TY - JOUR
AU - Gepstein,, Lior
AB - Abstract The emerging technology of optogenetics uses optical and genetic means to monitor and modulate the electrophysiological properties of excitable tissues. While transforming the field of neuroscience, the technology has recently gained popularity also in the cardiac arena. Here, we describe the basic principles of optogenetics, the available and evolving optogenetic tools, and the unique potential of this technology for basic and translational cardiac electrophysiology. Specifically, we discuss the ability to control (augment or suppress) the cardiac tissue’s excitable properties using optogenetic actuators (microbial opsins), which are light-gated ion channels and pumps that can cause light-triggered membrane depolarization or hyperpolarization. We then focus on the potential clinical implications of this technology for the treatment of cardiac arrhythmias by describing recent efforts for developing optogenetic-based cardiac pacing, resynchronization, and defibrillation experimental strategies. Finally, the significant obstacles and challenges that need to be overcome before any future clinical translation can be expected are discussed. Optogenetics , Opsins , Arrhythmias , Pacing , Defibrillation What’s new? Optogenetics is a new discipline that allows targeted light-induced modulation of the function of excitable tissues through the expression of light-sensitive proteins (ion channels and pump). Proof-of-concept in vitro and animal studies demonstrated the potential of optogenetics in the field of cardiac electrophysiology for cardiac pacing, resynchronization, and defibrillation. Several challenges remain on the road for clinical translation of cardiac optogenetics including in the need for advancements in gene delivery and light-emitting devices. Introduction The term optogenetics was first coined by Deisseroth et al.1 in 2006 and refers to the ability to control and monitor the activity of excitable cells by merging genetic and optical means. This ability is achieved through the expression of light-sensitive microbial proteins (opsins) in the targeted cells.2 The expressed opsins function as ion channels, ion pumps, or signalling receptors, and their selective light-dependent activation enables precise, localized, and low-energy control of cells, tissues, organs, and whole-organism activity.3 Today, over a decade after these initial discoveries, optogenetics has completely transformed neuroscience and the way we study the brain, enabling targeted modulation of the activity of specific opsin-expressing neuron populations by light and therefore tight spatiotemporal control of neuronal circuitry in the brain. In addition to allowing better understanding of the functions of the brain, optogenetic-based therapeutic applications are also beginning to emerge, albeit still at the preclinical stage, including the development of light-based treatment methods for epilepsy, depression, pain relief, and Parkinson’s disease.4,5 While most of the efforts in the optogenetic field focused on the brain, pioneering studies suggested that similar tools might also bring a unique value to cardiac electrophysiology.6–8 Consequentially, clinically relevant animal studies using viral gene delivery revealed the ability to modulate cardiac tissue electrical activity through light-induced opsins’ activation, enabling cardiac optogenetic-based pacing,9,10 resynchronization,10 and even defibrillation.11–13 In this review, we first described the biological foundations of optogenetics technology and its practical flexibility. We then discussed its application in the cardiac field: its assimilation in basic research, its clinical promises, and finally the remaining challenges for clinical uses. Biological foundations of optogenetics technology Optogenetics refers to the ability to monitor and control the activity of excitable cells using optogenetic sensors (genetically encoded fluorescent indicators) and optogenetic actuators (light-sensitive proteins and opsins), respectively. For both applications, a major prerequisite is the expression of the relevant proteins in the targeted cells using various gene delivery strategies. While sensor proteins (genetically encoded voltage or calcium indicators; GEVIs or GECIs) permit to monitor different cellular activities online (such as action potentials or calcium-handling properties), actuator proteins allow to perform controllable (light-based) high-resolution functional modulation of the tissues’ excitable properties. The use of optogenetic sensors have already proven valuable for high-throughput as well as long-term monitoring of the electrophysiological and calcium-handling properties of cardiac cells for various pathophysiological studies, disease modelling, and drug screening applications.14–16 Nevertheless, since the clinical use of optogenetic sensors is less obvious, we will focus in the current review only on the potential applications of optogenetic actuators. For cardiac electrophysiology, relevant optogenetic actuators primarily act by altering the membrane potential. These light-sensitive proteins, which are of microbial origins, function as ionotropic receptors whose ligand is light photons and form transmembrane channels or pumps. They are covalently bound to a light-sensitive organic cofactor, called retinal. Once activated by photons, the retinal changes its conformation from all-trans-retinal (ATR) to 13-cis-retinal, allowing ions to pass through the membrane-spanning