TY - JOUR
AU - Qiang, Liu, Zi
AB - Abstract Optogenetics is a cell-type specific and high spatial–temporal resolution method that combines genetic encoding of light-sensitive proteins and optical manipulation techniques. Optogenetics technology provides a novel approach for research on cardiac arrhythmia treatment, including pacing, recovering the conduction system, and achieving cardiac resynchronization with precise and low-energy optical control. Photosensitive proteins, which usually act as ion channels, pumps, or receptors, are delivered to target cells, where they respond to light pulses of specific wavelengths, evoke transient flows of transmembrane ion currents, and induce signal transmission. With the development of gene technology, the in vivo efficiency of optogenetics in cardiology has been trialed, and in vitro experiments have been performed to test its potential in cardiac electrophysiology. Challenges for applying optogenetics in large animals and humans include the effectiveness, safety, and long-term expression of photosensitive proteins, unscattered and unattenuated exogenous light stimulation, and the need for implantable miniature light stimulators. Photosensitive proteins, genetic engineering technology, and light equipment are essential for experiments in cardiac optogenetics. Optogenetics may provide an alternative method for evaluating the mechanism of cardiac arrhythmias, testing hypotheses, and treating cardiovascular diseases. Optogenetics, Photosensitive protein, Cardiac electrophysiology, Arrhythmia Introduction Optogenetics is a novel strategy for controlling biological behaviour with a combination of genetic engineering and light application in genetically modified cells expressing photosensitive proteins. The first description of archaeal photoreceptors date back to the early 1970s,1 and the term of optogenetics was coined in 2006.2 Optogenetics was recognized as the Method of the Year for 2010 by Nature Methods. During the past decades, optogenetics has been used widely in the neurosciences to dissect neural circuits and probe the connectivity of the brain; the therapeutic potential of optogenetics for epilepsy, depression, and Parkinson’s disease has been investigated3–7 in animal experiments. Optogenetics has recently been introduced to animal experiments in other fields; for instance, it has been used to find new methods to relieve chronic pain,8 to control tumourigenesis,9,10 and to restore movement to the paralysed.11 Recently, this revolutionary technique was introduced to a clinical trial for retinitis pigmentosa treatment.12 The precise optical tools have attracted the attention of cardiovascular researchers, including our team, to control heart rhythm, mimic cardiac arrhythmias, test cardiology hypotheses, and determine whether optogenetics could be applied in cardiovascular therapy. Optogenetics may provide new methods to treat the cardiac conduction system, restore pacemaking ability, correct conduction slowing of the heart, achieve cardiac resynchronization, mimic cardiac arrhythmias, and terminate cardiac arrhythmias in a spatiotemporal, cell-selective, and low-energy manner. The light-activated ion pump halorhodopsin from Natronomonas (NpHR) and the light-sensitive ion channel channelrhodopsin-2 H134R (ChR2) have been expressed in zebrafish to optically control the heart rate, alter the activation sequence, and induce disease-like states such as tachycardia, bradycardia, atrioventricular blocks, and cardiac arrest.13 In transgenic mice, ChR2 was expressed to control its heart muscles in vivo.14 Cardiomyocytes expressing photosensitive proteins can be stimulated to exert diverse biological functions such as excitatory or inhibitory transmembrane currents, changing intracellular concentrations, and controlling receptors by illuminating with specific wavelengths. Compared with conventional treatments such as drugs and electrical therapies, optogenetics may provide an alternative approach for basic cardiovascular research and treatment of cardiovascular disease without the limitations associated with electrical stimulation treatments,15–17 such as high-voltage shocks, low-specificity non-synchronous depolarization, and myocardial tissue damage. It is, therefore, necessary to discover novel photosensitive proteins or reform the existing proteins to perform optimal functions such as evoking larger or more rapid photocurrents, responding to light stimuli with deeper tissue penetrability, and selectively allowing ions to pass through. Ongoing developments in transgenic technology (viral preparation and transgenic animals), precise illumination devices, and versatile recording instrumentation are beneficial to the successful application of cardiac optogenetics in experimental or translational contexts. The purpose of this article is to introduce the key