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, causin....

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 o....

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