domain, thereby modulating membrane potential.17 Optogenetics palette Each opsin has its unique biophysical properties regarding mechanism of action, kinetics, selectivity to ions, light-sensitivity, and spectral response.18–27 For cardiac optogenetic applications, opsins from three major groups are currently used: (i) bacteriorhodopsins (BRs)18 including proton-pumps such as Arch and ArchT proteins; (ii) halorhodopsins (HRs) including chloride-pumps such as eNpHR3.0; and (iii) channelrhodopsins (ChRs),19 which are light-gated ionic channels including the most widely used opsin, ChR2, a non-selective light-sensitive cationic channel. Notice that the first two groups include pumps, actively transporting only a single ion per photon. Importantly, while being less affected by changes in the membrane potential, the photocurrents generated by HRs and BRs are significantly lower than those induced by light-sensitive ion channels such as ChRs. This provides a challenge for applications requiring roust hyperpolarization (traditionally achieved by HRs and BRs). The design of a new family of engineered chloride conducting hyperpolarizing ChRs,20–22 which are channels, may provide a possible solution to this challenge. Because of the great diversity in opsin characteristics, optogenetics offers several advantages for modulation of cardiac excitability. First, optogenetics allows both excitatory and inhibitory stimuli, as BRs and HRs induce hyperpolarization (thereby potentially suppressing excitability), while conventional ChRs induce depolarization and therefore can augment excitability. Second, it allows specific control of different light-sensitive proteins, even when co-expressed together in the same tissue, thanks to distinct sensitivities to different light wavelengths. These two features were highlighted in the study of Nussinovitch et al., who co-expressed both ChR2 and ArchT in cardiomyocyte cultures.28 It was then demonstrated that illumination of the cultures with 624 nm monochromatic light resulted in activation of the ArchT proton pump, induction of proton-based outward currents, and suppression of cardiomyocytes’ electrical activity. The same cultures, on the other hand, could be paced at a variety of beating rates by application of flashes of blue light (470 nm) activating the cationic channel ChR2. Finally, one could potentially control the activity of only specific cell types based on the specific tissue-type promoter used to control opsins’ expression. The available opsin diversity continues to grow as scientists discover new natural opsins and perform targeted modifications of existing variants. The quest for new opsin variants aims to: (i) improve opsins’ light-sensitivity; (ii) augment the magnitude of the generated photocurrents; (iii) modulate ion selectivity to produce novel and more effective ways to suppress electrical activity; and (iv) generate opsin variants that will be sensitive to red-shifted wavelengths to allow better tissue penetration. A description of available variants and their functional characteristics can be found in several reviews.23–27 Cardiac optogenetics Evidence for the ability to modulate cardiac excitability with light-sensitive proteins are accumulating. These studies, which primarily used ChR2, utilized a variety of experimental models at the in vitro cell/tissue, ex vivo organ, and in vivo animal levels as well as in silico computational modelling. Methods to express the light-sensitive protein in cardiac cells have focused on three different approaches. (i) The use of transgenic animals (primarily fruit-flies, zebrafish, and mice). (ii) Direct opsin transgene delivery to the targeted cardiomyocytes. This gene therapy strategy primarily utilizes viral vectors, with the most clinically relevant being adeno-associated viruses (AAVs). (iii) Indirect gene delivery using cell-grafts, which are initially genetically modified ex vivo to express the desired light-sensitive proteins. After transplantation, the opsin-carriers engineered cells (such as fibroblasts, HEK cells, and cardiomyocytes) can couple electrically with host cardiomyocytes and modulate their electrical behaviour during light-activation.8,28,30,31 In addition to the expression of the relevant opsins in the desired cells, the delivery of light to target tissues is also an important and challenging task but also offers unique opportunities. The most straightforward illumination designs focus on application of light (either relatively short flashes or continuous illumination) to the targeted tissue in a diffuse pattern (activating the entire tissue) or by using single- or multi-site point stimulation. To realize the full dynamic flexibility of optogenetic stimulations, recent studies adopted light patterning techniques from neuroscience to sculpture more complex light stimulations patterns on target tissues. An intriguing option is the use of a digital micromirror device (DMD), composed of rapidly moving micron-scale mirrors capable of directing light to desired targets within the excitation field to achieve complex activation patterns as well as high spatial resolution.31 In vitro models Much of the early work in cardiac optogenetics was conducted in in vitro cardiomyocyte cultures. These studies primarily used ChR2, which produces a robust depolarizing transmembrane current in response to blue light, leading to the development of action potentials in the genetically modified myocytes. Bruegmann et al.,7 for example, generated ChR2(H134R)-expressing mouse embryonic