procedures of optogenetics, describe current applications in cardiology, and evaluate the prospects of optogenetics in cardiac research. Photosensitive proteins in optogenetics Photosensitive proteins in optogenetics can be classified as genetically encoded fluorescent reporters (sensors), light-activated tools (actuators), and others (light-activated enzymes, KillerRed).18 Optical sensors such as voltage-sensitive fluorescent protein (VSFP)19,20 green calcium indicator protein (GCaMP),21 and red calcium indicator protein (R-GECO)22 offer cell-specific readouts or in vivo monitoring of electrical activity. Optical actuators, including voltage actuators and biochemical actuators such as microbial rhodopsins, ChRs, NpHR, archaerhodopsin-T (ArchT), and G-protein-coupled rhodopsins (melanopsin) usually act as ion channels, pumps, or signal receptors to produce physiological reactions in mammalian cells and tissues in response to optical signals. The microbial rhodopsins, ChRs, and NpHR are common tools in optogenetics research that generate excitatory or inhibitory transmembrane currents to control the activity of excitable cells (Figure 1). Different photosensitive proteins can produce different electrophysiological effects, enabling researchers to bidirectionally and reversibly manipulate biological functions, including membrane voltage and action potentials, by expressing one or more photosensitive proteins in target cells. Figure 1 View largeDownload slide Common light-sensitive proteins in optogenetics research. (A) ChR2 allows inward photocurrent of non-selective cations, which can evoke cell depolarization under 470 nm wavelength light. (B) NpHR, an anion pump (chloride), can produce membrane hyperpolarization and electrophysiological inhibition under 580 nm wavelength light. Figure 1 View largeDownload slide Common light-sensitive proteins in optogenetics research. (A) ChR2 allows inward photocurrent of non-selective cations, which can evoke cell depolarization under 470 nm wavelength light. (B) NpHR, an anion pump (chloride), can produce membrane hyperpolarization and electrophysiological inhibition under 580 nm wavelength light. Improved variants of ChRs with better photosensitivity, higher current production, and tools with red-shifted absorption spectrum that respond to 590–630-nm light with better tissue penetration are important to apply optogenetics research into large animals or humans. In cardiovascular research, photosensitive proteins such as ChRs and its variants, CatCh, ReaChR, and ChRimson, are more common than Arch-T, NpHR, anion channelrhodopsins (ACRs), KillerRed, and melanopsin (Table 1).13,20,22,–35 ChRs are light-gated, unselective cation channels that mediate inward currents at negative holding potentials. In mammalian cell research, ChR1 with slow light inactivation and a red-shifted visible absorption is less effective than ChR2.36,37 The ChR2 from Chlamydomonas reinhardtii has 737 amino acids, including 315 highly conserved ones that are sufficient for channel activity, and 7 transmembrane helices called opsins that are covalently linked to retinal, which form a light-switchable channel for cations. Under the optimum wavelength of 470 nm, ChR2 changes conformation and produces an inward transient flow of transmembrane current (protons, sodium, and calcium ions) to induce cell depolarization and action potentials that are timed within milliseconds after the light pulse. Changing the structure of proteins38 may improve the absorption properties of the chromophore, which utilizes the illumination most effectively under optimum light intensity. ChR2-H134R,39,40 a standard tool for optogenetics in cardiac research, may cause larger stationary photocurrents than ChR2(wild-type), enabling precise optogenetic pacing of cardiomyocytes, even at low temperatures and low calcium levels, without causing physical damage. The light activation is lost irregularly when the frequency of the light stimulation is more than 40 Hz. Recently, with the development of molecular engineering, more variants of ChRs have been discovered or synthesized, such as CatCh41 with increased light sensitivity (70-fold more light sensitive than wild-type ChR2) and calcium ion permeability, ChETATC22 with large photocurrents and rapid inactivation, and ReaChR and ChRimson25,38,42,43 with increased red-shifted absorption spectrum that enables the use of red light that can penetrate deeper into dense cardiac tissues. However, it is necessary to improve photosensitive proteins with increased absorption cross-section and quantum efficiency to maximize the use of light. Table 1 Photosensitive proteins in cardiovascular research Photosensitive proteins Spectrum λ (nm) Properties Reference ChR2-H134R 470 Larger stationary photocurrents 13, 23, 24 ReaChR 590–630 Opsins with red-shifted absorption spectrum 25, 26 Catch 