Name: Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System (Video) | JoVE Journal
Uploaded: 2018-05-22T14:00:00.000+00:00
Duration: 6 min 40 s
Description: Learn to analyze and modulate the intracardiac autonomic nervous system using an ex vivo Langendorff setup. This method assesses electrophysiology, arrhythmogenesis, and cAMP dynamics in decentralized hearts.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors would like to thank Hartwig Wieboldt for his excellent technical assistance, and the UKE Microscopy Imaging Facility (Umif) of the University Medical Center Hamburg-Eppendorf for providing microscopes and support. This research was funded bythe F&#246;rderverein des Universit&#228;ren Herzzentrums Hamburg e.V. and by the DZHK (German Centre for Cardiovascular Research) [FKZ 81Z4710141].

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.
<ol>
	<li>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 solution to prevent calcium precipitation. Filter the buffer with a pore size of 0.22 &#181;m.</li>
	<li>Add a pharmacological agent to the buffer to investigate its effect on cardiac electrophysiology and arrhythmogenesis as needed.</li>
	<li>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 &#730;C.</li>
	<li>Start the roller pump and fill the apparatus with the perfusion solution as soon as the correct temperature is reached.</li>
	<li>Adjust the pump rate before attaching the heart, so that no air bubbles are left in the cannula when mounting it to the apparatus.</li>
</ol>
2. Hard- and Software Preparation
<ol>
	<li>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.
	<ol>
		<li>Set the targeted perfusion pressure in the general settings to 80 mmHg.</li>
		<li>Start recording.</li>
	</ol>
	</li>
	<li>Use an electrophysiology catheter (Table of Materials) with platinum electrodes, an electrode surface of 0.5 x 0.5 mm, and an electrode spacing of 0.5 mm for the data recording and stimulation with a designated digital stimulus generator.
	<ol>
		<li>Place the catheter close to the area where the heart will be positioned after the attachment to the apparatus.</li>
		<li>Prepare the stimulation by selecting 2 electrodes from the catheter and use a cycle length of 100 ms.</li>
	</ol>
	</li>
</ol>
3. Preparation of the Heart
<ol>
	<li>Add the cold (~2 - 4 &#730;C) perfusion buffer (10 - 20 mL and 40 - 50 mL, so that the whole dish is covered) to the 2 Petri dishes (diameter of 6 cm and 10 cm) and the dish with the cannula and place them on ice directly next and under the microscope. Prepare a double knot around the cannula.</li>
	<li>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.</li>
	<li>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 by using Strabismus scissors and London forceps. Then place the heart under the microscope and carefully remove the thymus, the esophagus and trachea by using Spring scissors and Dumont SS forceps.</li>
	<li>Use the Spring scissors to cut a hole of 1.5 - 2 mm in the upper part of the right atrium for the insertion of the catheter. Cut a hole in the pulmonary artery. Then cut the aorta directly under the supraaortic branches and remove the tissue from the aorta, so that the knot can be attached easily.</li>
	<li>Keep the atrial fat pads, including the major atrially located ganglionated plexi, around the left atrium intact or completely remove them by careful dissection.</li>
	<li>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.</li>
	<li>Attach the cannula quickly to the Langendorff apparatus. Make sure that there are no bubbles left in the cannula.</li>
	<li>Switch the perfusion pressure to 80 mmHg, allowing a constant pressure perfusion.</li>
	<li>Insert the catheter carefully into the right atrium and right ventricle without touching or damaging the heart and attach the catheter to the cannula with tape.</li>
	<li>Start the stimulation with the prepared cycle length of 100 ms for an initial 20 min equilibration period.</li>
	<li>Close the chamber to allow a stable temperature.</li>
</ol>
4. Electrophysiological Parameters and Arrhythmogenesis