stem cells derived cardiomyocytes, which contracted in response to short blue-light flashes and accelerated their spontaneous beating rate following extended blue-light activation. Similarly, Abilez32 utilized lentiviral transduction to express ChR2 in human embryonic stem cells derived cardiomyocytes (hESC-CMs) and to transiently optogenetically pace the beating cell clusters. As discussed above, an alternative strategy may be the use of opsin-carrying cells that can influence the electrophysiological properties of neighbouring cardiomyocytes through electrotonic interactions. Such ‘opsin-donor’ ChR2-HEK cells were used by Jia et al.8 to generate ‘tandem cell units’ (TCUs) with adjacent cardiomyocytes, enabling to trigger light-induced electrical activity in adult canine and neonatal rat cardiomyocytes (NRCMs). Taking this approach a step forward, Nussinovitch and Gepstein30 demonstrated the ability to also suppress local cardiac electrical activity, in both NRCMs and hESC-CMs co-cultures by using engineered cell-grafts (HEK cells) transfected to express the light-sensitive hyperpolarizing proton-pump archaerhodopsin-3 (Arch3). Finally, the possibility of bi-directional light-based control of cardiomyocyte membrane potential (i.e., both depolarization and hyperpolarization) was also demonstrated using a combination of excitatory and inhibitory opsins (Figure 1).28 To this end, engineered fibroblasts (capable of electrical coupling with adjacent cardiomyocytes33), expressing both ChR2 and a variant of archaerhodopsin (ChR2-ArchT-fibroblasts),28 were seeded in NRCMs co-cultures (Figure 1A). Thanks to the non-overlapping absorption spectra of ChR2 and ArchT (blue and red ranges of the spectrum, respectively), cardiomyocytes could then be either paced (Figure 1C and D) or their electrical activity silenced (Figure 1B). Figure 1 View largeDownload slide Bi-directional optogenetic control of cardiac excitability in co-cultures containing engineered cells co-expressing ChR2 and Arch-T. (A) Neonatal rat cardiomyocytes cultures were diffusely seeded with engineered cells expressing both ChR2 (eGFP, middle panel) and Arch-T (red fluorescence, right) on top of multielectrode array (MEA) plates. (B) Electrode tracing showing complete silencing of electrical activity during red light illumination (red bar) activating ArchT pump. (C and D) Optogenetic pacing at different rates achieved by application of short pulses of blue light activating the ChR2 channel. (The figure was modified with permission from Nussinovitch et al.28). Figure 1 View largeDownload slide Bi-directional optogenetic control of cardiac excitability in co-cultures containing engineered cells co-expressing ChR2 and Arch-T. (A) Neonatal rat cardiomyocytes cultures were diffusely seeded with engineered cells expressing both ChR2 (eGFP, middle panel) and Arch-T (red fluorescence, right) on top of multielectrode array (MEA) plates. (B) Electrode tracing showing complete silencing of electrical activity during red light illumination (red bar) activating ArchT pump. (C and D) Optogenetic pacing at different rates achieved by application of short pulses of blue light activating the ChR2 channel. (The figure was modified with permission from Nussinovitch et al.28). In vivo models Transgenic animals The first reports of cardiac optogenetics involved the use of transgenic animal (zebrafish and mice) engineered to express light-sensitive proteins in all heart cells. For example, Arrenberg et al.6 generated a transgenic zebrafish model expressing both ChR2 and NpHR. Using the response of these light-sensitive zebrafish hearts to targeted illumination-based localized hyperpolarizations, the authors were able to locate pacemakers cells and thereby study the development of the conduction system at different developmental stages.6 They also demonstrated the ability to induce tachycardia, bradycardia, atrioventricular blocks, and cardiac arrest by optical means. More recently, Alex et al.34 exploited similar DMD-based optogenetic techniques to pace and interrogate conduction system development in transgenic Drosophila melanogaster. Taking this approach to the mammalian heart, Bruegmann et al.7 utilized transgenic mice with ChR2 expression and provided proof-of-concept evidence for optogenetics pacing in vivo. Using the open chest configuration, they could activate the heart and control its activation pattern by stimulating different locations. Gene therapy Since the transgenic animal strategy is obviously not relevant to human subjects, the next step in cardiac optogenetics involved the development of more clinically relevant approaches such as the use of viral vectors to deliver the opsin transgenes to the heart. To achieve this goal, two different approaches were used: a systemic approach where the viral vectors are delivered intravenously or a direct cardiac delivery approach. Systemic delivery can be simple and non-invasive, suitable when uniform expression of the transgene in the heart is desired.35 To prevent expression in non-cardiac tissues, it requires specific tropism of the vectors used to cardiac muscle and/or the use of cardiac-specific promoters. Some of the limitations of this approach are the dilution of the vector in the blood stream requiring higher viral titres to achieve adequate expression, the potential for off-target expression in non-cardiac tissues, the potential activation of an immune response, and the inability to achieve expression only at pre-determined specific myocardial areas. The