This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.
Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.
Early investigations of the spatial-temporal dynamics during arrhythmia revealed that the complex electrical patterns during cardiac fibrillation are driven by vortex-like rotating excitation waves1. This finding gave new insights into the underlying mechanisms of arrhythmias, which then led to the development of novel electrical termination therapies based on multi-site excitation of the myocardium2,3,4. However, treatments using electric field stimulation are non-local and may innervate all surrounding excitable cells, including muscle tissue, causing cellular and tissue damage, as well as intolerable pain. In contrast to electrical therapies, optogenetic approaches provide a specific and tissue-protective technique for evoking cardiomyocyte action potentials with high spatial and temporal precision. Therefore, optogenetic stimulation has the potential for minimal invasive control of the chaotic activation patterns during cardiac fibrillation.
The introduction of the light-sensitive ion channel channelrhodopsin-2 (ChR2) into excitable cells via genetic manipulation5,6,7, enabled the depolarization of the membrane potential of excitable cells using photostimulation. Several medical applications, including the activation of neuronal networks, the control of cardiac activity, the restoration of vision and hearing, the treatment of spinal cord injuries, and others8,9,10,11,12,13,14 have been developed. The application of ChR2 in cardiology has significant potential due to its millisecond response time15, making it well suited for the targeted control of arrhythmic cardiac dynamics.
In this study, multi-site photostimulation of intact hearts of a transgenic mouse model is shown. In summary, a transgenic alpha-MHC-ChR2 mouse line was established within the scope of the European Community's Seventh Framework Programme FP7/2007-2013 (HEALTH-F2-2009-241526) and kindly provided by Prof. S. E. Lehnart. In general, transgenic adult male C57/B6/J, expressing Cre-recombinase under control of alpha-MHC were paired to mate with female B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hye/J. Since the cardiac STOP cassette was deleted in the second-generation, the offspring showed a stable MHC-ChR2 expression and was used to maintain cardiac photosensitive colonies. All experiments were done with adult mice of both genders at an age of 36 – 48 weeks. The illumination is achieved using a 3 x 3 micro-LED array, fabricated as described in16,17 except that the silicon-based housing and the short optical glass fibers are not implemented. Its first usage in a cardiac application is found in18. A linear micro-LED array based on a similar fabrication technology has been applied as a penetrating probe for heart pacing19. The micro-LEDs are arranged in a 3 x 3 array at a pitch of 550 µm, providing both a high spatial resolution and a high radiant power on a very small area. The authors demonstrate in this work a versatile local multi-site photostimulation that may open the path for developing novel anti-arrhythmic therapy methods.
The following experimental protocol involves a retrograde Langendorff perfusion ex vivo, for which the cannulated aorta functions as perfusion inlet. Due to the applied perfusion pressure and the cardiac contraction the perfusate is flowing through the coronary arteries, which branch off the aorta. In the presented work, the heart is perfused using a constant pressure setup achieved by elevating the perfusate reservoirs to 1 m height, equivalent to 73.2 mmHg, which yields to a flow rate of 2.633 ± 0.583 mL/min. Two kinds of Tyrode's solution are used as perfusate during the experiment. Regular Tyrode's solution supports a stable sinus rhythm, whereas Low-K+ Tyrode's solution is mixed with Pinacidil to enable the induction of arrhythmia in murine hearts. The usage of a hexagonal water bath permits the observation of the heart through six different planar windows, allowing the coupling of several optical components with less distortion by refraction.
All experiments strictly followed the animal welfare regulation, in agreement with German legislation, local stipulations, and in accordance with recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). The application for approval of animal experiments has been approved by the responsible animal welfare authority, and all experiments were reported to our animal welfare representatives.