<ol>
	<li>Apply a programmed stimulation via the distal or proximal electrodes of the catheter at twice the atrial or ventricular pacing threshold to evaluate the electrophysiological parameters as described in the following steps.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 response10,11.</li>
	</ol>
	</li>
	<li>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 arrhythmogenesis10,11,12.</li>
</ol>
5. Epicardial Conduction Measurements
NOTE: Record unipolar epicardial electrograms by using a 128-channel, computer-assisted recording system with a sampling rate of 25 kHz for high-resolution mapping. Use a 32 multi-electrode array (MEA; inter-electrode distance: 300 &#181;m; 1.8 x 1.8 mm). Note that the data were bandpass filtered (50 Hz) and digitized with 12 bit and a signal range of 20 mV.
<ol>
	<li>Place the MEA in the designated area of the heart and add the grounding to another part of the heart13,14,15.</li>
	<li>Place an epicardial stimulation catheter close to the MEA and start a constant stimulation.</li>
	<li>Start recording after the confirmation of good contact of the electrodes by checking the signal quality and amplitude.</li>
	<li>Use an offline analysis for the determination of wave propagation velocity and dispersion in the conduction direction.</li>
</ol>
6. F&#246;rster Resonance Energy Transfer (FRET)-based Cyclic Adenosine Monophosphate (cAMP) Imaging
NOTE: For FRET-based measurements, harvest hearts from CAG-Epac1-camps transgenic mice16.
<ol>
	<li>Use a self-built imaging system around a stereomicroscope15,17.</li>
	<li>Place the stereomicroscope in front of the heart and adjust it for acuity.</li>
	<li>Excite the cAMP sensor with a light source [e.g., use a single-wavelength light emitting diode (440 nm)]. Split the emission light into donor and acceptor channels using a beam-splitter (for a cyan fluorescent protein/yellow fluorescent protein pair, use a 565dcxr dichroic mirror and D480/30 and D535/40 emission filters).</li>
	<li>Ensure a stable temperature by putting a plastic wrap around the chamber.</li>
	<li>Take images by using a scientific complementary metal-oxide-semiconductor (sCMOS) camera. Coordinate the light source and camera image capturing with an open source imaging software such as Micro-Manager.</li>
	<li>To start the image acquisition, push the Multi-D Acq. button and set up a time-lapse, which acquires an image every 10 s, with the appropriate exposure time, which depends on the strength of the fluorescent signal (around 100 ms).</li>
	<li>Use the previously described and available FRET online and FRET online 2 plugins17 to split the image into two channels, select the regions of interest, and monitor the ratio tracing.</li>
	<li>During the acquisition, perfuse the heart with the modified Krebs-Henseleit solution containing different substances, depending on the nature of the experiment.</li>
	<li>At the end of the experiment, switch off the acquisition by pushing the Stop button and save the stack of images.</li>
	<li>Analyze the FRET data offline using a dedicated analysis software that can split images into two identical sections for the donor and acceptor channels and can perform FRET analysis in multiple regions-of-interest. 
	NOTE: A dedicated plug-in (FRET offline) is needed, which is provided in Sprenger et al.17.
	<ol>
		<li>Start the software. Open the analysis by going to the Plugins menu, then click on MicroManager, and then on Open Micro-Manager File.</li>
		<li>Run the FRET offline plugin in order to split the time-lapse into two identical images for the CFP and YFP channels.</li>
		<li>Use the Freehand Selections tool to mark the region of interest in the YFP image. Press the Add button to add the selection to the Multi Measure window.</li>
		<li>Choose the region of interest in the Multi Measure window and press Multi to obtain a table with mean grey values for each frame and region. Copy and paste all the data into an Excel sheet.</li>
