Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Summary

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.

Abstract

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.

Introduction

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 myocardium^2,3,4. 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 manipulation^5,6,7, 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 others^{8,9,10,11,12,13,14} 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 in^16,17 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.

Protocol

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

1. Optical mapping setup
   NOTE: The optical setup, as well as the electrical setup, are shown in Figure 1. All components used in the optical and electrical setup are listed in detail in the Table of Materials.
   1. Use LED 1 and LED 2 for induction of arrhythmia and backup defibrillation. Choose high power LEDs with a wavelength λ_blue near 475 nm, which is the peak of the excitation wavelength of ChR2⁶. To further narrow the optical spectrum, use a 470 ± 20 nm bandpass filter.
      NOTE: In this work, LED 1 and LED 2 have a typical radiant flux of 3.9 to 5.3 W, according to the datasheet²⁰.
   2. Illuminate the epicardium for optical mapping with a high-power red LED (LED 3 in Figure 1), which emits light with a center wavelength of λ_red = 625 nm and a radiant flux of 700 mW²¹. The red light is filtered with a 628 ± 20 nm bandpass filter and reflected by a long pass dichroic mirror (DM) with a cutoff wavelength of λ_DM = 685 nm.
   3. Use an emission filter with λ_filter-cam = 775 ± 70 nm in front of the camera objective to only record the fluorescence emission of the cardiac activity. Use a fast objective that is well suited for low light applications.
      NOTE: The frequency of fibrillation of a mouse heart ranges from 20 to 35 Hz; therefore, use a fast enough camera to record with a frequency of 1 to 2 kHz, or even higher.
2. Micro-LED array
   NOTE: The micro-LED arrays applied here are realized using microsystems processing as further detailed elsewhere^16,17.
   1. Spin coat a 5 µm-thick polyimide (PI) layer onto 4-inch silicon substrates (single-side polished, 525-µm thick).
   2. Cure this PI layer at a maximum temperature of 450 °C under a nitrogen atmosphere. Keep the maximum temperature constant for 10 min.
   3. Deposit and pattern an image reversal photoresist (PR) using ultraviolet (UV) lithography and sputter deposit a 250-nm thin platinum layer (Pt).
   4. Thicken this Pt-based metallization by electroplating a 1 µm-thick gold (Au) layer with the patterned PR serving as a masking layer.
   5. Before spin-coating a second PI layer, expose the wafer with its first PI layer and the Au-electroplated metallization to an oxygen plasma that chemically activates the surface of the PI layer.
   6. Cure the second PI layer again at 450 °C, apply UV lithography to pattern a PR layer and open the contact pads of the array for the micro-LED chips and the interfacing printed circuit board (PCB) by reactive ion etching (RIE) using the patterned PR as a masking layer.
      NOTE: In this RIE process steps, it is recommended to apply 200 W and 100 W for 10 and 30 min, respectively, to define the contact pad openings as well as the outer shape of the two-dimensional (2D) micro-LED array.
   7. Strip the PR using solvents and plasma etching. Further thicken the contact pads by electroplating an additional 6 µm-thick gold layer.
   8. Attach the micro-LED chips to the contact pads using a flip-chip bonder.
   9. Activate the PI surface in an oxygen plasma and underfill the micro-LED chips with a solvent-free adhesive. Cure then the adhesive for 12 h at 120 °C.
   10. To encapsulate the micro-LED chips, perform another plasma treatment with Argon and apply a thin fluoropolymer layer manually. Pre-cure this layer at 80 °C for 1 h.
   11. Manually apply silicone as the final encapsulation layer after exposing the micro-LED array to an oxygen plasma, used to improve the silicon adhesion to the underlying fluoropolymer layer. Cure the silicone layer at 80 °C and 180 °C for 1 h each. These final curing steps also cure the fluoropolymer layer completely.
   12. Solder the contact pads of the PI substrate to a printed circuit board which carries strip connectors for interconnection of the array to an external instrumentation. Cover the solder pads on the PCB using an adhesive.
3. Electrical setup
   1. Use electrodes suited for recording an electrocardiogram (ECG), e.g., silver/silver-chloride electrodes or Monophasic Action Potential (MAP) electrodes and an ECG amplifier to monitor the electrical activity of the heart continuously. Furthermore, use an appropriate acquisition device (AD) to record all electrical signals obtained.
   2. Choose a well-suited driver for the high-power LEDs (LED 1, LED 2, and LED 3), which can manage the maximum current applied to each device. Use an arbitrary function generator (AFG) to control the output of the LED drivers accurately.
   3. Use a multi-channel LED driver to control the current flowing through the micro-LED array. An AFG with multiple outputs is as well suitable for this task.
      NOTE: It is advisable to choose LED drivers limiting the current to the maximum current of the micro-LED, otherwise the diodes might get damaged. One example of a multi-channel micro-LED driver is described in another work¹⁸. If necessary, the AFG or any other LED driver might be connected to a computer to remotely control the micro-LED settings. If this is the case connect the LED driver to the computer with the communication protocol of your choice, e.g., General Purpose Interface Bus (GPIB) or a serial connection.