470 Accelerated response time and enhanced Ca2+ permeability 27, 28 ChRimson 470 Opsins with red-shifted absorption spectrum 25 ChETATC 405 Large photocurrents and rapid inactivation 22 NpHR 525–650 An anion pump (Cl−) to induce hyperpolarization 13, 29 Arch-T 624 A proton pump, to induce lager hyperpolarizing photocurrents than NpHR 30–32 ACRs 470 Anion channelrhodopsins to produce lager inhibitory currents than Archs 31 KillerRed 540–600 A photoinducer of reactive oxygen species 33, 34 Melanopsin 470 A light-sensitive Gq-coupled receptor 35 VSFP 508 Voltage-sensitive fluorescent protein 20 R-GECO 568 Red-shifted calcium indicator 22 Photosensitive proteins Spectrum λ (nm) Properties Reference ChR2-H134R 470 Larger stationary photocurrents 13, 23, 24 ReaChR 590–630 Opsins with red-shifted absorption spectrum 25, 26 Catch 470 Accelerated response time and enhanced Ca2+ permeability 27, 28 ChRimson 470 Opsins with red-shifted absorption spectrum 25 ChETATC 405 Large photocurrents and rapid inactivation 22 NpHR 525–650 An anion pump (Cl−) to induce hyperpolarization 13, 29 Arch-T 624 A proton pump, to induce lager hyperpolarizing photocurrents than NpHR 30–32 ACRs 470 Anion channelrhodopsins to produce lager inhibitory currents than Archs 31 KillerRed 540–600 A photoinducer of reactive oxygen species 33, 34 Melanopsin 470 A light-sensitive Gq-coupled receptor 35 VSFP 508 Voltage-sensitive fluorescent protein 20 R-GECO 568 Red-shifted calcium indicator 22 Table 1 Photosensitive proteins in cardiovascular research Photosensitive proteins Spectrum λ (nm) Properties Reference ChR2-H134R 470 Larger stationary photocurrents 13, 23, 24 ReaChR 590–630 Opsins with red-shifted absorption spectrum 25, 26 Catch 470 Accelerated response time and enhanced Ca2+ permeability 27, 28 ChRimson 470 Opsins with red-shifted absorption spectrum 25 ChETATC 405 Large photocurrents and rapid inactivation 22 NpHR 525–650 An anion pump (Cl−) to induce hyperpolarization 13, 29 Arch-T 624 A proton pump, to induce lager hyperpolarizing photocurrents than NpHR 30–32 ACRs 470 Anion channelrhodopsins to produce lager inhibitory currents than Archs 31 KillerRed 540–600 A photoinducer of reactive oxygen species 33, 34 Melanopsin 470 A light-sensitive Gq-coupled receptor 35 VSFP 508 Voltage-sensitive fluorescent protein 20 R-GECO 568 Red-shifted calcium indicator 22 Photosensitive proteins Spectrum λ (nm) Properties Reference ChR2-H134R 470 Larger stationary photocurrents 13, 23, 24 ReaChR 590–630 Opsins with red-shifted absorption spectrum 25, 26 Catch 470 Accelerated response time and enhanced Ca2+ permeability 27, 28 ChRimson 470 Opsins with red-shifted absorption spectrum 25 ChETATC 405 Large photocurrents and rapid inactivation 22 NpHR 525–650 An anion pump (Cl−) to induce hyperpolarization 13, 29 Arch-T 624 A proton pump, to induce lager hyperpolarizing photocurrents than NpHR 30–32 ACRs 470 Anion channelrhodopsins to produce lager inhibitory currents than Archs 31 KillerRed 540–600 A photoinducer of reactive oxygen species 33, 34 Melanopsin 470 A light-sensitive Gq-coupled receptor 35 VSFP 508 Voltage-sensitive fluorescent protein 20 R-GECO 568 Red-shifted calcium indicator 22 Another excitatory photosensitive protein, melanopsin (Opn4),44,45 is a light-activated Gq-coupled receptor that exists on the ganglion cells of the retina and is involved in the regulation of circadian rhythms and pupillary light reflexes. Opn4 was recently used in a new method for investigating the spatiotemporal effects of Gq stimulation on the pacemaking of cardiomyocytes.35 Cardiomyocytes expressing Opn4 can be controlled by a light pulse of 470 nm to increase the heart rate and generate local pacemaker activity with Gq activation. Inhibitory proteins, NpHR, Arch, and ACRs, can be used to suppress cardiac electrical activity and shorten action potential duration by switching on illumination during repolarization.13,30,31 This could be a potential therapeutic strategy for tachycardia and long-QT syndrome. The NpHR,46 an anion pump (chloride) isolated from the Natronomonas pharaonic, can be activated by yellow light (bandwidth, 573–613 nm), causing membrane hyperpolarization and electrophysiological inhibition. Arch-T,30,31,46,47 a yellow–green photosensitive proton pump, can generate larger hyperpolarizing photocurrents than NpHR in response to light; they can also be used to suppress spontaneous or electrode-triggered activity in continuous monochromatic illumination (624 nm). Preliminary experimental evidence47 suggested that Arch-T might be a safe and effective optical tool for applications in neuroscience, as the changes in pH caused by Arch illumination are minimized. ACRs31 from cryptophyte algae conducts many ions per photocycle, in contrast to only one ion being transported by inhibition of pumps, which explains why ACRs require less than