		<li>Click on the CFP Image Stack. Perform the same actions as in step 6.10.4 for the CFP stack and paste the mean grey values into the same Excel sheet.</li>
	</ol>
	</li>
	<li>Correct the raw data offline for the bleed-through factor of the donor into the acceptor channel17.
	<ol>
		<li>Use the following formula, where B is the bleed-through factor: 
		Ratio = (YFP - B x CFP) / CFP</li>
		<li>Determine the bleed-through factor B by imaging a heart expressing CFP only and measure the percentage of the donor fluorescence in the YFP channel (B = YFP / CFP).</li>
	</ol>
	</li>
</ol>
7. Neuromorphology
NOTE: Analyze the intracardiac autonomic nervous system by using whole-mount immunostainings of intact murine hearts. Note that the majority of intracardiac ganglia are localized in the epicardial adipose tissue close to the pulmonary veins.
<ol>
	<li>Use different stainings for the depiction of neurofilament (general neuronal structures; chicken anti-NF-H, 1:3,000), tyrosine hydroxylase (TH, sympathetic neuronal structures; rabbit &#945; TH, 1:1,000), and choline acetyltransferase (ChAT, parasympathetic neuronal structures; goat &#945; ChAT, 1:50).</li>
	<li>After the perfusion in the Langendorff apparatus, fix the mouse hearts in 10 mL of formalin for 24 h and store them in phosphate-buffered saline (PBS) at 4 &#730;C.</li>
	<li>Bleach the hearts in Dent&#39;s bleach (4:1:1 methanol: hydrogen peroxide solution 30% (w/w) in H2O: dimethyl sulfoxide (DMSO)) for 1 week at 4 &#730;C and rehydrate them subsequently to PBS in a series of descending methanol in PBS (100%, 75%, 50%, 25%; 1 h each)18.</li>
	<li>Perform the following incubations in a 24-well-plate format with a gentle agitation at 4 &#730;C:
	<ol>
		<li>Permeabilize the hearts in 1% Triton-X-100/PBS (PBS-T) for 3x 1 h at room temperature before blocking them overnight in a blocking buffer [5% bovine serum albumin (BSA) / PBS-T + 0.2% sodium azide].</li>
		<li>Dilute the antibodies as follows: goat &#945; ChAT (1:50), rabbit &#945; TH (1:1,000), chicken &#945; neurofilament (1:3,000); secondary antibodies for fluorescent labelling (1:500; or according to the manufacturer&#39;s instructions); biotinylated secondary antibodies for chromogenic labelling (1:200; or according to the manufacturer&#39;s instructions).</li>
	</ol>
	</li>
	<li>Incubate the specimens in primary antibodies diluted in a blocking buffer for 1 week at 4 &#730;C.</li>
	<li>Wash the hearts for 3 x 15 min in PBS-T before the secondary antibody incubation in a blocking buffer for 4 days.</li>
	<li>Wash the hearts for 3 x 15 min in PBS-T and store them in a mounting medium for fluorescent staining for 3 h at room temperature or use an avidin-biotin complex detection kit according to the manufacturer&#39;s instructions.</li>
	<li>Pre-incubate the hearts for 1 h in a commercial buffer for 3,3&#39;-diaminobenzidine (DAB), before developing them under a visual control in a DAB-containing buffer according to the manufacturer&#39;s instructions.</li>
	<li>Store the specimens in double distilled water.</li>
	<li>For paraffin sections, dehydrate and embed the hearts in paraffin.
	<ol>
		<li>Cut 4-&#181;m thick sections and deparaffinize them according to the laboratory&#39;s routine procedures. Whether and how antigen retrieval needs to be performed needs to be established for every individual setup since it depends on the antibody combination.</li>
		<li>Permeabilize the sections for 10 min in 0.2% Triton-X-100/Tris-buffered saline (TBS), followed by 3 x 5 min washes in TBS.</li>
		<li>Block them with 3% BSA/TBS for 1 h at room temperature.</li>
		<li>Incubate them overnight at 4 &#730;C [primary antibodies: goat &#945; ChAT (1:50), rabbit &#945; TH (1:500), chicken &#945; neurofilament (1:1,000)] or 2 h at room temperature (fluorescence-labelled secondary antibodies, 1:500) in 1% BSA/TBS with 3 x 5 min washes TBS in between.</li>
		<li>Add 1 &#181;g/mL of bisbenzimide H33342 trihydrochloride to the secondary antibody solution or use a different nuclear staining method.</li>
		<li>Mount the slides in a mounting medium for fluorescent staining.</li>
	</ol>
	</li>
</ol>