   

2. Experimental procedures

1. Solution preparation
   1. Prepare Tyrode's solution: 130 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 24 mM NaHCO₃, 1.8 mM CaCl₂, 1.2 mM KH₂PO₄, 5.6 mM Glucose, 0.1% BSA/Albumin.
   2. Prepare Low-K⁺ Tyrode's solution: Low-K⁺ Tyrode's is made in the same way as regular Tyrode's solution except that only half the amount of KCl is added (2 mM instead of 4 mM KCl).
      ​NOTE: For an experiment lasting 3 h usually 2-3 L of Low-K⁺ Tyrode's (additionally mixed with Blebbistatin (Step 2.1.5) if optical mapping is performed) and 1-2 L of regular Tyrode's are sufficient.
   3. Add Pinacidil to the Low-K⁺ Tyrode's solution to ease the process of arrhythmia induction, as described in²², to obtain a 100 mM concentration. Wear protective laboratory gloves when handling Pinacidil.
   4. Prepare 1 mL of 50 µM DI-4-ANBDQPQ with regular Tyrode's solution. Protect the dye from light to prevent photobleaching.
   5. Make a 10 mM stock solution of Blebbistatin. For optical mapping, mix Blebbistatin with the 100 mM Pinacidil-Tyrode's-solution (Step 2.1.3) to obtain a 5 µM solution. Wear protective laboratory gloves when handling Blebbistatin.
      NOTE: Keep both the dye and the Blebbistatin solution aside until optical mapping begins.
2. Langendorff perfusion
   NOTE: The setup consists of two reservoirs for the two Tyrode's solutions. They are connected to a bubble trap via tubes with three-way cocks. The heart is later attached to the bubble trap by a Luer lock connector, and it is then suspended in a hexagonal water bath. The water bath is, in turn, connected to a waste container to collect the used Tyrode's solution.
   1. Clean all tubes before every experiment with fully demineralized water.
   2. Aerate both Tyrode's solutions with Carbogen (5% CO₂ and 95% O₂) for 30 min at room temperature before beginning of the experiment. Adjust the pH value of the Tyrode's solutions to 7.4 with NaOH.
   3. Fill 500 mL of each Tyrode's solution in the corresponding reservoir and de-aerate the tubes as well as the bubble trap by running Tyrode's solution through the perfusion system until no more trapped air bubbles are seen in the tubes or in the bubble trap.
   4. Continue aerating the Tyrode's solutions during the whole experiment in the reservoirs with Carbogen to ensure that the pH of the perfusate remains stable later during perfusion.
   5. Heat the perfusion system to 37 °C with a water heat pump. Keep the perfusate temperature constant within the water bath by using an additional heating element such as a waterproof heating cable.
      NOTE: During the experiment, it is crucial to refill the Tyrode's reservoirs before they run empty. Otherwise, air bubbles can enter the heart, which can clog the vessels and lead to ischemia.
3. Mouse Preparation
   1. Inject subcutaneously 0.1 mL of 500 I.E. Heparin 30 min before heart isolation procedure.
   2. Fill a 6 cm Petri dish and a 2 mL syringe with ice-cold Tyrode's solution. Place under the stereoscopic microscope.
   3. Perform short time anesthesia of mice by a saturated Isoflurane environment for 2 min and immediate cervical dislocation afterwards.
      ​NOTE: In order to verify sufficient anesthesia a check for the negative inter-toe reflex is absolutely necessary.
   4. Open the chest, remove the heart, as described elsewhere²³, and place it into the 6 cm Petri dish with ice-cold Tyrode's solution. Cardiac beating will be diminished due to temperature drop.
   5. Do the fine preparation under a stereoscopic microscope, as detailed elsewhere²³. Attach the aorta onto the blunt needle and fix the vessel with suture material.
   6. As a control, inject ice-cold Tyrode's solution through the needle into the heart and check that the heart is tightly mounted. This step also rinses the remaining blood out of the heart.
   7. Transfer the mounted heart to the perfusion system. Ensure that the perfusate is flowing to prevent air from entering the heart while connecting the needle with the bubble trap. Check that the heart is covered with Tyrode's solution in the water bath. Steps 2.3.4, 2.3.5, and 2.3.7 are illustrated in Figure 2.
