Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Summary

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Abstract

Since its invention in the late 19^th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly—or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. Förster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Introduction

The Langendorff ex vivo heart perfusion system continues to be a relevant tool for performing a broad spectrum of physiological, biochemical, morphological, and pharmacological studies in centrally denervated hearts^1,2,3,4,5 since its invention in the late 19^th century⁶. To date, this system is still widely used for various topics (e.g., ischemia reperfusion) or to study cardiac pharmacological effects^7,

Protocol

All procedures involving animals were approved by the local authorities of the State of Hamburg, the University of Hamburg Animal Care and Use Committees.

1. Preparation of the Langendorff Apparatus

NOTE: A commercially available Langendorff perfusion system is used.

1. Prepare a modified Krebs-Henseleit solution (119 mM of sodium chloride, 25 mM of sodium bicarbonate, 4.6 mM of potassium chloride, 1.2 mM of potassium phosphate monobasic, 1.1 mM of magnesium sulfate, 2.5 mM of calcium chloride, 8.3 mM of glucose, and 2 mM of sodium pyruvate). Add a mixture of 95% oxygen/5% carbon dioxide to the perfusion....

Results

Figure 1 shows an image of the Langendorff setup including 2 multi-electrode arrays (MEAs). Before the experiment, the intracardiac catheter is positioned close to the cannula to facilitate a quick and easy insertion in the right atrium/right ventricle and to ensure a short time period until the equilibration can start. The lower part of the chamber can be heightened (see the arrows in Figure 1) so that the chamber is fully close.......

Discussion

In this manuscript, the well-known Langendorff ex vivo heart perfusion system is presented as a tool to study the impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis by using different mapping and stimulation techniques including endocardial and epicardial approaches.