<ol><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrograde+heart+perfusion%3A+the+Langendorff+technique+of+isolated+heart+perfusion%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.</a> Journal of Molecular and Cellular Cardiology. 50 (6), 940-950 (2011).</li>
<li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolated+blood+and+perfusion+fluid+perfused+heart%5BTitle%5D)+AND+%22Pharmacological+Research%22%5BJournal%5D)">The isolated blood and perfusion fluid perfused heart.</a> Pharmacological Research. 41 (6), 613-627 (2000).</li>
<li>Hearse, D. J., Sutherland, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+models+for+the+study+of+cardiovascular+function+and+disease%5BTitle%5D)+AND+%22Pharmacological+Research%22%5BJournal%5D)">Experimental models for the study of cardiovascular function and disease.</a> Pharmacological Research. 41 (6), 597-603 (2000).</li>
<li>Valentin, J. P., Hoffmann, P., De Clerck, F., Hammond, T. G., Hondeghem, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+the+predictive+value+of+the+Langendorff+heart+model+%28Screenit+system%29+in+assessing+the+proarrhythmic+potential+of+drugs%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Review of the predictive value of the Langendorff heart model (Screenit system) in assessing the proarrhythmic potential of drugs.</a> Journal of Pharmacological and Toxicological Methods. 49 (3), 171-181 (2004).</li>
<li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+heart+perfusion+according+to+Langendorff-still+viable+in+the+new+millennium%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Isolated heart perfusion according to Langendorff-still viable in the new millennium.</a> Journal of Pharmacological and Toxicological Methods. 55 (2), 113-126 (2007).</li>
<li>Langendorff, O. Investigation of the living mammalian heart. Pflügers Archiv. 61, 291-332 (1895).</li>
<li>Matsumoto-Ida, M., Akao, M., Takeda, T., Kato, M., Kita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+2-photon+imaging+of+mitochondrial+function+in+perfused+rat+hearts+subjected+to+ischemia%2Freperfusion%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Real-time 2-photon imaging of mitochondrial function in perfused rat hearts subjected to ischemia/reperfusion.</a> Circulation. 114 (14), 1497-1503 (2006).</li>
<li>Rassaf, T., Totzeck, M., Hendgen-Cotta, U. B., Shiva, S., Heusch, G., Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+nitrite+contributes+to+cardioprotection+by+remote+ischemic+preconditioning%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Circulating nitrite contributes to cardioprotection by remote ischemic preconditioning.</a> Circulation Research. 114 (10), 1601-1610 (2014).</li>
<li>Schechter, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+isolated+working+heart+system+for+large+animal+models%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">An isolated working heart system for large animal models.</a> Journal of Visualized Experiments. 88 (88), 51671(2014).</li>
<li>Stockigt, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+beta-adrenoceptor+knockout+slows+conduction+and+reduces+inducible+arrhythmias+in+the+mouse+heart%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Total beta-adrenoceptor knockout slows conduction and reduces inducible arrhythmias in the mouse heart.</a> PLoS One. 7 (11), e49203(2012).</li>
<li>Berul, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+phenotyping+in+genetically+engineered+mice%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">Electrophysiological phenotyping in genetically engineered mice.</a> Physiological Genomics. 13 (3), 207-216 (2003).</li>
<li>Curtis, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Lambeth+Conventions+%28II%29%3A+guidelines+for+the+study+of+animal+and+human+ventricular+and+supraventricular+arrhythmias%5BTitle%5D)+AND+%22Pharmacology+%26+Therapeutics%22%5BJournal%5D)">The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias.</a> Pharmacology & Therapeutics. 139 (2), 213-248 (2013).</li>
<li>Schrickel, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+heterogeneity+of+myocardial+conduction+and+severe+cardiac+electrical+instability+in+annexin+A7-deficient+mice%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Enhanced heterogeneity of myocardial conduction and severe cardiac electrical instability in annexin A7-deficient mice.</a> Cardiovascular Research. 76 (2), 257-268 (2007).</li>
<li>Rudolph, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloperoxidase+acts+as+a+profibrotic+mediator+of+atrial+fibrillation%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation.</a> Nature Medicine. 16 (4), 470-474 (2010).</li>
<li>Jungen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+cardiac+cholinergic+neurons+enhances+susceptibility+to+ventricular+arrhythmias%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Disruption of cardiac cholinergic neurons enhances susceptibility to ventricular arrhythmias.</a> Nature Communications. 8, 14155(2017).</li>
<li>Calebiro, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+cAMP-signals+triggered+by+internalized+G-protein-coupled+receptors%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.</a> PLoS Biology. 7 (8), e1000172(2009).</li>
<li>Sprenger, J. U., Perera, R. K., Götz, K. R., Nikolaev, V. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FRET+microscopy+for+real-time+monitoring+of+signaling+events+in+live+cells+using+unimolecular+biosensors%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors.</a> Journal of Visualized Experiments. (66), e4081(2012).</li>
<li>Alanentalo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nature Methods. 4 (1), 31-33 (2007).</li>
<li>Whittington, N. C., Wray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+red+blood+cell+autofluorescence+for+immunocytochemistry+on+fixed+embryonic+mouse+tissue%5BTitle%5D)+AND+%22Current+Protocols+in+Neuroscience%22%5BJournal%5D)">Suppression of red blood cell autofluorescence for immunocytochemistry on fixed embryonic mouse tissue.</a> Current Protocols in Neuroscience. 81, 2.28.1-2.28.12 (2017).</li>
<li>Fukuda, K., Kanazawa, H., Aizawa, Y., Ardell, J. L., Shivkumar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+innervation+and+sudden+cardiac+death%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Cardiac innervation and sudden cardiac death.</a> Circulation Research. 116 (12), 2005-2019 (2015).</li>
<li>Wengrowski, A. M., Wang, X., Tapa, S., Posnack, N. G., Mendelowitz, D., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+release+of+norepinephrine+from+cardiac+sympathetic+neurons+alters+mechanical+and+electrical+function%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Optogenetic release of norepinephrine from cardiac sympathetic neurons alters mechanical and electrical function.</a> Cardiovascular Research. 105 (2), 143-150 (2015).</li>
<li>Rivinius, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+cardiac+chronotropic+function+in+patients+after+heart+transplantation%3A+effects+of+ivabradine+and+metoprolol+succinate+on+resting+heart+rate+in+the+denervated+heart%5BTitle%5D)+AND+%22Clinical+Research+in+Cardiology%22%5BJournal%5D)">Control of cardiac chronotropic function in patients after heart transplantation: effects of ivabradine and metoprolol succinate on resting heart rate in the denervated heart.</a> Clinical Research in Cardiology. , (2017).</li>
<li>Ajijola, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Augmentation+of+cardiac+sympathetic+tone+by+percutaneous+low-level+stellate+ganglion+stimulation+in+humans%3A+a+feasibility+study%5BTitle%5D)+AND+%22Physiological+Reports%22%5BJournal%5D)">Augmentation of cardiac sympathetic tone by percutaneous low-level stellate ganglion stimulation in humans: a feasibility study.</a> Physiological Reports. 3 (3), e12328(2015).</li>
</ol>

