Stimulation of the Intracardiac Nervous System to Assess Susceptibility to Arrhythmia

Abstract

Source: Jungen, C., et al. Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System. J. Vis. Exp. (2018).

The video demonstrates an ex vivo technique for stimulating the intracardiac nervous system to assess the heart’s susceptibility to arrhythmia. Using a perfusion apparatus, an isolated mouse heart is perfused with an oxygenated buffer. The intracardiac nervous system is then stimulated at a high frequency to induce arrhythmias.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

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). To prevent calcium precipitation, add a mixture of 95% oxygen/5% carbon dioxide to the perfusion solution. Filter the buffer with a pore size of 0.22 µm.
2. Add a pharmacological agent to the buffer to investigate its effect on cardiac electrophysiology and arrhythmogenesis as needed.
3. Start the water bath and place the perfusion solution including a mixture of 95% oxygen/5% carbon dioxide in it. Adjust the temperature of the water bath, so that the perfusion solution temperature directly before the cannula is ~37 ˚C.
4. As soon as the correct temperature is reached, start the roller pump and fill the apparatus with the perfusion solution.
5. Adjust the pump rate before attaching the heart, so that no air bubbles are left in the cannula when mounting it to the apparatus.

2. Hard- and Software Preparation

1. Connect a digital data acquisition system and its corresponding software to the Langendorff apparatus for a continuous recording of the perfusion pressure, flow rate, and heart rate.
   1. Set the targeted perfusion pressure in the general settings to 80 mmHg.
   2. Start recording.
2. For data recording and stimulation with a designated digital stimulus generator, use an electrophysiology catheter with platinum electrodes, an electrode surface of 0.5 x 0.5 mm, and an electrode spacing of 0.5 mm.
   1. Place the catheter close to the area where the heart will be positioned after the attachment to the apparatus.
   2. Prepare the stimulation by selecting 2 electrodes from the catheter and use a cycle length of 100 ms.

3. Preparation of the Heart

1. Add the cold (~2 - 4 ˚C) perfusion buffer (10 - 20 mL and 40 - 50 mL, so that the whole dish is covered) to the 2 Petri dishes (diameters of 6 cm and 10 cm) and the dish with the cannula, and place them on ice directly next to and under the microscope. Prepare a double knot around the cannula.
2. Rapidly excise the heart after cervical dislocation by opening the thorax using Mayo scissors and narrow pattern forceps. Then grip the aorta and vena cava above the diaphragm using the London forceps and excise the heart-lung block by cutting all vessels and connective tissue close to the spine with the Strabismus scissors.
3. Transfer the heart-lung block into the first dish (6 cm diameter) filled with the ice-cold buffer and carefully remove the lungs without damaging the heart using Strabismus scissors and London forceps. Then, place the heart under the microscope and carefully remove the thymus, esophagus, and trachea using Spring scissors and Dumont SS forceps.
4. Use the Spring scissors to cut a 1.5 - 2 mm hole in the upper part of the right atrium for the catheter's insertion. Cut a hole in the pulmonary artery. Then cut the aorta directly under the supra-aortic branches and remove the tissue so that the knot can be attached easily.
5. Keep the atrial fat pads, including the major atrially located ganglionated plexi, around the left atrium intact or completely remove them by careful dissection.
6. Transfer the heart to the dish with the cannula and place it under the microscope. Pull the aorta over the cannula with the Dumont SS forceps and tie the prepared knot tightly around the aorta. Make sure that the cannula is not placed too deep in the aorta so that the aortic valve and the coronary vessels are left free.
7. Attach the cannula quickly to the Langendorff apparatus. Make sure that there are no bubbles left in the cannula.
8. Switch the perfusion pressure to 80 mmHg, allowing a constant pressure perfusion.
9. Insert the catheter carefully into the right atrium and right ventricle without touching or damaging the heart, and attach it to the cannula with tape.
10. Start the stimulation with the prepared cycle length of 100 ms for an initial 20 min equilibration period.
11. Close the chamber to allow a stable temperature.

4. Electrophysiological Parameters and Arrhythmogenesis

1. Apply programmed stimulation via the catheter's distal or proximal electrodes at twice the atrial or ventricular pacing threshold to evaluate the electrophysiological parameters, as described in the following steps.
   1. Determine the sinus node recovery time as the maximum return cycle length after 10 s of fixed-rate pacing at an S1S1 cycle length of 120 ms, 100 ms, and 80 ms.
   2. Determine the Wenckebach point as the longest S1S1 cycle length (8 stimuli; S1S1: 100 ms; 2 ms stepwise reduction) with a loss of 11 AV nodal conduction. Determine the atrioventricular nodal refractory periods as the longest S1S2 (12 stimuli; S1S1: 120 ms, 110 ms, and 100ms; one short coupled extrastimulus with a 2 ms stepwise S1S2 reduction) with a loss of AV nodal conduction.
   3. Determine the atrial and ventricular refractory periods as the longest S1S2 (12 stimuli; S1S1: 120 ms, 110 ms, and 100ms; one short coupled extrastimulus with a 2 ms stepwise S1S2 reduction) with an absent atrial or ventricular response.
2. Perform a programmed extrastimulation (S1S1: 120 ms, 100 ms, and 80 ms, followed by up to 3 extra beats; 60 - 20 ms with a 2 ms stepwise reduction) or burst pacing protocols (5 s at S1S1: 50 - 10 ms with a 10 ms stepwise reduction) in line with the Lambeth Conventions to evaluate the ventricular arrhythmogenesis.

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% oxygen / 5% carbon dioxide)	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
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
Plastic Wrap	Parafilm M, Bemis NA, based in Neenah, WI, United States		Consumable Materials
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