Optogenetics involves the measurement and manipulation of cellular activity using genetically encoded light-sensitive proteins (Deisseroth et al., 2006; Miesenböck, 2009). Originally developed as a set of tools for neuroscience to activate or silence neuronal circuits and observe neuronal activity (Li et al., 2005; Nagel et al., 2005), optogenetic “reporters” (for measurement of membrane potential (Siegel and Isacoff, 1997; Sakai et al., 2001; Ataka and Pieribone, 2002) or intracellular calcium [Ca²⁺] (Miyawaki et al., 1997, 1999; Baird et al., 1999)) and ‘actuators' [for modulation of membrane potential (Nagel et al., 2002, 2003; Boyden et al., 2005)] have now been utilized in cardiac research for over a decade (Entcheva and Kay, 2021). Cardiac optogenetics has had a wide-range of applications, including: (i) all-optical studies of cardiac electrophysiology and high-throughput drug screening; (ii) cell-specific measurement or control to investigate cardiac sub-populations (e.g., myocytes, Purkinje cells, fibroblasts, neurons, and immune cells); (iii) manipulation of cardiac ion channels, G protein- coupled receptor signaling, and energetics; (iv) control of action potential morphology or excitation waves; and (v) cardiac pacing, cardioversion/defibrillation, or arrhythmia termination/ablation. Some of the pioneering studies that applied optogenetics to the heart were performed in zebrafish (Danio rerio). Here we provide an overview of the use of zebrafish for cardiac optogenetic studies, highlighting their advantages, limitations, and future potential [for a more general consideration of cardiac optogenetics, please see the recent review by Entcheva and Kay (Entcheva and Kay, 2021)].

The zebrafish has become an important integrative animal model for cardiac research, based on its particular advantages as an experimental tool (Table 1) (Gut et al., 2017; Stoyek and Quinn, 2018). The zebrafish offers a fully sequenced genome, which can be easily altered using standard genetic techniques at relatively low cost (in terms of time, effort, and money) (Rafferty and Quinn, 2018; Stoyek et al., in press), and almost every cardiac gene has a human ortholog with analogous function (Howe et al., 2013). This high degree of genetic similarity has permitted researchers to recapitulate a variety of human cardiac diseases in the zebrafish (Bowley et al., in press), which can be studied in a high throughput manner (Kithcart and MacRae, 2018). Functionally, the zebrafish heart has comparable heart rate, action potential morphologies, ion channels (Ravens, 2018), and Ca²⁺-handling proteins (van Opbergen et al., 2018b) to human. Furthermore, it has been shown that cardiac regulatory pathways and mechanisms of both intracardiac (MacDonald et al., 2017) and extracardiac (Stoyek et al., 2016) origin are similar to human, and like the cardiac electrophysiology of the zebrafish (Nemtsas et al., 2010), are often more so than rodents.

Of course, as with any experimental model, there are also limitations to the zebrafish's use (Table 1). The zebrafish heart is small, has only two chambers (one atrium and one ventricle, rather than the four chambers found in human), and generates relatively low pressures (Hu et al., 2001). While over 70% of human genes have at least one zebrafish ortholog, 24% of genes have more than one ortholog (due to a duplication of the zebrafish genome), which can confer redundancy in gene function and confound results of genetic manipulations (Howe et al., 2013). Functionally, while cardiac electrophysiology appears strikingly similar to humans (Vornanen and Hassinen, 2016; Ravens, 2018), there are important differences in cellular calcium cycling (Genge et al., 2016; van Opbergen et al., 2018b). Zebrafish cardiomyocytes have a lack of transverse tubules (Brette et al., 2008), and even though sarcoplasmic reticulum Ca²⁺ levels are much higher in the zebrafish, release of Ca²⁺ from the sarcoplasmic reticulum following excitation (Ca²⁺-induced Ca²⁺ release) appears to be limited (due in part to a low sensitivity of ryanodine receptors to Ca²⁺) (Bovo et al., 2013). As a result, sarcolemmal Ca²⁺ influx is responsible for ~80% of the Ca²⁺ transient in zebrafish cardiomyocytes (compared to 25% in human) (Bovo et al., 2013), although this remains somewhat controversial, as others have shown a strong dependence of contractile force on sarcoplasmic reticulum Ca²⁺ release (Haustein et al., 2015) and the existence of Ca²⁺ sparks with characteristics similar to mammals (Llach et al., 2011). Zebrafish also have a higher sodium-Ca²⁺ exchanger current than in mammals, such that its reverse-mode has been shown to trigger sarcoplasmic reticulum Ca²⁺ release (Zhang et al., 2011).