1. Experiment preparation and materials
2. Experimental procedures
The protocol allows the induction of ventricular arrhythmias in intact murine hearts using photostimulation pulses generated by LED 1 and LED 2 (Figure 1) with a frequency find between 25 Hz and 35 Hz and a pulse duration Wind between 2 ms and 10 ms. Please notice that the aim of such rapid light pulses is not to capture the cardiac rhythm but rather to unbalance the cardiac activity so that erratic electrical waves can be generated, which then facilitate an arrhythmia. The advantage of inducing arrhythmia with light over induction with electrical stimulation is that no artifacts are provoked in the ECG, providing the possibility to post-analyze the acquired signal without restrictions and even evaluate the electrical response of the heart during rapid pacing, this fact also provides the possibility to observe the heart behavior during photo-defibrillation. This is not possible with electrical induction or defibrillation methods. Nevertheless, if the setup used does not allow the usage of external high-power LEDs, for example, because of place constraints, an additional pacing electrode can be placed onto the heart to induce arrhythmia, as shown elsewhere3,22,24.
Once fibrillation is induced the arrhythmia must last at least 5 seconds to ensure that it sustains, afterwards the micro-LED-based defibrillation attempts are started. Since the main parameters of cardiac arrhythmia, such as basic cycle length or dominant frequency, amplitude, and morphology, are continually changing and as it is up to date not possible to predict which photodefibrillation parameters provide the best outcome, it was of significant interest to understand whether there is a relationship between the frequency, pulse width, area of photostimulation and termination rate. Therefore, a series of experiments with different frequencies fdef, number of micro-LEDs, and pulse durations Wdef were tested, and the success rate for N = 11 mice was extracted, as shown in Figure 3.
It could be demonstrated that pulses of 1 to 20 ms durations can defibrillate with different success rates (Figure 3). Since the light intensity LIµLED was kept constant during every photostimulation pulse, as mentioned in Step 2.4.6, and the success rate of three micro-LEDs against nine is notably lower, the presented results suggest that the area covered on the heart, the number of micro-LEDs, and thus the total radiant flux applied are crucial factors in achieving defibrillation. Considering that every micro-LED on the array is a Lambertian light source and that they are positioned directly onto the surface of the heart so that the approximate distance to the tissue is zero, it can be assumed that the irradiance contour of the illuminated area on the heart when using a single micro-LED is equivalent to AµLED = 0.059 mm², as also shown in25 for flat rectangular LEDs. Furthermore, although some photons might leave the micro-LED laterally from the edges, the contribution of those to the total light intensity is considered so small that their effect can be neglected. To quantify the irradiated light of the array, the authors measured the radiant flux from the micro-LED array with a commercial power meter and calculated the light intensity which reaches the heart as shown in Table 1. From Table 1 it can also be read that the radiant flux increases with the number of used micro-LEDs, but the light intensity remains constant due to the illumination profile implications mentioned before.
Interestingly, it can also be observed that the success rate of nine LEDs with Wdef = 1 ms (Figure 3a) and Wdef = 20 ms (Figure 3d) at a defibrillation frequency fdef = 18 Hz and fdef = 20 Hz are comparably high. Considering that the average frequency of the induced arrhythmias is 22.55 ± 4.03 Hz, this fact might indicate that for ChR2 murine hearts, the success rate increases significantly the closer the pacing frequency is to the arrhythmia frequency. This is also shown in numerical simulations26. However, this cannot be easily generalized because the dominant frequency of complex arrhythmias is constantly changing. To illustrate this, Figure 4 shows two different defibrillation attempts with fdef = 14 Hz.. At the beginning of the ECG segment in Figure 4a) and according to the morphology of the ECG signal a ventricular fibrillation (VF) is shown. When the micro-LED photostimulation starts, the fibrillation is turned into a more ordered pattern which is more likely to be a ventricular tachycardia (VT). Whenever the micro-LED array is turned off, the original chaotic VF waves take over again. Thus, the arrhythmia is not terminated. Although in this example the VF cannot be terminated with the given parameters, it does get disturbed and it can be changed to a more regular pattern (VT). Figure 4b Segment 1 shows that the dominant frequency of 24 Hz slightly increases until photostimulation begins and the VF is turned into a VT in Segment 2, where the dominant frequency drops to 14 Hz. Furthermore, Figure 4c shows a VT which can be terminated with the same fdef as in Figure 4a, but with a different Wdef. First, the micro-LED photostimulation changes the morphology of the arrhythmia, to finally terminate it with 1:1 pacing capture from the 19th pulse onwards. These results might imply that the photodefibrillation parameters, for example Wdef, must adapt to the morphology change of the arrhythmia over time. The experiments leading to these results were conducted without using Blebbistatin because of the resulting change in action potential duration (APD)27. Therefore, no optical mapping was performed in these series.