use of such a systemic gene delivery approach was demonstrated by the work of Vogt et al.9 who injected AAV9 vectors carrying a ChR2 variant [ChR2(H134R) fused to mCherry] transgene into the left jugular vein of mice. This systemic delivery resulted in uniform expression of ChR2 throughout the entire heart and ultimately enabled optical ventricular pacing of the transduced hearts both ex vivo and in vivo.9 The alternative approach to the systemic delivery focuses on direct delivery of the relevant transgenes to the heart.35 A number of approaches were developed to this end in the field of cardiac gene therapy, including intracoronary delivery or direct intramyocardial injection, with the latter strategy being the only approach tested so far for optogenetics. In contrast to the systemic injection, intramyocardial injection offers the ability to reduce viral dose, to increase transduction efficiency by eliminating the need to penetrate endothelial barriers, and potentially to reduce immune response. More importantly, by targeting specific myocardial areas it allows an addition layer of control on the spatial effects induced by the optogenetic stimuli. The disadvantages of this approach include the potential for patchy and inhomogeneous expressions patterns and greater difficulties in achieving global cardiac expression (which is required for certain applications such as cardiac defibrillation) and the adverse effects that may be associated with the invasive procedure. Nussinovitch and Gepstein10 demonstrated the feasibility of the intramyocardial delivery approach by utilizing the AAV9 vector system to deliver the ChR2 transgene to a distinct intarmyocardial site at the rat's left-ventricular apex. Two weeks later the authors could optogenetically pace the hearts at different beating frequencies using flashes of blue light in both the ex vivo (isolated Langendorff-perfused rat heart model, Figure 2A) and in vivo (Figure 2B) settings. Optical mapping confirmed that the source of the pacemaker activity was the site of ChR2 transgene delivery. Figure 2 View largeDownload slide In vivo optogenetic pacing. (A) Optogenetic pacing of the isolated perfused rat heart. The ChR2 transgene was delivered to the rat heart by localized injection of the AAV vectors to the apex area. Two weeks later the heart was studied using the Langendorff isolated heart preparation. Shown are representative electrograms recorded during spontaneous rate (∼150 bpm) and during focused blue-light illumination at increasing frequencies (160–300 flashes/min). (B) Closed chest setting for optogenetic pacing by an optical-fibre, connected to an external LED system. The body-surface electrocardiogram tracings demonstrate the ability of the blue-light flashes (at 240 and 260 flashes/min) to pace the rat heart. Blue bars represent the timing of light stimuli. (The figures were modified with permission from Nussinovitch et al.10). bpm, beats per minute. Figure 2 View largeDownload slide In vivo optogenetic pacing. (A) Optogenetic pacing of the isolated perfused rat heart. The ChR2 transgene was delivered to the rat heart by localized injection of the AAV vectors to the apex area. Two weeks later the heart was studied using the Langendorff isolated heart preparation. Shown are representative electrograms recorded during spontaneous rate (∼150 bpm) and during focused blue-light illumination at increasing frequencies (160–300 flashes/min). (B) Closed chest setting for optogenetic pacing by an optical-fibre, connected to an external LED system. The body-surface electrocardiogram tracings demonstrate the ability of the blue-light flashes (at 240 and 260 flashes/min) to pace the rat heart. Blue bars represent the timing of light stimuli. (The figures were modified with permission from Nussinovitch et al.10). bpm, beats per minute. Computer simulations Another important research platform used in the cardiac optogenetics field is computational simulations. First, computer-modeling studies of the function of different opsins were performed. Williams et al.,36 for instance, developed an empirically validated computational model of the ChR2-H134R channel that could be applied specifically in different cardiomyocyte subtypes (i.e. human ventricular, atrial, and Purkinje cell models). The next stage involved the development of multiscale tissue- and organ-level models to simulate various cardiac optogenetic approaches.29,37,38 To this end, Boyle et al.,29 developed a comprehensive framework for incorporating optogenetic phenomena in detailed multiscale simulations of cardiac electrophysiology and demonstrated the potential for optogenetics pacing in human, canine, and rabbit hearts. They then compared the effects of the two different opsin delivery strategies (cell vs. direct gene delivery), demonstrated that the most efficient optical stimulation would involve targeting Purkinje cells, and quantified and discussed the limitation of light tissue penetration. Finally, they modelled the ability to deliver depolarizing or hyperpolarizing signals as a way to induce or suppress abnormal automatic foci. Similarly, Karathanos et al.,38 constructed a human ventricular computerized model based on MRI scans. Then, by simulating systemic gene delivery, they were able to examine the theoretical feasibility of terminating sustained ventricular fibrillation via light-induced excitation of opsins expressed throughout the myocardium and evaluated the ideal opsin properties and light source configurations that would