Tobias Bruegmann

Optogenetic control of heart muscle in vitro and in vivo

Electrical stimulation is the standard technique for exploring electrical behavior of heart muscle, but this approach has considerable technical limitations. Here we report expression of the light-activated cation channel channelrhodopsin-2 for light-induced stimulation of heart muscle in vitro and in mice. This method enabled precise localized stimulation and constant prolonged depolarization of cardiomyocytes and cardiac tissue resulting in alterations of pacemaking, Ca2+ homeostasis, electrical coupling and arrhythmogenic spontaneous extrabeats.

Systemic gene transfer enables optogenetic pacing of mouse hearts

AIMS Optogenetic pacing of the heart has been demonstrated in transgenic animals expressing channelrhodopsin-2 (ChR2). However, for the clinical use of optogenetics to treat cardiac arrhythmias, gene transfer to non-transgenic hearts is required. The aim of this study was to describe a reliable method for gene transfer of ChR2 into a sufficient percentage of cardiomyocytes to overcome the electrical sink of all the coupled non-expressing cardiomyocytes during optical pacing of the whole heart in vivo. METHODS AND RESULTS Adeno-associated virus (AAV) with cardiac tropism for expression of ChR2 in fusion with mCherry was systemically injected into wild-type mouse hearts. Bright mCherry fluorescence was detected in the whole heart 4-10 weeks later. Single-cell dissociation revealed that on average 58% cardiomyocytes were mCherry-positive. These showed light-induced inward currents, action potentials, and contractions. Pulsed illumination of the left ventricle induced ventricular pacing in vivo in 74% of mice, and higher light intensities were required for reduced pulse duration or size of illumination. Non-responding hearts showed low AAV expression, and the threshold for optical pacing was estimated to be 35-40% ChR2-expressing cardiomyocytes. Optical pacing in vivo was stable over extended periods without negative effects on normal sinus rhythm and ECG parameters after termination of stimulation indicating sufficient cardiac output during pacing. CONCLUSIONS Gene transfer generates sufficient ChR2 photocurrent for reliable optogenetic pacing in vivo and lays out the basis for future optogenetic pacemaker and pain-free defibrillation therapies.

Optogenetic defibrillation terminates ventricular arrhythmia in mouse hearts and human simulations

Ventricular arrhythmias are among the most severe complications of heart disease and can result in sudden cardiac death. Patients at risk currently receive implantable defibrillators that deliver electrical shocks to terminate arrhythmias on demand. However, strong electrical shocks can damage the heart and cause severe pain. Therefore, we have tested optogenetic defibrillation using expression of the light-sensitive channel channelrhodopsin-2 (ChR2) in cardiac tissue. Epicardial illumination effectively terminated ventricular arrhythmias in hearts from transgenic mice and from WT mice after adeno-associated virus-based gene transfer of ChR2. We also explored optogenetic defibrillation for human hearts, taking advantage of a recently developed, clinically validated in silico approach for simulating infarct-related ventricular tachycardia (VT). Our analysis revealed that illumination with red light effectively terminates VT in diseased, ChR2-expressing human hearts. Mechanistically, we determined that the observed VT termination is due to ChR2-mediated transmural depolarization of the myocardium, which causes a block of voltage-dependent Na+ channels throughout the myocardial wall and interrupts wavefront propagation into illuminated tissue. Thus, our results demonstrate that optogenetic defibrillation is highly effective in the mouse heart and could potentially be translated into humans to achieve nondamaging and pain-free termination of ventricular arrhythmia.

Optogenetic control of contractile function in skeletal muscle

Optogenetic stimulation allows activation of cells with high spatial and temporal precision. Here we show direct optogenetic stimulation of skeletal muscle from transgenic mice expressing the light-sensitive channel Channelrhodopsin-2 (ChR2). Largest tetanic contractions are observed with 5-ms light pulses at 30 Hz, resulting in 84% of the maximal force induced by electrical stimulation. We demonstrate the utility of this approach by selectively stimulating with a light guide individual intralaryngeal muscles in explanted larynges from ChR2-transgenic mice, which enables selective opening and closing of the vocal cords. Furthermore, systemic injection of adeno-associated virus into wild-type mice provides sufficient ChR2 expression for optogenetic opening of the vocal cords. Thus, direct optogenetic stimulation of skeletal muscle generates large force and provides the distinct advantage of localized and cell-type-specific activation. This technology could be useful for therapeutic purposes, such as restoring the mobility of the vocal cords in patients suffering from laryngeal paralysis.

Optogenetic activation of Gq signalling modulates pacemaker activity of cardiomyocytes.

AIMS Investigation of Gq signalling with pharmacological agonists of Gq-coupled receptors lacks spatio-temporal precision. The aim of this study was to establish melanopsin, a light-sensitive Gq-coupled receptor, as a new tool for the investigation of spatial and temporal effects of Gq stimulation on pacemaking in cardiomyocytes at an early developmental stage. METHODS AND RESULTS A vector for ubiquitous expression of melanopsin was tested in HEK293FT cells, which showed light-induced production of inositol-1,4,5-trisphosphate and elevation of intracellular Ca(2+) concentration. Mouse embryonic stem cells were stably transfected with this plasmid and differentiated into spontaneously beating embryoid bodies (EBs). Cardiomyocytes within EBs showed melanopsin expression and illumination (60 s, 308.5 nW/mm(2), 470 nm) of EBs increased beating rate within 10.2 ± 1.7 s to 317.1 ± 16.3% of baseline frequency. Illumination as short as 5 s was sufficient for generating the maximal frequency response. After termination of illumination, baseline frequency was reached with a decay constant of 27.1 ± 2.5 s. The light-induced acceleration of beating frequency showed a sigmoid dependence on light intensity with a half maximal effective light intensity of 41.7 nW/mm(2). Interestingly, EBs showed a high rate of irregular contractions after termination of high-intensity illumination. Local Gq activation by illumination of a small region in a functional syncytium of cardiomyocytes led to pacemaker activity within the illuminated area. CONCLUSIONS Light-induced Gq activation in melanopsin-expressing cardiomyocytes increases beating rate and generates local pacemaker activity. We propose that melanopsin is a powerful optogenetic tool for the investigation of spatial and temporal aspects of Gq signalling in cardiovascular research.