The authors have nothing to disclose.

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...

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 hearts1,2,3,4,5 since its invention in the late 19th century6. To date, this system is still widely used for various topics (e.g., ischemia reperfusion) or to study cardiac pharmacological effects7,8, and is a basic tool in cardiovascular research. The longevity of this method results from several advantages (e.g., measurements are performed without the influence of the central nervous system or other organs, systemic circulation, or circulating hormones). If needed, pharmaceuticals can be added in a controlled manner to the perfusion buffer or applied to specific structures directly. Experiments are reproducible, and a relatively high number of experiments can be performed in a short period of time. The (in part) open nature of the setup can make temperature regulation difficult and needs to be taken into account. Although the Langendorff system is also used in larger species9, smaller animals are primarily used as the experimental setup is less complex, and a greater biological variability (e.g., transgenic mouse models) can be used.
In the experimental setup of this protocol, the influence of the intracardiac autonomic nervous system on basic electrophysiological parameters, ventricular arrhythmogenesis, epicardial conduction, and cyclic adenosine monophosphate (cAMP) dynamics is evaluated. A large number of intracardiac ganglia, which are mainly located in the atrial fat pads and are now well known to control cardiac electrophysiology independent from central neural control, are either left intact or manually removed with careful mechanical dissection. A pharmacological modulation of the autonomic nervous system is performed either globally by adding pharmaceuticals to the perfusion buffer or locally by targeted modulation of the atrial ganglia. After the experiments, the hearts are well suited for an immunohistological assessment as all blood cells have been removed due to the continuous perfusion, which can increase the quality of staining.
The overall goal of the described techniques is to offer novel perspectives for detailed studies regarding the impact of the autonomic nervous system on cardiac electrophysiology and arrhythmogenesis in the mouse heart. A reason to use this technique is that it is possible to study and alter the autonomic nervous system without the impact of the central nervous system. One major advantage is the easy employment of pharmacological experiments, in which potential pro- or antiarrhythmic properties of old and new agents can be tested. In addition, transgenic and knockout mouse models of various cardiac diseases are available to investigate the mechanisms underlying arrhythmias, heart failure, or metabolic diseases. This approach has enhanced our understanding of how the autonomic nervous system on the atrial level can impact ventricular cardiac electrophysiology and the induction of arrhythmias.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Sodium hydrogencarbonate</td><td>Sigma-Aldrich</td><td>401676</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>M1880</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Sodium pyruvate bioXtra</td><td>Sigma-Aldrich</td><td>P8574</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Carbogen (95% O&lt;sub&gt;2&lt;/sub&gt; / 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany</td><td>Modified Krebs-Henleit solution</td><td></td></tr><tr><td>Sterile filter steritop-GP 0.22</td><td>EMD Millipore</td><td>SCGPT05RE</td><td>Modified Krebs-Henleit solution</td></tr><tr><td>Atropine sulfate</td><td>Sigma-Aldrich</td><td>A0257</td><td>Neuromodulation</td></tr><tr><td>Hexamethonium chloride</td><td>Sigma-Aldrich</td><td>H2138</td><td>Neuromodulation</td></tr><tr><td>Nicotine free base 98-100%</td><td>Sigma-Aldrich</td><td>N3876</td><td>Neuromodulation</td></tr><tr><td>Formalin solution neutral buffered 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Whole mount staining</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859</td><td>Whole mount staining</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Whole mount staining</td></tr><tr><td>Hydrogen peroxide solution 30% (w/w) in H2O</td><td>Merck, KGA, Darmstadt, Germany</td><td>H1009</td><td>Whole mount staining</td></tr><tr><td>Dimethyl sulfoxide</td><td>Merck, KGA, Darmstadt, Germany</td><td>D8418</td><td>Whole mount staining</td></tr><tr><td>Phosphate-buffered saline tablets</td><td>Gibco / Invitrogen</td><td>18912-014</td><td>Whole mount staining</td></tr><tr><td>Triton-x-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Whole mount staining</td></tr><tr><td>Albumin bovine fraction V</td><td>Biomol, Hamburg, Germany</td><td>11924.03</td><td>Whole mount