maximize therapeutic efficacy. These and other works provide valuable clues regarding potential advantages and limitations of clinical optogenetic applications in the human heart. Towards clinical applications Work in the cardiac field has demonstrated the feasibility and the growing interest in using optogenetics in basic and translational research. Nevertheless, whether such a technology could become a clinical therapeutic tool is still unclear due to several conceptual and technological challenges. In the next sections, we will discuss the potential different clinical applications of cardiac optogenetics, provide the initial feasibility animal data for each application, and discuss the important conceptual and technical challenges facing clinical translation of these strategies. Optogenetics pacemakers The most straightforward clinical application of the optogenetics technology is for cardiac pacing as a potential alternative to electronic pacemakers. The motivation to seek such alternatives stems from the different limitations modern electronic pacemakers still possess such as electrochemical reactions at the electrode–tissue interface, the requirement for repeated battery replacements, risk of infections, and lead and device malfunctions. While not all of the aforementioned shortcomings could be addressed by optogenetics pacing (for example, optogenetic pacemakers will probably still carry the risk of infection as they also require hardware implantation in the form of light-emitting devices), they possess some important theoretical advantages. For example, in contrast to electronic pacers, which are limited by the number and locations of the pacing wires used, the combination of diverse opsin delivery strategies and light illumination patterns should allow pacing from any number of sites and the generation of complex pacing activation patterns. As mentioned above, the notion of optogenetic-based cardiac pacing was successfully applied in vitro, in vivo using a viral gene therapy approach,9,10 and in isolated ex vivo perfused hearts.9,10 In all the aforementioned strategies, illumination with flashes of blue light allowed to pace the studied cardiac tissues at different rates. One unique advantage of optogenetics vs. conventional electrode-based pacing is the ability to direct the pacing source to a predesigned site and to specific/selected types of cells. This spatial flexibility and discriminative power stem from the fact that only opsin expressing cells that are being illuminated can become pacemakers. This enables multilevel control: (i) during the time of transgene delivery (designing the spatial distribution of opsin expression); (ii) during illumination, pinpointing the illumination path and pattern to the desired pacing site; and (iii) through the use of cell-specific promoters enabling cardiac cell-specific subtype expression. For example, the α-MHC promoter was used for general opsin expression in cardiomyocytes but not in non-myocyte cardiac cells.39 Further specification can be achieved by using promoter-specific selective expression in ventricular-, atrial-, or nodal-like cells using the MLC2v, SLN, and SHOX2 genes enhancer/promoter elements, respectively.40 Finally, Zaglia et al.39 achieved Purkinje cells selectivity by using the Cx40 promoter. The latter approach could allow selective expression only in the His-Purkinje system, resulting in low-energy requirements for optogenetic pacing and potentially also for selective His bundle pacing to achieve more physiological ventricular activation pattern. Optogenetics-based cardiac resynchronization therapy Work in the last decade led to a number of innovative experimental stem cell and gene therapy strategies for biological pacemaking.41 One of the important limitations of these strategies, however, is the inability to pace the heart from multiple sites in a co-ordinated manner. This limitation stems from the inability of the engrafted cells or the genetically modified cells at remote cardiac sites to communicate with each other. Consequentially, these approaches, in essence, are limited to single-site pacing, analogous to single-chamber (AAI or VVI) pacers. In contrast, using light as an external mean to provide pacemaking activity can enable to co-ordinate the pacemaking function of multiple sites. This could allow, for example, sequential optogenetics pacing of the atria and ventricles in a similar manner to dual chamber (DDD) DDD pacing where both the atrium and ventricle can be sensed and paced. Another area where the ability to co-ordinate the pacing functions of multiple sites can be crucial is for the development of an optogenetic-based cardiac resynchronization therapy (CRT) approach. Cardiac resynchronization therapy using biventricular pacing has become an important treatment modality for heart failure patients with ventricular dyssynchrony as indicated by a wide QRS. This approach may be restricted; however, by limitations on the number of pacing wires and their location, potentially confining the efficacy of this procedure in some cases. In this context, the use of diffuse illumination or multi-site optogenetic pacing can be used as a potentially more effective source to the traditional electrical CRT strategy. The feasibility of using optogenetics as a non-electrical approach for CRT was initially evaluated in vitro where diffused illumination effectively synchronized activation in the NRCM co-culture model, resulting in significant shortening of the total culture activation