Frequency-dependent drug screening using optogenetic stimulation of human iPSC-derived cardiomyocytes

Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.

Optogenetic termination of atrial fibrillation in mice

Aims The primary goal in the treatment of symptomatic atrial fibrillation/flutter (AF) is to restore sinus rhythm by cardioversion. Electrical shocks are highly effective, but have to be applied under analgo-sedation and can further harm the heart. In order to develop a novel pain-free and less harmful approach, we explored herein the optogenetic cardioversion by light-induced depolarization. Methods and results Hearts from mice expressing Channelrhodopsin-2 (ChR2) and the AF-promoting loss-of-function Connexin 40 Ala96Ser mutation were explanted and perfused with low K+ Tyrodes solution and an atrial KATP-channel activator. This new protocol shortened atrial refractoriness as well as slowed atrial conduction and thereby enabled the induction of sustained AF. AF episodes could be terminated by epicardial illumination of the atria with focussed blue light (470 nm, 0.4 mW/mm2) with an efficacy of ∼97% (n = 17 hearts). In > 80% of cases, light directly terminated the AF episode with onset of illumination. Because similar illumination intensity was able to locally inhibit atrial activity, we propose that a light-induced block of electrical activity is responsible for reliable AF termination. The success rate was strongly depending on the illuminated area, applied light intensity and duration of illumination. Importantly, we were also able to demonstrate optogenetic termination of AF in vivo, using epicardial illumination through the open chest (n = 3 hearts). To point towards a translational potential, we systemically injected an adeno-associated virus to express ChR2 in wild type hearts. After 6-8 months, we found robust ChR2 expression in the atria, enabling light-mediated AF termination in six of seven mice tested. Conclusion We provide the first evidence for optogenetic termination of atrial tachyarrhythmia in intact hearts from transgenic as well as wild type mice ex and in vivo. Thus, this report could lay the foundation for the development of implantable devices for pain-free termination of AF.

Optogenetic cardiac pacemakers: Science or fiction?

Optogenetics is a novel technology that allows cell-specific controlling of membrane potential with light by expressing light-sensitive proteins in cells of interest [1]. This method is extensively used for optical stimulation of specific cell types in well-defined brain regions in vivo. Optogenetic cardiac pacing in vivo has also been demonstrated in adult mouse hearts [2] and embryonic zebrafish hearts [3] using transgenic animals expressing the blue light-sensitive cation channel Channelrhodopsin 2 (ChR2). In this issue of Trends in Cardiovascular Medicine, Boyle et al. [4] review the existing literature on optogenetic control of heart muscle in vitro, in silico, and in vivo and discuss the potential clinical use to treat cardiac arrhythmia. Because optogenetic stimulation has distinct advantages over electrical stimulation such as low energy consumption, cell-specific stimulation, uniform deor hyperpolarization, and high spatial precision, optogenetic cardiac pacing or defibrillation can be envisioned in the future. It is important to note that before raising hope for future therapies, the potential, effectiveness, advantages, and disadvantages of optogenetic pacing or defibrillation have to be investigated in vivo in native non-transgenic hearts. Therefore, the most important issue to be solved is how to make native hearts light sensitive. The authors discuss two fundamentally different approaches [4], either transplantation of cells expressing optogenetic proteins (cell delivery) or viral gene transfer of optogenetic proteins into native cardiomyocytes (gene delivery). In comparison to transgenic animals, both approaches will result in a lower percentage and patterned distribution of ChR2-expressing cells within the myocardium. Because all cardiomyocytes are well coupled through gap junctions, the three-dimensional ventricle, which represents a large

Optical control of L-type Ca 2+ channels using a diltiazem photoswitch

L-type Ca2+ channels (LTCCs) play a crucial role in excitation–contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic β-cell function and cardiac activity under optical control.A photoswitchable probe to control Ca2+ influx through L-type Ca2+ channels is useful in pancreatic β cells and can be employed to modulate beating rate in explanted hearts.

Spot light on skeletal muscles: optogenetic stimulation to understand and restore skeletal muscle function

Damage of peripheral nerves results in paralysis of skeletal muscle. Currently, the only treatment option to restore proper function is electrical stimulation of the innervating nerve or of the skeletal muscles directly. However this approach has low spatial and temporal precision leading to co-activation of antagonistic muscles and lacks cell-type selectivity resulting in pain or discomfort by stimulation of sensible nerves. In contrast to electrical stimulation, optogenetic methods enable spatially confined and cell-type selective stimulation of cells expressing the light sensitive channel Channelrhodopsin-2 with precise temporal control over the membrane potential. Herein we summarize the current knowledge about the use of this technology to control skeletal muscle function with the focus on the direct, non-neuronal stimulation of muscle fibers. The high temporal flexibility of using light pulses allows new stimulation patterns to investigate skeletal muscle physiology. Furthermore, the high spatial precision of focused illumination was shown to be beneficial for selective stimulation of distinct nearby muscle groups. Finally, the cell-type specific expression of the light-sensitive effector proteins in muscle fibers will allow pain-free stimulation and open new options for clinical treatments. Therefore, we believe that direct optogenetic stimulation of skeletal muscles is a very potent method for basic scientists that also harbors several distinct advantages over electrical stimulation to be considered for clinical use in the future.