Considering its use specifically for cardiac optogenetic studies, the zebrafish has a further advantage over other animal models, in that the entire zebrafish heart can be optically accessed in vivo in the transparent, externally developing embryo (van Opbergen et al., 2018a) or in situ in the relatively small, isolated adult heart (Stoyek et al., 2018), in a manner not possible in mammals. While other non-mammalian models may have a similar advantage (e.g., Drosophila melanogaster [Wolf et al., 2006) and Xenopus laevis (Warkman and Krieg, 2007)], they are limited in other ways. For instance, while Drosophila have been highly utilized for studies of cardiac genetics (Wolf et al., 2006), it is an invertebrate, and differences in the morphology of its heart—which is a tube—limits its applicability for functional studies (Rotstein and Paululat, 2016). The heart of Xenopus, on the other hand, is in some ways more anatomically similar to humans than zebrafish—for instance, it has a pulmonary circulation—but there is a limited genetic tool box for their transgenesis (Ishibashi et al., 2008).

Ultimately, the similarities of zebrafish to human, and its particular experimental advantages, have resulted in it being a popular experimental model for optogenetic investigations, both for neuroscience [the brain and nervous system can also be optically accessed in the whole animal (Del Bene and Wyart, 2012; Simmich et al., 2012; Portugues et al., 2013)] and for cardiovascular research (Table 2), which is the focus of this review.

The electrical activity of the heart has been optically monitored for decades, well before the emergence of modern optogenetics and the use of functional fluorescent proteins. In the 1970s, Salama and Morad published the first reports of the use of voltage sensitive fluorescent dyes to record cardiac action potentials (Salama and Morad, 1976; Morad and Salama, 1979). Since that time, optical mapping of membrane potential and intracellular Ca²⁺ in the whole heart or isolated tissue and cells has become a “standard” technique in many research labs (Herron et al., 2012; Jaimes et al., 2016; Berenfeld and Efimov, 2019), including studies using zebrafish (Sabeh et al., 2012; Lin et al., 2020). The use of optogenetic reporters (genetically encoded voltage and Ca²⁺ indicators, GEVIs and GECIs, respectively) have additional advantages as they allow for organ-, organelle-, and cell-specific measurements and for in vitro and in vivo longitudinal studies. There is now a wide array of GEVIs and GECIs available for use in the heart, with a range of excitation and emission spectra, light sensitivity and signal intensity, temporal dynamics, and other properties that dictate their specific use (Figure 1) (Kaestner et al., 2014, 2015; Koopman et al., 2017).

Optogenetic actuators are light-activated proteins that generate a transmembrane ion flux. The discovery and cloning of the cation-selective ion channel channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii in 2003 (Nagel et al., 2003) has led to the development of an extensive toolkit that includes depolarizing (excitatory) and hyperpolarizing (inhibitory) opsins, which are activated across a wide spectrum of wavelengths, and may be used for manipulation of cardiac membrane potential (Figure 1) (Schneider-Warme, 2018; Ferenczi et al., 2019; Entcheva and Kay, 2021). As for the cardiac application of GEVIs and GECIs, one of the first reports of the use of optogenetic actuators in the heart was in zebrafish (Arrenberg et al., 2010). This involved the use of both ChR2 [Tg(E1b:Gal4^s1101t; UAS:ChR2(H134R)-eYFP^s1990t)] and the chloride-specific ion channel halorhodopsin from Natronomonas pharaonis (NpHR) (Zhang et al., 2007) [Tg(E1b:Gal4-VP16^s1101t; UAS:NpHR-mCherry^s1989t)], to locate and control cardiac pacemaker cells in intact 1–5 dpf zebrafish embryos. In NpHR-expressing zebrafish, maps were generated at each day post-fertilization by sequentially illuminating small, overlapping regions of the heart, and measuring the heart rate response, or the incidence of cardiac arrest or arrhythmia (Figure 5A). It was found that: (i) at 1 dpf, the heart stopped beating when a region at the venous pole was illuminated, indicating the location of the pacemaker; (ii) at 2 dpf, the pacemaker region was more confined to the sinoatrial ring, with illumination of large adjacent areas having no effect, and atrioventricular block (of varying degree, depending on light intensity) occurred with illumination of the atrioventricular canal; and (iii) at 3 dpf, the pacemaker region was more defined, being confined to the dorsal right quadrant of the sinoatrial ring. It was further found that in 4 dpf embryos, pulsed photo-stimulation of the sinoatrial ring at a frequency of 2.7–4.7 Hz with ChR2 was able to control heart rate.