Another set of experiments was performed for optical mapping using the red-shifted potentiometric dye (Step 2.1.4). Optical mapping with high-speed cameras allows to observe propagating excitation waves on the surface of the heart during sinus rhythm (Figure 5) and complex tachyarrhythmias28. Since the fractional change of the potentiometric dye is very low, the obtained videos were post-processed using a mathematical programming language. The first step to improve the quality of the optical signals is to remove noise applying a Gaussian smoothing filter with a standard deviation of σ = 1, followed by a bandpass filter with corner frequencies fhigh = 0.1 Hz and flow = 70 Hz. The stopband at fhigh removes slow changes in the signal which are not related to the sinus frequency of the heart which lies between 3 Hz < fsinus < 8 Hz, while the stopband flow removes high frequency noise which is captured by the camera. It is important to note that both blue light emissions from LED 1, LED 2 and from the micro-LED array can cause cross talk and a very high interference signal in optical mapping. In addition, it was observed that not even a very narrow bandpass filter in front of the camera, with wavelength λfilter-cam, as mentioned in Step 1.2.3, would filter out the influence of the blue light. This might be caused partly by the excitation response of the dye itself. Be therefore very careful when choosing the optics for optical mapping. For the means of video analysis, all frames in which blue light has been recorded had to be neglected so that in many cases it is not possible to visualize the heart during photostimulation, as also mentioned in another study29.
Figure 1: Schematic of the electrical and optical setup. (a) LED 1 and LED 2 provide a blue light source used for induction of arrhythmia and backup defibrillation. LED 3 is used as an excitation light source for the red-shifted dye DI-4-ANBDQPQ. The red light is directed to the heart by means of the dichroic mirror DM. The emission light shown in dark red is recorded by the high-speed camera through an emission filter, as mentioned in the text. LED 2 and ECG electrodes are not shown for simplicity. (b) One segment of the recorded ECG signal shown in red. Dark blue shows the light pulses from LED 1 and LED 2 at a frequency find = 35 Hz and Wind = 4 ms used to induce fibrillation. Immediately after finishing the light stimulus, ventricular fibrillation (VF) can be observed. The micro-LED-based photostimulation shown in light blue (fdef = 16 Hz, Wdef = 20 ms) successfully terminates the arrhythmia. Please click here to view a larger version of this figure.
Figure 2: Heart preparation. (a) Open chest of a mouse showing the intact heart and the surrounding organs. (b) Explanted heart immersed in ice-cold Tyrode's solution for further preparation. (c) Mouse heart properly attached to a blunt needle. (d) Murine heart suspended in Tyrode's solution. Please click here to view a larger version of this figure.
Figure 3: Experimentally extracted success rates. Success rates for 30 micro-LED-based photostimulation pulses using three, six and nine LEDs at different pulse durations Wdef and frequencies fdef for N = 11. Error bars shown with standard error of mean S.E.M. Please click here to view a larger version of this figure.