one-thousandth of the light that the proton pump archaerhodopsin-3 (Arch) requires to produce inhibitory currents in cultured neonatal rat ventricular cardiomyocytes. Inhibitory proteins are rarely used in cardiac applications, possibly due to the risk of inducing cardiac arrests and ventricular arrhythmias. More experiments are needed to explore the electrophysiological function of inhibitory proteins in the heart in vivo. Expressing inhibitory and excitatory proteins in the same cells enables bilateral and precise control of cardiac rhythms with illumination using different wavelengths of light, which may provide a flexible method to mimic cardiac arrhythmias. The membrane photosensitive protein, KillerRed,33,48 usually acts as a photoinducer of reactive oxygen species to produce cytotoxicity and trigger cell degeneration and death. Disruption of neuronal function and ablation of many neuron types such as sensory neurons, interneurons, and motor neurons have been demonstrated in Caenorhabditis elegans.49 KillerRed may be an unconventional way to implement cardiac ablation in ectopic excited foci, cardiac nerves, or hypertrophic myocardium; however, it may be a pioneering method for treating cardiac arrhythmias and hypertrophic cardiomyopathy. Recently, KillerRed was used to modulate the heart rate, change cardiac contractility, and generate a model of cardiac deficiency in zebrafish with an intense green light (540–600 nm).32,33 In summary, optimizing the structure of photosensitive proteins with different properties43 (photocycle duration, opening duration, or action spectum) could contribute to effective manipulation of the electrical activity of cells, tissues, and entire organs. Optogenetics combines the sensors and actuators of photosensitive proteins to enable precise cell-to-cell research of the mechanism behind cardiac arrhythmias and macroscopic investigation of cell electrophysiology in the whole heart. Optogenetics in cardiac research In the past decade, optogenetics revolutionized neuroscience by enabling spatiotemporal control of neural excitation to study brain function in normal and pathological conditions. Optogenetics was applied in a clinical trial to treat retinitis pigmentosa,12 which was a milestone for the clinical application of optogenetics. Initial reports on cardiac optogenetics in 2010 showed that optogenetics can be applied to cardiovascular research to control the heart rhythm, alter the heart contractility, and terminate arrhythmias.13 In the following sections, we review the development of optogenetics in cardiology, from cardiomyocytes (in vitro) to the whole heart (in vivo; Figure 2). We also discuss the limitations of the clinical application of optogenetics for cardiovascular disease. Figure 2 View largeDownload slide A schematic diagram of cardiac optogenetics in vivo with ChR2. Viral vehicle (AAV9), packaged with a single-stranded DNA containing light-sensitive protein gene, fluorescent protein gene, and promoter gene (A), was systemically injected into mice/rat or injected into the myocardium (B). The heart expressed light-sensitive proteins (ChR2) was stimulated by light-emmitting diode/laser light (C), which can produce inward photocurrents and evoke action potential (D). Figure 2 View largeDownload slide A schematic diagram of cardiac optogenetics in vivo with ChR2. Viral vehicle (AAV9), packaged with a single-stranded DNA containing light-sensitive protein gene, fluorescent protein gene, and promoter gene (A), was systemically injected into mice/rat or injected into the myocardium (B). The heart expressed light-sensitive proteins (ChR2) was stimulated by light-emmitting diode/laser light (C), which can produce inward photocurrents and evoke action potential (D). In vitro The potential for the in vitro application of excitatory and inhibitory photosensitive proteins in cardiomyocytes was shown in cells using the patch clamp technique, and manipulation of the whole heart was evaluated in the Langendorff perfused heart with optical stimulation from a laser or light-emitting diode. Computer technology provided an alternate platform to overcome the limitations of experimental optogenetics in cardiomyocytes or whole hearts, enabling researchers to simulate and predict the electrophysiology of hearts expressing photosensitive proteins in multiscale computational models. In vitro research provides a foundation for the development of experimental in vivo optogenetics for clinical applications. Different photoeffects in different cell types Optogenetics has become a topic of keen interest in cardiac arrhythmia research. The expression of ChR2 and NpHR was shown in transgenic zebrafish myocardium, and simulated models were developed for tachycardia, bradycardia, atrioventricular block, and cardiac arrest.13 A non-viral method was developed with a tandem cell unit