time.28 The feasibility of this approach was also evaluated in the in vivo heart where multiple AAV-ChR2 injections (Figure 3A) and diffuse light pulses enabled synchronous multisite activation in Langendorff-perfused rat hearts.10 This can be appreciated in the optical-mapping results depicted in Figure 3, where ventricular activation maps are shown during right-ventricular electrical pacing (simulating electrical dyssynchrony, Figure 3B) and during multisite optogenetics pacing (Figure 3C). The latter configuration resulted in narrowing of the recorded QRS complex (Figure 3D) and to significant shortening of total ventricular activation time (to values similar to baseline sinus-rhythm, Figure 3E). Figure 3 View largeDownload slide Multi-site optogenetic pacing for cardiac resynchronization. Optical mapping of the isolated perfused rat heart during single- and multi-site optogenetics pacing. (A) The ChR2 transgene was delivered to the rat heart by localized injection of the AAV vectors to three sites in the left ventricular myocardium (sites identified by red circles). (B and C) Isochronal maps obtained during RV single-site electrical pacing (simulating dyssynchrony, (B) and during multi-site optogenetic pacing (C). Note that the latter strategy resulted in more synchronous (significantly shorter total activation time) when compared to electrical pacing. (D) Electrocardiogram traces. Multi-site optogenetic pacing results in shortening of QRS duration (lower panel) in comparison to single site electrical pacing (upper panel). (E) Quantification of total ventricular activation time (as determined by optical mapping) during sinus rhythm, during right ventricular point electrical stimulation (simulating dyssynchrony), and during multi-site optogenetics pacing. Note shortening of activation time with multi-site pacing to levels comparable with sinus rhythm. (The figure was modified with permission from Nussinovitch et al.10). Figure 3 View largeDownload slide Multi-site optogenetic pacing for cardiac resynchronization. Optical mapping of the isolated perfused rat heart during single- and multi-site optogenetics pacing. (A) The ChR2 transgene was delivered to the rat heart by localized injection of the AAV vectors to three sites in the left ventricular myocardium (sites identified by red circles). (B and C) Isochronal maps obtained during RV single-site electrical pacing (simulating dyssynchrony, (B) and during multi-site optogenetic pacing (C). Note that the latter strategy resulted in more synchronous (significantly shorter total activation time) when compared to electrical pacing. (D) Electrocardiogram traces. Multi-site optogenetic pacing results in shortening of QRS duration (lower panel) in comparison to single site electrical pacing (upper panel). (E) Quantification of total ventricular activation time (as determined by optical mapping) during sinus rhythm, during right ventricular point electrical stimulation (simulating dyssynchrony), and during multi-site optogenetics pacing. Note shortening of activation time with multi-site pacing to levels comparable with sinus rhythm. (The figure was modified with permission from Nussinovitch et al.10). Optogenetic treatment of tachyarrhythmias In addition to optogenetic-based cardiac pacing and resynchronization, the same technologies could potentially also be used to treat tachyarrhythmias (either re-entrant or focal) through hyperpolarization-based suppression of excitability or via alternative depolarization-based mechanisms. Hyperpolarization-based suppression of excitability This approach is commonly used to suppress neuronal excitability and involves the use of light-sensitive chloride (HR) or proton (archaerhodopsin) pumps to induce membrane hyperpolarization. The feasibility of such strategies for suppression of cardiomyocyte excitability was recently described in feasibility studies both in vitro (in co-culture studies using Arch or ArchT, Figure 1B)28,30 and in the zebrafish model using HR6 as described above. The results of these studies suggest a number of different clinical scenarios in which optogenetics suppression of excitability could become a useful tool for the treatment of tachyarrhythmias. The first could be the use of light-induced hyperpolarization for induction of localized conduction blocks, as a functional ‘molecular ablation’ approach. The second application could be the complete electrical isolation of cardiac tissue that could prove useful, for example, for pulmonary vein isolation for the treatment of atrial fibrillation. The third potential application could be the suppression of the activity of ectopic foci. Finally, the ubiquitous expression of hyperpolarizing light-sensitive protein throughout the heart may allow generalized painless hyperpolarization-based defibrillation. Nevertheless, in contrast to the robust effects of using ChR2 to augment cardiac excitability and of light-induced Arch/Halo based hyperpolarization to silence neuronal activity, the suppressive effects induced by Arch/Halo on cardiomyocytes were rather mild and may not be sufficient to achieve the desired antiarrhythmic effects described above. As mentioned above, this may stem from the fact that in contrast to light-sensitive ion channels such as ChR2, Arch/Halo are pumps, and therefore produce significantly lower photocurrents. Moreover, inducing resting membrane hyperpolarization to a certain extent in cardiomyocytes may paradoxically augment excitability, a phenomenon termed ‘anode-break excitation’. This