Figure 4: Manipulation of the cardiac rhythm by means of photostimulation. (a) Segment of an ECG recording of a non-terminated arrhythmia. (b) Spectrogram of the ECG shown in panel a. The power spectral density (PSD) of Segment (1) shows an arrhythmia with a dominant frequency of 24 Hz. Segment (2) photostimulation with the shown parameters. It can be observed that the dominant frequency drops to 14 Hz. Segment (3) Unsuccessful termination and return to arrhythmic behavior with a dominant frequency of 24 Hz. (c) ECG of a successful defibrillation attempt. (d) Spectrogram of the successful termination displayed in panel c. Segment (1) shows a ventricular tachycardia (VT) with dominant frequency of 23 Hz. Segment (2) photostimulation using the shown settings. Segment (3) displays a successful termination, which leads to a normal sinus rhythm with a fundamental frequency of 3.5 Hz and the resulting harmonics. Please click here to view a larger version of this figure.
Figure 5: Optical mapping of the whole heart. The change of fluorescence intensity during one single beat of the heart in normal sinus rhythm is shown. The heart was positioned facing the camera so that the right and left ventricle are visible (RV, LV). The asterisk shows the pixel at which the action potential shown on top was taken. Please click here to view a larger version of this figure.
Number of microLED | Irradiated area Aµled [mm2] | Radiant flux φ [mW] | Light Intensity LI [mW mm-2] |
3 | 0.178 | 5.9 ± 0.47 | 33.11 ± 2.66 |
6 | 0.356 | 11.91 ± 0.84 | 33.42 ± 2.37 |
9 | 0.535 | 17.85 ± 0.61 | 33.39 ± 1.14 |
Table 1: Measured radiant flux of the micro-LED array and the corresponding light intensity.
A successful treatment of cardiac tachyarrhythmias is key to cardiac therapy. However, the biophysical mechanisms underlying arrhythmia initiation, perpetuation and termination are not fully understood. Therefore, cardiac research aims to optimize electrical shock therapy towards a more gentle termination of arrhythmias, thereby increasing the quality of life of patients28,29,30,31. Low energy electrical approaches promise a significant reduction of severe side effects, however might still induce unwanted muscle excitation. Cardiac optogenetics could overcome this limitation and provide not only a tissue-gentle termination technique but also a flexible platform to investigate the arrhythmia-specific targeted control of vortex-like excitation waves in the intact murine heart and in cell cultures32,33.
Given that motivation, a robust photostimulation setup, as well as a protocol were designed and implemented, both offering a highly adaptable optical system, which could easily be extended to three-dimensional panoramic optical mapping studies34.
It could be shown that cardiac arrhythmias can successfully be terminated with different success rates depending on the parameters which are chosen for photostimulation, for example the illuminated area on the heart. The presented results suggest that increasing the irradiated surface recruited a critical number of cardiomyocytes extinguishing the chaotic activity by conduction block as also shown in22. In this study, the energy required to photodefibrillate is E = 10.69 ± 0.37 mJ (using nine micro-LEDs, 30 pulses and pulse width Wdef = 20 ms). This turns out to be lower than earlier reported in 22,24 with E22 = 228.8 mJ and E24 = 153.6 mJ, where a bigger area22 or the entire heart24 were illuminated, respectively. Nevertheless, compared to the approach shown in 35, where a well delimited patterned area is illuminated with 10 photodefibrillation pulses resulting in E35 = 1.8 mJ, the photodefibrillation energy in the present study is notably higher. In contrast to the three other approaches, a success rate over 90% could not be reached with the presented protocol. One possible reason for the reduced performance despite a higher photodefibrillation energy might be that the complexity of the underlying arrhythmia is not being considered. With regard to the results presented in 35, where a high termination rate is accomplished by illuminating a small area on the heart, and simultaneously measuring the spatial-temporal dynamics of an arrhythmia, the presented approach can certainly be further improved by considering feedback-control, which responds with a different pattern of micro-LED illumination depending on the current state of the heart. Moreover, it was also demonstrated that although arrhythmias cannot always be terminated with the current method, the intrinsic complex dynamics can be disturbed during photostimulation leading to a more ordered temporal state. As shown in36, the termination rate is significantly different when addressing monomorphic (more ordered) and polymorphic (less ordered) arrhythmias. Hence the logical step towards a better defibrillation rate might be to influence the cardiac dynamics during a VF episode, turn the arrhythmia into a less complex pattern and terminate with another set of pulses, building in this way a two-step photostimulation approach.