   8. Ensure that the heart starts beating within a few minutes. Let the heart adapt to the perfusion setup for 15 to 20 min, then switch to low-K⁺ Tyrode's solution with Pinacidil (Step 2.1.3) respectively low-K⁺ Tyrode's solution with Pinacidil and Blebbistatin (Step 2.1.5) if optical mapping is to be performed.
4. Arrhythmia induction and optical defibrillation
   1. Place one of the ECG electrodes as close as possible to the heart surface to ensure good signal quality. Suspend the second ECG electrode in the Tyrode's solution. Make sure that the ECG acquired is being recorded by the AD of choice.
   2. Place the micro-LED array on the area of interest of the study, for example, onto the left ventricle.
   3. Change the perfusion to low-K⁺ Tyrode's with Pinacidil and perfuse the heart for 15 to 30 min.
   4. To induce arrhythmia, illuminate the heart with LED 1 and LED 2 with a train of 20 to 50 light pulses with a frequency f_ind of 25 to 35 Hz, pulse duration W_ind of 2 to 15 ms, and light intensity LI_{opt_ind} of 2.8 mW mm^-2.
   5. Repeat the process until arrhythmia is induced.
      NOTE: Arrhythmias are easy to identify in the ECG signal because the frequency and morphology of the signal differ from normal sinus rhythm. Should the arrhythmia terminate within the next 5 s, classify it as self-terminated, and start a new induction attempt.
   6. Once a sustained arrhythmia is visually detected, apply a burst of pulses with different widths W_def and frequencies f_def, using three, six, or nine micro-LEDs of the array at a pulsing current I_pulse of 15 mA yielding to a light intensity LI_µLED = 33.31 ± 2.05 mW mm^-2.
   7. Should the arrhythmia keep ongoing after five micro-LED array-based defibrillation trials, classify the attempt as unsuccessful and start backup defibrillation.
   8. For backup defibrillation, use LED 1 and LED 2 using the same timing parameters as set for the micro-LED array.
      ​NOTE: Because the heart is exposed to ischemic and metabolic stress over the whole experimental period, it is possible that termination attempts of arrhythmia are unsuccessful even with backup defibrillation. Whenever this happens, change the perfusion solution to the regular Tyrode's and let the heart recover for 5 to 10 min. When the ECG returns to sinus rhythm, repeat the protocol from Step 2.4.3 again.
5. Optical Mapping
   1. Perfuse the heart with the Blebbistatin solution prepared in Step 2.1.5 and wait until mechanical uncoupling occurs. This is accomplished when the heart stops beating, but an ECG signal is still measurable.
      NOTE: Mixing the Blebbistatin solution to the mentioned concentration and keeping the heart perfused with this solution maintains the cardiac mechanical activity uncoupled from the electrical activity during the whole experiment.
   2. Give the 1 mL voltage dye DI-4-ANBDQPQ (prepared in Step 2.1.4) as a bolus in the bubble trap of the Langendorff perfusion. Wait for 5 to 10 min to allow the dye to perfuse the heart uniformly.
      ​NOTE: Avoid photobleaching of the dye by turning off the red light whenever no recording is being made. If the signal-to-noise ratio of the recording becomes too small (acquired signal is too noisy), repeat steps 2.1.4 and 2.5.2.
   3. Focus the camera onto the heart surface, turn on LED 3, and apply 1.27 mW mm^-2 optical power.
   4. Turn off the laboratory lights and start recording. Ensure that an optical signal is being acquired by comparing the frequency of the obtained signal to the frequency of the recorded ECG. This ensures that the obtained optical signal is purely related to the electrical activity of the heart.
      NOTE: Since the fluorescence light emitted by the dye is very week, optical mapping is done in a dark room. This avoids signal interference from any other light sources.

Results

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 f_ind between 25 Hz and 35 Hz and a pulse duration W_ind 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....

Discussion

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 patients^28,29,30,31. Low energy ...

Materials

Name	Company	Catalog Number	Comments
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-Di02-25x36	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