by electrically coupling cardiomyocytes with non-excitable HEK293 cells to express ChR2.50 In another study, heterocellular coupling was used to evaluate cardiac electrical activity controlled by optogenetics, and ChR2/ArchT-fibroblasts and Arch3-HEK293 cells were cocultured with neonatal rat cardiomyocytes and human embryonic stem cells.30,32 Other researchers infected neonatal rat ventricular myocytes with an adenovirus vector for expressing ChR2-H134R in fusion with the eYFP fluorescence protein under the control of common promoter, cytomegalovirus; they observed an optimal multiplicity of infection (25) based on maximum expression and minimum cell death.23 Structural–functional relations were tested for gene and cell therapy in cardiac tissue, and the myocardium excitability was restored by optogenetics.51 In other studies, atrial cardiomyocytes were transduced with lentiviral vectors to express a light-activated calcium ion-translocating ChR, Catch, which showed high potential for treating atrial fibrillation by inducing depolarizing currents.27,28 The pathological responses of inflammatory activation in cardiac fibroblasts after myocardial injury and cardiac remodelling could affect the interactions between injured and normal cardiomyocytes, and the abnormal connection might reduce or block conduction, causing abnormal automaticity.52 Cardiac fibroblasts infected with an adenovirus vector for expressing ChR2, when fused with common promoter CAG, showed low expression of ChR2 and were tolerant to high viral doses.23,53 More specific promoters such as hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), Connexin40 (Cx40), Contactin-2 (CNTN2), α-myosin heavy chain (α-MHC), and cardiac troponin T (cTnT) may be fused selectively with photosensitive proteins in specific cardiac cell types—cardiomyocytes (α-MHC, cTnT), cardiac fibroblasts (HCN4), conduction system cells (Contactin-2), and gap junction cells (Cx40)—to specifically control or image the heart. This approach could potentially decrease or terminate malignant arrhythmias caused by the presence of remodelled tissues in the vicinity of an infarct after a myocardial infarction. Cardiomyocyte transplants, which are being developed as a treatment for end-stage heart disease, may help maintain the electrical stability of the injured heart, but has high technical requirements.54 The use of optogenetics began in gene therapy research, in which murine stem cells differentiated into cardiomyocytes.14,55,56 ChR2 and NpHR1.0 were introduced into human induced-pluripotent stem cell cardiomyocytes (hiPSC-CM) via a lentivirus vector, and electrophysiological activation was controlled by optogenetics.57 The use of optogenetics and iPSC-CMs may also provide an option for initiating patient-specific drug testing.58 Different cell types have diverse optical excitation thresholds.14,23,58 Purkinje cells need less illumination intensity for excitation than ventricular myocytes, due to their unique features such as slow spontaneous diastolic depolarization, high current densities in many major ion channels, distinctive ion channel kinetic properties, and low source–sink mismatch due to network geometry.59 Cellular research shows that photosensitive proteins can be expressed in cardiac cells and controlled by light pulses of specific wavelengths. This establishes the theoretical foundation to convert the study of optogenetics from cardiomyocytes (in vitro) into the whole heart (in vivo). Multiscale computational models Multiscale computational models have been used in cardiac electrophysiology research, including the prediction of electrophysiological mechanisms and the exploration of therapeutic applications.55,60 In a study of the efficacy of optogenetics in prolonging cardiac action potentials in a model of short-QT syndrome in human atria, it was concluded that61 (i) optogenetics accurately modulated and restored the action potential shape and duration in atrial myocytes, (ii) the main limitation for the use of optogenetics was poor penetration in cardiac tissues due to light attenuation and scattering, and (iii) light stimulation may be important for the development of optogenetics-based tools for treating cardiac arrhythmias. In a computational model of the fibrillating human ventricles, arrhythmia was effectively terminated25 by either blue light illumination when the heart was modified to express ChR2+ (a theoretical ChR2-H134R variant with augmented light sensitivity comparable to that shown for CatCh27,28) or red light illumination when ventricular myocytes expressed ChR2-RED (another theoretical ChR2-H134R variant with red-shifted spectral sensitivity comparable to ReaChR25,26 and ChRimson25). The authors’ rationale for using theoretical opsin variants was that biophysically detailed mathematical representations of current in depolarizing opsins