In regard of the perfusion protocol, the most critical steps are found in the correct extraction and preparation of the heart as well as in the correct adjustment of the optical mapping optics. Involving optical mapping strictly requires the proper selection of dye spectra, appropriate excitation light-sources and well-chosen optical filters for the camera29. Otherwise, the recorded optical signals might be too noisy and also could contain cross-talk of photostimulation with dye excitation. Subsequent analysis would therefore necessitate post-processing of signals with several analytical filters and image smoothing often resulting in worsening.
Another crucial step in this protocol is the correct and precise placement of the micro-LED array. Since the interconnecting lead between the micro-LED array and the driver is very thin and flexible, it is sometimes challenging to ensure that the array will be located at approximately the same location on the heart surface for each experiment. To facilitate positioning and to fix the acquired position of the micro-LED array, a holder was designed and printed in 3D, allowing the array to be attached to a micromanipulator. This gives more control over the movement of the array in the Tyrode's solution. Depending on the material chosen for the interconnecting lead of the micro-LED array, the use of a holder might not be necessary.
Besides, another critical step of the protocol is the addition of pro-arrhythmia drugs, like e.g., Pinacidil37. Since several chemical compounds are well known for changing the physiological response of the heart, this should be considered when analyzing and interpreting the results. As far as optical mapping is concerned, the proposed protocol uses Blebbistatin as a mechanical uncoupler. This has the advantage of removing motion artifacts during recording, but it can also prolong the APD27. To overcome this drawback, analyzing methods of motion tracking during recording could be considered38,39. This way, the normal physiological condition of the heart would be preserved, and a high-quality signal can be obtained.
Although it was proven that the presented protocol can be used for multi-site photo-defibrillation, it still has some limitations. It has been found that in some cases fibrillation cannot be terminated by the micro-LED-based photostimulation but only be disturbed, resulting in frequency changes. One hypothesis is that the meandering waves on the heart are only being displaced from the left ventricle, regenerating themselves in other parts of the heart. Compared with other methods such as global illumination24, the present method offers a lower success rate due to a smaller coverage of the heart. Though, we are confident that with the proper hardware-based recognition method of spiral activity, improvement of termination success rate is feasible.
In conclusion, the presented photostimulation system establishes a powerful experimental tool for multiple cardioversion approaches and manipulation studies of cardiac arrhythmia. The knowledge learned in this system will be used to investigate and evaluate new potential (photo) defibrillation protocols in clinically relevant large animal models.
The authors have nothing to disclose.
The authors would like to thank Marion Kunze and Tina Althaus for their excellent technical support during experiments. The research leading to the results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement number HEALTH-F2-2009-241526. Support was also provided by the German Center for Cardiovascular Research, DZHK e.V. (Project MD28), partner site Goettingen, the German Research Foundation CRC 1002 (project C03), and the Max Planck Society. This work was partly supported by BrainLinks-BrainTools, Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086).