Since the time of that pioneering study, another prominent family of chloride-specific light-activated ion channels has been developed, the Guillardia theta anion channelrhodopsin 1 and 2 (GtACR1 and GtACR2) (Govorunova et al., 2015), which have been shown to silence neuronal AP generation (including in zebrafish) (Malyshev et al., 2017; Mauss et al., 2017; Mohamed et al., 2017; Forli et al., 2018). The first cardiac application of GtACR1 involved zebrafish [using hearts isolated from 3 months post-fertilization adults with cardiac-specific GtACR1 expression, Tg(cmlc2:GtACR1-eGFP)], which in combination with experiments in genetically transfected single rabbit ventricular myocytes demonstrated that GtACR1 activation causes depolarization of ventricular myocytes when applied during the resting (diastolic) phase of the AP (and if suprathreshold, results in excitation), but causes repolarization when applied during the (systolic) plateau (resulting in shortening of the AP). This biphasic response relates to the reversal potential of chloride in ventricular myocytes, which is somewhere between −40 and −33 mV (Clemo et al., 1999), so that the flow of negative ions switches from outward (causing depolarization) to inward (causing repolarization) as cells are excited. As a result, pulsed illumination can be used to pace the heart, while sustained illumination can arrest the heart in a depolarized state (Figure 5B) (Kopton et al., 2018). This indicates that while GtACR1 does not address the need for optogenetic silencing through a physiological means (i.e., hyperpolarization), it is a potentially attractive tool for exciting cardiomyocytes by transient light-induced depolarization.

Unlike the prevalent use of optogenetics in zebrafish to study the nervous system (Del Bene and Wyart, 2012; Simmich et al., 2012; Portugues et al., 2013), there have been relatively few cardiac optogenetic studies performed in zebrafish (summarized in Table 2), yet those studies have been fundamental in progressing the application of optogenetic technologies to the heart. With the continual improvement of optogenetic techniques (Entcheva and Kay, 2021), the use of zebrafish for integrative (patho-)physiological cardiac structure-function studies holds great promise. Future research will be driven by technological advances such as high-speed, cell-accurate, three-dimensional mapping (Mickoleit et al., 2014; Weber et al., 2017; Sacconi et al., in press), more effective methods for cell-specific spatial and temporal gene expression (Reade et al., 2017; LaBelle et al., 2021), and novel optogenetic actuators and reporters with enhanced expression, fluorescence, and kinetics, combined with improved light delivery (Entcheva and Kay, 2021). As the field continues to develop, the zebrafish may be invaluable for cardiac optogenetic studies directly related to its strengths as an experimental model (Table 1; i.e., development, genetic screening, drug discovery, cardiotoxicity testing, disease modeling, all-optical studies of electrophysiology and cell signaling, and anti-arrhythmic strategy development). It may also be a powerful tool for fundamental investigations of the hetero-cellular heart (e.g., structure-function interactions of myocytes, fibroblasts, intracardiac neurons, and immune and endothelial cells) and in helping to overcome hindrances related to the clinical translation of optogenetic techniques (e.g., genetic transfection, immune responses, phototoxicity) (Richter and Bruegmann, 2020). This will be enhanced by the large number of currently available transgenic lines, genetic material, and tools (easily found through online resources and central repositories), facilitated by the open zebrafish community willing to share them (Rafferty and Quinn, 2018; Stoyek et al., in press).

While a majority of cardiac optogenetic studies in the zebrafish have been performed in the early stages of development, technological advances in fluorescence imaging approaches and methods for spatially-resolved light stimulation have the promise to enable studies to be performed in the adult isolated whole heart and in vivo. This will be aided by the continuing development of transgenic lines that lack pigment and are thus largely transparent throughout their lifespan (i.e., “casper” [White et al., 2008) and “crystal” (Antinucci and Hindges, 2016)], and can be used as a background on which to express optogenetic reporters and actuators along with mutations of interest.

Optogenetics is a powerful and highly successful (Deisseroth, 2010) set of techniques that has been instrumental in recent developments in neuroscience research (Deisseroth, 2015; Kim et al., 2017), and more recently also for cardiac research (Entcheva and Kay, 2021). Zebrafish provide specific advantages as an experimental model for optogenetic cardiac investigations (Gut et al., 2017; Stoyek and Quinn, 2018), and have been instrumental in its early development, suggesting a bright future for this little fish. With the increasing sophistication of optogenetic methods, the zebrafish represents an experimental model with great potential for cardiac optogenetic studies. Hopefully more cardiac researchers will soon begin to see the light.

JB wrote the manuscript. MS and TQ revised the manuscript. All authors approved the final version.

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-04879 to TQ), the Heart and Stroke Foundation of Canada (G-18-0022185 to TQ), and the Canadian Institutes of Health Research (MOP 342562 to TQ).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.