other than ChR2-H134R do not yet exist. Ventricular myocytes require higher irradiance than the conduction system and atrial myocytes; the conduction system and atrial myocytes were more sensitive to optical action potential modulation due to their lower depolarized plateau. The oscillatory frequency of Purkinje cells expressing ChR2-H134R can be controlled accurately by constant light pulses at extremely low irradiance, using a computational model; however, constant electrical stimulation is not feasible due to electrochemical limitations.17,62 Organ level with Langendorff perfused heart Optogenetics pacing in isolated whole heart takes optogenetics a step closer to application in in vivo cardiac research. Stimulation of whole rat heart with blue light at three different light intensities and three different flash durations showed a set of minimal pairs of amplitude and duration (>90% capture efficiency), 5 ms flashes with 4.06 mW/mm2, and 10 ms flashes with 1.95 mW/mm2.63 The pacing threshold in different anatomical locations (atrium, ventricle, and ventricular septum) of the Langendorff perfused mouse hearts was compared. The results showed that the pacing thresholds were not significantly different between the mice expressing equivalent ChR2; the pacing threshold was also highly related to the amount of ChR2 expressed.24 Therefore, it is important to ensure sufficient expression of ChR2 in cardiomyocytes using an optimized gene delivery vehicle. Recent research has shown that ventricular arrhythmias (ventricular tachycardia and fibrillation) can be terminated by continually or regularly illuminating a ChR2-expressed heart to induce transmural depolarization and blocking voltage-dependent sodium ion channels without strong electrical shocks.25,64,65 A comparison of four patterns of optogenetic stimulation (single-point, single-barrier, triple-barrier, and whole left ventricle) with different optical patterns revealed that applying discrete stimulation patterns could effectively reduce the energy required to defibrillate the heart.65 Optogenetics may provide a precise method to study the initiation of cardiac arrhythmias by controlling cardiac macroscopic propagating waves recorded by a macroscope (Olympus MVX10) and camera (Andor Neo sCMOS), and aberrant re-entrant waves may cause deadly tachycardias and fibrillation.66 Although optogenetics has been applied successfully for research on cardiac pacing and defibrillation with excitatory photosensitive proteins, inhibitory photosensitive proteins that can induce cardiac arrest have been used infrequently for mammalian heart studies. In vivo With the development of optogenetics in cardiology, some methods have been used effectively in in vivo research, including viral delivery of photosensitive proteins fused with fluorescent proteins into the myocardium (direct injection or systemic intravenous injection) and transgenic animals (especially Cre recombinase transgenic mice). The method of the research in vitro, such as transgene vehicle, recording apparatus, and illuminated parameters, is difficult to transform to the research in vivo or in patients, and more studies are needed to overcome this challenge. Optogenetics pacing of the heart has been demonstrated in rats and mice expressing ChR2 in vivo. In one study, cardiac rhythm was controlled in the heart expressing ChR2-GFP with different frequencies of light pulse flashes by opening the chest and directly injecting the adeno-associated virus 9 (AAV9) into the myocardium.63 mCherry fluorescence can be detected in the whole heart 4–10 weeks after injecting it into the external jugular vein (2 × 1011 gc AAV9-CAG-hChR2 [H134R]-mCherry).24 The minimal transfection percentage of cardiomyocytes for optogenetic pacing is 30–40% of cells expressing ChR2, and the successful optogenetic pacing rate was similarly high after short (4–6 weeks) and long (6–10 weeks) incubation periods. An in vivo experiment in optogenetics showed that efficient systemic injection of AAV and sufficient expression of ChR2 may ensure the successful control of cardiomyocytes, and direct injection of virus into the myocardium may cause partial expression of ChR2 at the injection site and not at remote myocardial areas. The longest ChR2 expression period in optogenetic research is 1 year, when the photocurrent produced by illumination was sufficient to control the cardiac rhythm after systemic AAV9 injection.64 Extrasystole requires the simultaneous depolarization of at least 1300–1800 working cardiomyocytes or 90–160 Purkinje fibres in Cre recombinase transgenic mice with the cardiomyocyte-specific promoter, α-MyHC, or the Purkinje fibre-specific promoter, Cx40.67 The in vivo application of optogenetics in cardiac research suggested that it might be a novel method for treating cardiac arrhythmias. In one study, ectopic endodermal