may explain the lack of reports describing the use of these pumps in small/large animal models. The design of a new family of engineered hyperpolarizing opsins,20–22 which are channels rather than pumps and have already been shown to suppress activity in cardiomyocytes in vitro,22 may potentially tackle the aforementioned limitations and provide exciting tools for the future. Depolarization-based anti-arrhythmic mechanisms Light-induced depolarization may also be utilized to treat or prevent tachyarrhythmias through a number of mechanisms. Since slow and abnormal conduction, such as occurs at the infarct border zone, may play a critical role in the pathogenesis of re-entrant arrhythmias, restoring rapid conduction may prevent the development of such arrhythmias. Optogenetics approaches could theoretically be used to homogenize and synchronize conduction in such areas through simultaneous light-induced activation of all relevant cardiomyocytes. The feasibility of this approach was recently demonstrated in an in vitro co-culture model of slow conduction and conduction blocks, where diffuse illumination was used to simultaneously activate the entire syncytium.28 Whether such an approach can be used in more thicker three-dimensional cardiac muscle models and eventually in the infarct border-zone, given the issue of limited blue-light tissue penetration, has to be determined in future studies. The second approach focuses on applying diffused and continuous light-induced depolarization to achieve ‘optogenetic defibrillation’. This approach could potentially provide a pain-free alternative to the electric shocks used by existing devices, while also consuming significantly less energy. This may be specifically important when dealing with a non-fatal disorder such as atrial fibrillation. Recent studies provided proof-of-concept evidence for the validity of such optogenetics-based defibrillation strategies. Bingen et al.42 showed using optical mapping and MEA recording that fibrillatory activation patterns induced in rat atrial myocyte monolayers expressing calcium-translocating ChR2 (CatCh) could be reliably terminated using long (500 ms) blue-light pulses. The mechanism underlying arrhythmias termination was thought to involve reduction of overall excitability, increase in functional core size, and drift of phase singularities due to light-induced depolarization. More recent computer modelling studies, using a MRI-based patient-specific heart model of ventricular fibrillation, provided proof-of-concept simulations for the ability to achieve optogenetics-based defibrillation via long-pulses of light-induced excitations of ChR2 expressed throughout the myocardium.38 An increased probability for success was observed when using opsin variants with better tissue penetration, such as with red-shifted spectral sensitivity (ChR2-RED). More recently, different research groups demonstrated the feasibility of the aforementioned approach also in the mammalian heart. Bruegmann et al.11 successfully terminated ventricular arrhythmias in transgenic mouse hearts using different illumination protocols. They also showed the efficacy of optogenetic defibrillation in clinically relevant settings, using a post-myocardial infarction model and using an AAV9 gene delivery system.11 Similarly, Nyns et al.13 used AAV delivery to express red-activated ChR (ReaChR) in mouse hearts. The authors were then able to terminate ventricular arrhythmias induced in the ex vivo isolated heart configuration with diffused 470 nm illumination. Crocini et al.12 examined the efficacy of different illumination patterns to terminate ventricular arrhythmias in transgenic mouse cannulated hearts following ischaemia. They demonstrated how specific illumination patterns could be as potent as diffused illumination. Finally, Watanabe et al.43 developed an anatomical re-entry ventricular tissue slice model and demonstrated that the circuit can be manipulated by optogenetic induction of a local and reversible conduction block in the re-entrant pathway, allowing effective termination of the arrhythmia. Future clinical challenges Despite the exciting results described above, cardiac optogenetics is still at its infancy. For example, cell-delivery strategies for optogenetic applications have been restricted to in vitro culture models with no experimental proofs yet using whole hearts or in the in vivo setting. This may stem from the challenges associated with the field of cardiac cell therapy in general, as well for the specific requirements for optogenetic applications such as for the development of tight electrical coupling between host and grafted cells. As discussed above, the status of the optogenetics field is far more advanced for the gene therapy strategies with proof-of-concept evidence existing in several animal models. Nevertheless, in contrast to similar applications in the brain, optogenetics strategies in the heart were yet to be described in chronic, freely moving, animals. Finally, several obstacles need to be overcome before any clinical translation can be expected. One significant obstacle is associated with the fact that the technical, regulatory, and conceptual challenges that have to be addressed are not limited only to one area. Thus, issues related both to the development of a clinical cell/gene therapy procedure requiring long-lasting, robust and safe transgene expression as well as the need for development of a device capable of achieving appropriate light access to the area of interest