Several parts of the protocol are crucial for the setup. First, it is important to use a preparation technique in which the atrial fat pads stay intact or are removed quickly without injuring.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium chloride	Sigma-Aldrich	S3014	Modified Krebs-Henleit solution
Sodium hydrogencarbonate	Sigma-Aldrich	401676	Modified Krebs-Henleit solution
Potassium chloride	Sigma-Aldrich	P5405	Modified Krebs-Henleit solution
Potassium phosphate monobasic	Sigma-Aldrich	P5655	Modified Krebs-Henleit solution
Magnesium sulfate heptahydrate	Sigma-Aldrich	M1880	Modified Krebs-Henleit solution
Calcium chloride dihydrate	Sigma-Aldrich	C7902	Modified Krebs-Henleit solution
Glucose	Sigma-Aldrich	G5767	Modified Krebs-Henleit solution
Sodium pyruvate bioXtra	Sigma-Aldrich	P8574	Modified Krebs-Henleit solution
Carbogen (95% O2 / 5% CO2)	SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany	Modified Krebs-Henleit solution
Sterile filter steritop-GP 0.22	EMD Millipore	SCGPT05RE	Modified Krebs-Henleit solution
Atropine sulfate	Sigma-Aldrich	A0257	Neuromodulation
Hexamethonium chloride	Sigma-Aldrich	H2138	Neuromodulation
Nicotine free base 98-100%	Sigma-Aldrich	N3876	Neuromodulation
Formalin solution neutral buffered 10%	Sigma-Aldrich	HT501128	Whole mount staining
Tris(hydroxymethyl)aminomethane	Sigma-Aldrich	252859	Whole mount staining
Methanol	Sigma-Aldrich	34860	Whole mount staining
Hydrogen peroxide solution 30% (w/w) in H2O	Merck, KGA, Darmstadt, Germany	H1009	Whole mount staining
Dimethyl sulfoxide	Merck, KGA, Darmstadt, Germany	D8418	Whole mount staining
Phosphate-buffered saline tablets	Gibco / Invitrogen	18912-014	Whole mount staining
Triton-x-100	Sigma-Aldrich	T8787	Whole mount staining
Albumin bovine fraction V	Biomol, Hamburg, Germany	11924.03	Whole mount staining
Chicken anti neurofilament	EMD Millipore	AB5539	Whole mount staining
Rabbit anti tyrosine hydroxylase	EMD Millipore	AB152	Whole mount staining
Goat anti choline acetyltransferase	EMD Millipore	AP144P	Whole mount staining
Donkey α rabbit IgG Alexa 488	Thermo Fisher Scientific	A21206	Whole mount staining
Donkey α goat IgG Alexa 568	Thermo Fisher Scientific	A11057	Whole mount staining
Donkey α chicken IgY Alexa 647	Merck, KGA, Darmstadt, Germany	AP194SA6	Whole mount staining
Biotin-conjugated donkey α rabbit igG	R&D Systems	AP182B	Whole mount staining
Biotin-conjugated donkey α goat igG	R&D Systems	AP192P	Whole mount staining
Biotin-conjugated goat α chicken igY	R&D Systems	BAD010	Whole mount staining
Vectashield mounting medium	Vector laboratories, Burlingame, CA, USA	H-1000	Immunohistochemistry
Vectastain ABC kit	Vector laboratories, Burlingame, CA, USA	PK-4000	Immunohistochemistry
Steady DAB/Plus	Abcam plc, Cambridge, UK	ab103723	Whole mount staining
HistoClear	DiaTec, Bamberg, Germany	HS2002	Immunohistochemistry
BisBenzimide H33342 trihydrochloride (Hoechst)	Sigma-Aldrich, St. Louis, MO, USA	B2261	Immunohistochemistry
Vectashield HardSet mounting medium	Vector laboratories, Burlingame, CA, USA	VEC-H-1400	Immunohistochemistry
Perfusion system	HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH, March-Hugstetten, Germany	73-4343	Langendorff apparatus
Data acquisition system and corresponding software for catheter and physiological parameter	Powerlab 8/30 & Labchart, ADInstruments, Dunedin, New Zealand	PL3508 PowerLab 8/35	Langendorff setup
Octapolar catheter	CIB’ER Mouse, NuMed Inc., Hopkinton, NY, USA	custom	Langendorff setup
Stimulus generator	STG4002, Multi Channel Systems, Reutlingen, Germany	STG4002-160µA	Stimulation setup
Stimulation software	Multi Channel Systems, Reutlingen, Germany	MC_Stimulus II	Stimulation setup
Data acquisition system and corresponding software for epicardial electrograms	ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany	USB-ME128-System	MEA setup
Multi-electrode array	MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany	EcoFlexMEA36	MEA setup
Multi-electrode array recording software	Multi Channel Systems, Reutlingen, Germany	MC_Rack	MEA setup
Spring scissors	Fine Science Tools GmbH, Heidelberg, Germany	15003-08	Heart Preparation
Strabismus Scissors	Fine Science Tools GmbH, Heidelberg, Germany	14575-09	Heart Preparation
Mayo Scissors	Fine Science Tools GmbH, Heidelberg, Germany	14110-15	Heart Preparation
Dumont SS Forceps	Fine Science Tools GmbH, Heidelberg, Germany	11203-25	Heart Preparation
London Forceps	Fine Science Tools GmbH, Heidelberg, Germany	11080-02	Heart Preparation
Narrow Pattern Forceps	Fine Science Tools GmbH, Heidelberg, Germany	11003-13	Heart Preparation
Plastic Wrap	Parafilm M, Bemis NA, based in Neenah, WI, United States		Consumable Materials
Stereomicroscope	Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany		FRET
LED	CoolLED, Andover, UK	pE-100	FRET
DualView	Photometrics, Tucson, AZ, USA	DV2-SYS	FRET
DualView filter set	Photometrics, Tucson, AZ, USA	05-EM	FRET
optiMOS scientific CMOS camera	Qimaging, Surrey, BC, Canada	01-OPTIMOS-R-M-16-C	FRET
Imaging software	Micro-Manager; Vale Lab, University of California San Francisco, CA, USA		FRET
Analysis Software	Image J software; Public Domain, NIH, USA		FRET