cells were induced and nodal signalling was resolved at different stages of embryonic development by re-engineered blue light-activated EL222 system TAEL, a new optogenetics expression system used in zebrafish that has a large dynamic range and rapid activation kinetics and may enable optical control of gene expression.68 Other researchers have developed Pnmt-Cre mice to demonstrate the functional dissection of cardiomyocyte subpopulations in Pnmt-expressing neuroendocrine cells and explore the physiological roles of Pnmt-expressing neuroendocrine cells in normal heart functions and their potential applications in selective cardiac repair and regeneration strategies.69 Gene transfer to non-transgenic animals, minimally invasive and high-efficiency light sources, and suitable readout methods are essential for in vivo research on cardiac optogenetics.70 Before optogenetics can be clinically applied, safety issues must be resolved, including the immunity induced by the virus, the duration of photosensitive protein expression, the damage caused by light, and the optimal implant device. Modulation of cardiac activity by manipulating the sympathetic and parasympathetic nervous systems The autonomic nervous system (ANS) plays a major role in the pathgenesis of cardiac arrhythmias and sudden death. Distinguishing the interplay between sympathetic and parasympathetic systems will lead to a better understanding of different types of cardiac arrhythmias. Previous studies have shown that manipulating the sympathetic and parasympathetic nervous systems is a promising method to modulate the cardiac activity and control the blood pressure. Locus coeruleus noradrenergic neurons inhibit brainstem cardiac vagal neurons that generate parasympathetic activity in the heart of Cre/loxp recombinase transgenic animals expressing ChR2 in the locus coeruleus noradrenergic neurons.71 In addition, ChR2 was expressed in transgenic murine catecholaminergic sympathetic neurons to study the cardiac electrophysiology and examine the changes in contractile force, heart rate, and cardiac electrical activity,72 enabling a direct method to probe the relationship between sympathetic neurons and the heart. Optogenetics technology provides a new method for regulating the ANS, which has the potential to decrease the occurrence of cardiac arrhythmias. The method of delivering photosensitive proteins to the heart in vivo Transgenic technology for gene insertion may include transgenic animals, viral transduction, and cell transplant; AAV-based gene transfer of optogenetics may be easier to apply in patients than in other gene delivery vehicles.73 The AAV-mediated transgenic delivery has been used in gene therapy research and has the potential for clinical application.74 Adeno-associated virus 9 transduction was first applied clinically in a trial to treat retinitis pigmentosa12; further studies are required to evaluate the long-term effects and steady expression in the retina. Different types of AAVs show different viral tropism, and some published studies that have analysed the specific tropism of the AAV serotype in different tissues show that AAV9 is the most cardiotropic serotype in gene transfer studies in vivo.75,76 Cardiac-specific promoters (α-MHC, cTnT) can enhance the cardiac-specific of AAV9 to improve the precision of optogenetics technology, but the transgene expression may lower than the non-specific promoter. The capacity of the AAV is limited, which can consist of a linear ≈4.7 kb single-stranded DNA genome. AAV9 may be a viable vehicle for long-term gene expression in cardiomyocytes, as it is associated with minimal induction of immune responses.77 Improving the viral structure and delivery route may increase the efficiency of transfection. The efficacy of cardiac gene therapy may be improved using cardiac-specific cis-regulatory modules that are highly expressed specifically in the heart.78 Direct myocardial injections may increase the efficiency of local transfection but decrease the efficiency at remote myocardial sites, which is a limitation of target cell activation/inhibition in these remote areas. Multipoint injection may expand the transfected area but also increase myocardial injury. Although systemic intravenous injections require higher infectious titres and wastes viruses in non-target tissues, it transfers AAV to the whole heart and minimizes physical damage. For the application of AAV in large animals such as dogs, pigs, or primates, systemic intravenous injection may have numerous challenges such as the long-term in vivo expression of photosensitive proteins and the damage caused by high viral titres. Magnetofection is a technique based on the principle of magnetic targeting to overcome extracellular barriers, and it has been used to produce highly efficient and enduring expression of transfection. The