorescent imaging of optical APs (FluoVolt) and Ca2+‐transients (Fluo‐4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200‐nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin‐sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l‐type calcium and sodium‐calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+‐transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin‐verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage‐gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+‐transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). Conclusions The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage‐gated calcium channels) and SSD. Tetrodotoxin‐sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l‐type calcium and sodium‐calcium exchanger currents.

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An infrared optical pacing system for screening cardiac electrophysiology in human cardiomyocytes

Cited by 29 publications

References 64 publications

Optical Imaging of Isolated Murine Ventricular Myocytes

Optical Imaging of Isolated Murine Ventricular Myocytes

A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates

Excitation of murine cardiac myocytes by nanosecond pulsed electric field

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