Fluorescent, Bioluminescent, and Optogenetic Approaches to Study Excitable Physiology in the Single Cardiomyocyte

Abstract

This review briefly summarizes the single cell application of classical chemical dyes used to visualize cardiomyocyte physiology and their undesirable toxicities which have the potential to confound experimental observations. We will discuss, in detail, the more recent iterative development of fluorescent and bioluminescent protein-based indicators and their emerging application to cardiomyocytes. We will discuss the integration of optical control strategies (optogenetics) to augment the standard imaging approach. This will be done in the context of potential applications, and barriers, of these technologies to disease modelling, drug toxicity, and drug discovery efforts at the single-cell scale.

Comparative Analysis of Genetic Variations in the Nav1.5 Sodium Channel Subunits that Underlie Brugada Syndrome Using Patient-Specific iPSC-CMs

“…The BrS-CMs also presented a prolonged AP duration, which seems to be an overlapping feature of long QT type 3 and BrS (26) (27). Moreover, BrS-CMs exhibited multiple abnormal calcium-related activities, including EAD-like and DAD-like events, which are thought to be a surrogate for polymorphic VT (28,29). Collectively, both Na v 1.5 gene mutants in our study impaired electrophysiological function and triggered adverse events related to BrS clinical manifestations.…”

Feasibility of Using Adjunctive Optogenetic Technologies in Cardiomyocyte Phenotyping – from the Single Cell to the Whole Heart

“…Optogenetics was pioneered in 2002 initially in neural tissue [7], and became increasingly popular once improved light-sensitive microbial opsin channels (called channelrhodopsins (ChR2)) were produced [8]. Cardiac applications were first introduced in 2010 [9,10] and are now rapidly being adopted by the experimental community as an alternative method for pacing and cardioverting tissue with light [11,12]. This approach enables optical pacing, and overcomes many of the inherent limitations of direct electrical stimulation.…”

“…Although transgenic approaches may facilitate more homogeneous transgene expression, they tend to do so at the expense of lower protein levels compared to transient methods, which therefore makes cells harder to capture with optical stimulation. This, and a number of other technical considerations for successful optogenetic application in single cells have recently been reviewed [12]. Finally, in the drug screening context it is possible that some of the "hits" may be exerting their effects through the optogenetic component.…”

“…Images of cell activity can be obtained using exogenously applied dyes or proteins which are sensitive to membrane voltage, cell calcium or other ion species, or directly measure cardiac contraction using dark field, phase or other light refraction-based techniques. It is beyond the scope of this article to review the indicator literature, but a recent review is available [12], and a few caveats are worth highlighting. First, while optical probes show relative changes in membrane voltage or ion concentration, obtaining absolute measurement of these parameters involves ratiometric techniques as well as extensive calibration [16], which is not the case for sharp or patch electrode methods.…”

“…High throughput systems either depend on using a stage top incubation system, an enclosure that covers most of the microscope, use systems that fit within a standard incubator, or build systems that combine optics and environmental control in one unit. The optogenetic tools themselves also demonstrate thermal sensitivity, working better at body, rather than room temperature [12].…”