feasibility of long-term transfection was shown in vivo for a DNA plasmid vector in neurons using magnetofection, and this technique is a potential alternative to overcome the safety concerns of viral vectors and the restrictions of the genetic material size.79 Advances in genetic technology may enable the in vivo delivery of photosensitive proteins with high efficiency and accuracy. Future directions Optical equipment in neurosciences, such as lasers and light-emitting diodes, has been widely used for controlling target cells; however, these equipments could impede the natural movement of experimental animals and the fibre-optical have light scattering or attenuation. A flexible optofluidic neural probe, an ultrathin light-emitting diode, was implanted to maintain the shape of the beam to successfully reduce light scattering and attenuation. Recently, the wireless stimulator system80,81 combined a wirelessly controlled interface with a small implantable light-emitting diode to study the deep brain structure and efficiently manipulate neuronal activity in freely moving mice; this system may be introduced in cardiac research. A bioluminescence technique that requires no implantation of optical equipment may enable non-invasive optogenetics with chemogenetic control. The development of luminopsins, a fusion of the proteins luciferase and opsin, may enable the control of behaviour in freely moving animals using light from wild-type luminopsins and activating ChRs.82 In the future, optogenetics may provide new developments in cardiovascular research on electrophysiology, arrhythmias, cell signalling, and drug discovery and, potentially, may provide an innovative and accurate method to treat cardiovascular diseases by precisely controlling the heart rate, achieving synchronized contraction, and attempting cardiac ablation. Optogenetics with dynamic space–time patterns for optical stimulation and multisite electrical recording can be combined to study the mechanism of cardiac arrhythmias by reflecting the direction of the conduction of electrical activity. To investigate the quantitative details or spatial microdomains of cardiac excitation–contraction coupling and its modulation mechanisms such as complex signal pathways or autonomic nerves, we need more precise and multifunctional tool, the gain function of mutants of photosensitive proteins should be created, and the new proteins need to be discovered with different optical-control characteristics. These proteins could be utilized as light-sensitive probes in different cell subtypes of the heart and used for studying the differentiation of embryonic cells, manipulating cardiovascular functions in subcellular levels, and investigating pathogenesis and therapeutic strategies. There are multiple challenges for the application of optogenetics in heart research, including (i) safe and effective gene delivery; (ii) long-term expression of photosensitive proteins; (iii) optimization of devices such as optical device, sufficient optical power, light penetration, fibre or wireless devices, and detectors; (iv) unscattered and unattenuated exogenous light stimulation; and (v) location of the implanted stimulator and direction of illumination without interference from lung activity. A convenient, accurate, and bidirectional control method will provide a novel approach to study the mechanism behind arrhythmia and test cardiology hypotheses, and in the future, it may become a safe and effective strategy for the treatment of cardiovascular diseases. Acknowledgements We thank Guoxing Zheng, from Electronic Information School Wuhan University, for his suggestion for writing of the development of optical technology and his recommendation to include a section on light properties; Yi Lu, from Shenzhen Institutes of Advanced Technology of Chinese Academy of Sciences, for his modification of this manuscript; and Xiaobin He, from Wuhan Institute of Physics and Mathematics of Chinese Academy of Sciences, for his correction of the writing of gene delivery with viral vehicle and his recommendation to include a section on construction of virus and its properties. Funding This work was supported by the National Natural Science Foundation of China (81260052) and the Natural Science Foundation of Hubei Province of China (2014CKB497, 2015BKA339). Conflict of interest: none declared. References 1 Hildebrand E , Dencher N. Two photosystems controlling behavioural responses of Halobacterium halobium . Nature 1975 ; 257 : 46 – 8 . 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TI - Cardiac optogenetics: a novel approach to cardiovascular disease therapy
JF - Europace
DO - 10.1093/europace/eux345
DA - 2018-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/cardiac-optogenetics-a-novel-approach-to-cardiovascular-disease-9r63zFRd3A
SP - 1741
VL - 20
IS - 11
DP - DeepDyve
ER -