staining</td></tr><tr><td>Chicken anti neurofilament</td><td>EMD Millipore</td><td>AB5539</td><td>Whole mount staining</td></tr><tr><td>Rabbit anti tyrosine hydroxylase</td><td>EMD Millipore</td><td>AB152</td><td>Whole mount staining</td></tr><tr><td>Goat anti choline acetyltransferase</td><td>EMD Millipore</td><td>AP144P</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; rabbit IgG Alexa 488</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; goat IgG Alexa 568</td><td>Thermo Fisher Scientific</td><td>A11057</td><td>Whole mount staining</td></tr><tr><td>Donkey &amp;alpha; chicken IgY Alexa 647</td><td>Merck, KGA, Darmstadt, Germany</td><td>AP194SA6</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated donkey &amp;alpha; rabbit igG</td><td>R&amp;amp;D Systems</td><td>AP182B</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated donkey &amp;alpha; goat igG</td><td>R&amp;amp;D Systems</td><td>AP192P</td><td>Whole mount staining</td></tr><tr><td>Biotin-conjugated goat &amp;alpha; chicken igY</td><td>R&amp;amp;D Systems</td><td>BAD010</td><td>Whole mount staining</td></tr><tr><td>Vectashield mounting medium</td><td>Vector laboratories, Burlingame, CA, USA</td><td>H-1000</td><td>Immunohistochemistry</td></tr><tr><td>Vectastain ABC kit</td><td>Vector laboratories, Burlingame, CA, USA</td><td>PK-4000</td><td>Immunohistochemistry</td></tr><tr><td>Steady DAB/Plus</td><td>Abcam plc, Cambridge, UK</td><td>ab103723</td><td>Whole mount staining</td></tr><tr><td>HistoClear</td><td>DiaTec, Bamberg, Germany</td><td>HS2002</td><td>Immunohistochemistry</td></tr><tr><td>BisBenzimide H33342 trihydrochloride (Hoechst)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B2261</td><td>Immunohistochemistry</td></tr><tr><td>Vectashield HardSet mounting medium</td><td>Vector laboratories, Burlingame, CA, USA</td><td>VEC-H-1400</td><td>Immunohistochemistry</td></tr><tr><td>Perfusion system</td><td>HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH, March-Hugstetten, Germany</td><td>&amp;nbsp;73-4343</td><td>Langendorff apparatus</td></tr><tr><td>Data acquisition system and corresponding software for catheter and physiological parameter</td><td>Powerlab 8/30 &amp;amp; Labchart, ADInstruments, Dunedin, New Zealand</td><td>PL3508 PowerLab 8/35</td><td>Langendorff setup</td></tr><tr><td>Octapolar catheter</td><td>CIB&amp;rsquo;ER Mouse, NuMed Inc., Hopkinton, NY, USA</td><td>custom</td><td>Langendorff setup</td></tr><tr><td>Stimulus generator</td><td>STG4002, Multi Channel Systems, Reutlingen, Germany</td><td>STG4002-160&amp;micro;A</td><td>Stimulation setup</td></tr><tr><td>Stimulation software</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>MC_Stimulus II</td><td>Stimulation setup</td></tr><tr><td>Data acquisition system and corresponding software for epicardial electrograms</td><td>ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany</td><td>USB-ME128-System</td><td>MEA setup</td></tr><tr><td>Multi-electrode array</td><td>MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany</td><td>EcoFlexMEA36</td><td>MEA setup</td></tr><tr><td>Multi-electrode array recording software</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>MC_Rack</td><td>MEA setup</td></tr><tr><td>Spring scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>15003-08</td><td>Heart Preparation</td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>14575-09</td><td>Heart Preparation</td></tr><tr><td>Mayo Scissors</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>14110-15</td><td>Heart Preparation</td></tr><tr><td>Dumont SS Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11203-25</td><td>Heart Preparation</td></tr><tr><td>London Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11080-02</td><td>Heart Preparation</td></tr><tr><td>Narrow Pattern Forceps</td><td>Fine Science Tools GmbH, Heidelberg, Germany</td><td>11003-13</td><td>Heart Preparation</td></tr><tr><td>Plastic Wrap</td><td>Parafilm M, Bemis NA, based in Neenah, WI, United States</td><td></td><td>Consumable Materials</td></tr><tr><td>Stereomicroscope</td><td>Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany</td><td></td><td>FRET</td></tr><tr><td>LED</td><td>CoolLED, Andover, UK</td><td>&lt;em&gt;p&lt;/em&gt;E-100</td><td>FRET</td></tr><tr><td>DualView</td><td>Photometrics, Tucson, AZ, USA</td><td>DV2-SYS</td><td>FRET</td></tr><tr><td>DualView filter set</td><td>Photometrics, Tucson, AZ, USA</td><td>05-EM</td><td>FRET</td></tr><tr><td>optiMOS scientific CMOS camera</td><td>Qimaging, Surrey, BC, Canada</td><td>01-OPTIMOS-R-M-16-C</td><td>FRET</td></tr><tr><td>Imaging software &amp;nbsp;</td><td>Micro-Manager; Vale Lab, University of California San Francisco, CA, USA</td><td></td><td>FRET</td></tr><tr><td>Analysis Software</td><td>Image J software; Public Domain, NIH, USA</td><td></td><td>FRET</td></tr></tbody></table>

impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system

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

Since its invention in the late 19th 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&#8212;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. F&#246;rster 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.

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...

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.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

impact-intracardiac-neurons-on-cardiac-electrophysiology

Research

JoVE Journal

Medicine

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.

Stimulation of the Intracardiac Nervous System to Assess Susceptibility to Arrhythmia

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart.  A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds' mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology

This protocol illustrates a method for optogenetic stimulation of intrinsic cardiac neurons in transgenic mouse hearts. The approach described is used for investigating the kinetics of sudden cardiac neuron activation in ex vivo perfused hearts and the interactions between cholinergic and catecholaminergic activity.

Optogenetic Activation of Intrinsic Cardiac Autonomic Neurons in Excised Perfused Mouse Hearts

We describe a protocol for spinal multichannel extracellular recording alongside cardiac function recording and analyzing the cardiac-locked spinal dorsal horn neurons. This method offers a temporally synchronized framework for studying spinal mechanisms underlying thoracic visceral functional changes induced by acupuncture.

Studying the Coding Profiles of Somatic Stimulation on Cardiac-locked Neuronal Responses in the Rat Spinal Dorsal Horn

This protocol outlines advanced material fabrication and ex vivo rat heart methods for optical and electrical bidirectional biointerfacing, enabling precise cardiac stimulation, recording, and infarction modeling for bioelectronics research.

Bidirectional Electrical and Optoelectronic Interfaces in Healthy and Ischemic Ex Vivo Rat Hearts

Learn to analyze and modulate the intracardiac autonomic nervous system using an ex vivo Langendorff setup. This method assesses electrophysiology, arrhythmogenesis, and cAMP dynamics in decentralized hearts.

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

Explore More Videos