should be addressed. To address the latter problem, two possible solutions were suggested: the use fibre-optics for light delivery to the heart44 or implantation of miniaturized devices, which include light sources and photodiodes.45 The former strategy might result in pacemaker/implantable cardioverter device-like tool, which utilize fibre-optics rather than electrodes. Since blood may absorb light significantly, the preferred location of such optical fibres may be on the epicardium. Fiberoptics-based systems were recently reviewed by Klimas and Entcheva.44 The latter strategy aims to implant the light sources directly in the targeted site. Kim et al.46 developed small-scale devices that can serve as a wireless solution for optogenetics in the challenging mediastinal environment. Xu et al.45 developed three-dimensional elastic membranes, individually designed and printed for an optimized fit. These membranes can completely envelop the heart and the micro-LEDs embedded within them can provide a proper solution for precise light stimulation. More recently, the same group reported the development of implantable wireless optogenetic devices that combines tiny soft neural interfaces with fully implantable stretchable wireless radio power and control systems.47,48 These small devices were demonstrated to be able to optogenetically modulate the spinal cord and peripheral nervous system following implantation. Importantly, since the blue light used to activate ChR2 is characterized by relatively poor tissue penetration, it may not be sufficient to target the transduced cells in the human heart. Improved versions of opsins (with superior photosensitivity and higher photocurrents) and responsiveness to light wavelengths with better tissue-penetrance (for example red light shifted opsins such as the Chrimson49 and the ReaChR50) may be required for effective optogenetic applications in patients. An additional question relates to the potential immunogenicity of optogenetics procedures. Relevant issue are the potential immune response to the viral vectors used for delivery of the transgenes to the heart (primarily AAVs) and the foreign nature of the proteins used that are of microbial origin. As reviewed by Bera and Sen,51 evidence from pre-clinical and clinical studies suggests that the use of AAV vectors in the heart is safe and is associated with persistent transgene expression with minimal pathogenicity and cytotoxicity. Yet, some patients possess pre-existing antibodies against certain AAV serotypes (due to previous infections) that can neutralize the used virions. The prevalence of such antibodies could be as high as 70% in certain populations for specific AAV serotypes and can be even detected at an early age.52 Suggested future solutions for this problem in the field of cardiac gene therapy range from using higher viral titres or empty decoy caspids, through clearing the host’s immunoglobulins by plasmapheresis or applying mild immunosuppression, to switching AAV serotype or engineering less immunogenic AAV variants.52 Another related question is whether the light-sensitive protein themselves can elicit an immune response, since the opsins used (type I) are mainly of microbial origins. Despite this fact, evidence for immunogenicity of these opsins is rare, for whatever reason. Most data on this issue comes from studies using ChR2 for neuroscience applications and primarily in studies in the retina. Sugano et al.,53 for example, injected AAV serotype 2 (AAV2) carrying the ChR2 transgene into the vitreous body in the rat eye and could not detect any indication for immune cell infiltration. Similarly, Doroudchi et al.,54 delivered the ChR2 gene using AAV8 serotype to the mouse retina in order to restore visual function. Immune response was evaluated both locally and systemically by re-injecting the vector to the mouse ear. No signs of inflammation, immune cell infiltration, or humoral responses were noted. Despite these promising results, it should be noted that these studies were primarily performed in immune-privileged organs and did not challenge the human immune system. While immune rejection issues were not noted in the cardiac optogenetic studies to date, further long-term studies should specifically focus on this issue. Conclusion Optogenetics technology is deeply rooted in neuroscience as a powerful research tool with several potential clinical applications. Initial efforts have already introduced similar optogenetic tools to the cardiac arena and demonstrated the ability for bidirectional control (augmenting or suppressing) of cardiac excitability. The combined use of optical methods to perturb and to observe cardiac electrophysiology at the cellular, tissue, and organ levels offers new tools for precise feedback control of cardiac electrical activity at a resolution, not available previously with pharmacological and electrical stimulation, and may thus have important implications for basic and translational cardiac electrophysiology. Furthermore, the same tools may allow the development of novel clinical therapeutic strategies for various cardiac arrhythmias, including for optogenetic-based cardiac pacing, resynchronization, and defibrillation strategies. 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TI - Cardiac optogenetics: the next frontier
JO - Europace
DO - 10.1093/europace/eux371
DA - 2018-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/cardiac-optogenetics-the-next-frontier-L24MR03zLK
SP - 1910
VL - 20
IS - 12
DP - DeepDyve
ER -