Chemical Components | |||
Blebbistatin | TargetMol | T6038 | 10 mM stock solution |
BSA/Albumin | Sigma-Aldrich | A4919 | |
Calcium Chloride | Sigma-Aldrich | C1016 | CaCl2 |
Carbogen | Westfalen | 50 l bottle | |
DI-4-ANBDQPQ | AAT Bioquest | 21499 | Dye for Optical Mapping |
Glucose | Sigma-Aldrich | D9434 | C6H12O6 |
Heparin | LEO Pharma | Heparin-Natrium Leo 25.000 I.E./5 ml, available only on prescription | |
Hydrochlorid Acid | Merck | 1.09057.1000 | HCl, 1 M stock solution |
Isoflurane | CP Pharma | 1 ml/ml, available only on prescription | |
Magnesium Chloride | Merck | 8.14733.0500 | MgCl2 |
Monopotassium Phosphate | Sigma-Aldrich | 30407 | KH2PO4 |
Pinacidil monohydrate | Sigma-Aldrich | P154-500mg | 10 mM stock solution |
Potassium Chloride | Sigma-Aldrich | P5405 | KCl |
Sodium Bicarbonate | Sigma-Aldrich | S5761 | NaHCO3 |
Sodium Chloride | Sigma-Aldrich | S5886 | NaCl |
Sodium Hydroxide | Merck | 1.09137.1000 | NaOH, 1 M stock solution |
Electrical Setup | |||
Biopac MP150 | Biopac Systems | MP150WSW | data acquisition and analysis system |
Custom-built ECG, alternative ECG100C | Biopac Systems | ECG100C | Electrocardiogram Amplifier |
Custom-built water bath heater using heating cable | RMS Heating System | HK-5,0-12 | Heating cable 120W |
Hexagonal water bath | |||
LED Driver Power supply | Thorlabs | KPS101 | 15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for One K- or T-Cube. |
LEDD1B LED Driver | Thorlabs | LEDD1B | T-Cube LED Driver, 1200 mA Max Drive Current |
MAP, ECG Electrode | Hugo Sachs Elektronik | BS4 73-0200 | Mini-ECG Electrode for isoalted hearts |
micro-LED Driver e.g. AFG | Agilent Instruments | A-2230 | Arbitrary function generator (AFG) |
Signal Generator | Agilent Instruments | A-2230 | AFG |
micro-LED Array Components | |||
Epoxid glue | Epoxy Technology | EPO-TEK 353ND | Two component epoxy |
Fluoropolymer | Asahi Glass Co. Ltd. | Cytop 809M | Fluoropolymer with high transparency |
Image reversal photoresist | Merck KGaA | AZ 5214E | Image Reversal Resist for High Resolution |
LED chip | Cree Inc. | C460TR2227-S2100 | Blue micro-LED |
Photoresist | Merck KGaA | AZ 9260 | Thick Positive Photoresists |
Polyimide | UBE Industries Ltd. | U-Varnish S | Polyimide Solution |
Silicone | NuSil Technology LLC | MED-6215 | Low viscosity silicone elastomer |
Solvent free adhesive | John P. Kummer GmbH | Epo-Tek 301-2 | Epoxy resin with low viscosity |
Optical Mapping | |||
Blue Filter | Chroma Technology Corporation | ET470/40x | Blue excitation filter |
Camera | Photometrics | Cascade 128+ | High performance EMCCD Camera |
Camera Objective | Navitar | DO-5095 | Navitar high speed fixed focal length lenses work with CCD and CMOS cameras |
Dichroic Mirror | Semrock | FF685-Di02-25×36 | 685 nm edge BrightLine® single-edge standard epi-fluorescence dichroic beamsplitter |
Emmision Filter | Semrock | FF01-775/140-25 | 775/140 nm BrightLine® single-band bandpass filter |
Heatsink | Advanced Thermal Solutions | ATSEU-077A-C3-R0 | Heat Sinks – LED STAR LED Heatsink, 45mm dia., 68mm, Black/Silver, Unthreaded Baseplate Hardware |
LED 1 and LED 2 | LED Engin Osram | LZ4-00B208 | High Power LEDs – Single Colour Blue, 460 nm 130 lm, 700mA |
LED 3 | Thorlabs | M625L3 | 625 nm, 700 mW (Min) Mounted LED, 1000 mA |
Lenses | LED Engin Osram | LLNF-2T06-H | LED Lighting Lenses Assemblies LZ4 LENS NARROW FLOOD BEAM |
Photodiode for power meter | Thorlabs | S120VC | Standard Photodiode Power Sensor |
Power Meter | Thorlabs | PM100D | Compact Power and Energy Meter |
Red Filter | Semrock | FF02-628/40-25 | BrightLine® single-band bandpass filter |