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>

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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 myocardium. Second, a properly sized opening has to be cut in the right atrium for an easy insertion of the octapolar catheter into the right atrium and right ventricle. The catheter should slip easily into the right ventricle without generating any pressure. During the attachment of the catheter to the cannula, the catheter should not dip deeper into the ventricle, to avoid cardiac injury. Third, temperature control is a crucial part of all Langendorff setups1,2,5. The thermal chamber is closed during the arrhythmia testing, ensuring a stable temperature. But for MEA or FRET recordings, the chamber needs to be at least partly open to allow measurements. Either recording time should be kept to a minimum, or other techniques to reduce temperature loss, like putting a plastic wrap around the chamber during longer measurements, should be performed. Fourth, MEAs should be placed at the same anatomical locations in all experiments. Good surface contact, which is confirmed by large amplitudes in the real-time analysis, can be achieved by using two MEAs on opposite sites so that a counterbalance is produced. Fifth, FRET measurements are influenced by movement. To reduce spontaneous movement, the heart is paced at a stable frequency by the intracardiac catheter. For additional stabilization, a tube with a slight vacuum can stabilize the apex.
One advantage of the Langendorff system is that the hearts can be used subsequently for immunohistological assessments of the cardiac nervous system. The continuous perfusion removes most red blood cells which have a high level of autofluorescence19, improving the quality of staining. After formalin fixation, the hearts can be stored in a temperature controlled (4 &#730;C) environment in phosphate-buffered saline for up to a year without noticeable changes in staining quality.
The most important feature of this setup is that all measurements are performed in a centrally denervated heart. The predominantly parasympathetic atrial intracardiac ganglia are the last relay station within the heart20 as the sympathetic ganglion stellatum is located intrathoracically and is therefore removed during preparation. Although the intracardiac neurons get no central input, it has been shown that they are still active in a physiological way as the photoactivation of cardiac sympathetic nerves increases the heart rate and cardiac contractile force21. In line with these findings supporting the functional importance of intracardiac neurons in the centrally denervated heart, we recently demonstrated their impact on ventricular function and arrhythmogenesis15.
One advantage of this centrally denervated setup is that it allows the researcher to study the communication between different intracardiac regional neural networks (e.g., the interaction between atrium and ventricle)15. These differences might be relevant for patients after heart transplantation in whom treatment with the selective sinus node modulator ivabradine improves survival, compared to treatment with the beta-blocker metoprolol succinate22. In a future step, direct electrical stimulation of parasympathetic (vagus nerve) or sympathetic structures (Ggl. stellatum23) will help to improve our knowledge of the interaction between the extra- and intracardiac autonomic nervous system.
It is important to keep in mind that parasympathetic and sympathetic fibers are mostly co-localized so that current therapies like catheter ablation of atrial or ventricular arrhythmias will inevitably modify both structures. In the here described setup, local pharmaceutical modification of targeted structures (e.g., specific stimulation of parasympathetic ganglia) can be studied. Besides targeted modifications, global perfusion with different pharmaceuticals (e.g., beta-blockers) is easily possible, so that potential proarrhythmic or antiarrhythmic properties of various agents can be studied. Using this setup, interventions and different techniques can be tested during the stimulation or inhibition of different parts of the intracardiac autonomic nervous system, revealing information of the impact of specific parts of the autonomic nervous system on cardiac function and arrhythmogenesis. Further, the murine setup allows studying the cardiac autonomic nervous system in states of disease like myocardial infarction, heart failure or diabetes.
In conclusion, the simple and well-known Langendorff ex vivo heart perfusion system provides a flexible basis to modify and study the impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis.

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

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 closed and guarantees a stable temperature.
Figure 2 depicts different representative cardiac stainings. In Figure 2A a hematoxylin and eosin (H&#38;E) staining of a paraffin section is presented. In the exemplary enlargement (Figure 2B), an immunohistochemical staining of one atrial ganglion demonstrates the predominantly parasympathetic cells (red, ChAT-positive) compared to less numerous sympathetic cells (green, TH positive). In Figure 2C-E an immunohistochemical staining of neural (Figure 2C, green, neurofilament) and sympathetic fibers (Figure 2D, red, TH) as well as the overlay of the two images (Figure 2E) depicts how neural fibers traverse from the atria via the coronary sinus towards the posterior ventricles.
Figure 3 shows the murine heart connected to the cannula of the Langendorff apparatus with an inserted octapolar catheter in the right atrium and right ventricle and an epicardial multi-electrode array (MEA) placed on the anterior left ventricle (Figure 3A). Ventricular arrhythmia susceptibility testing via the electrodes in the RV is presented in Figure 3B. The induction of a ventricular tachycardia in hearts occurred more frequently after partial atrial denervation. In the enlarged MEA (Figure 3C) the schematic layout of the electrodes is presented. It is important to ensure a stable epicardial contact of all electrodes. In Figure 3D the offline-analyzed epicardial conduction recorded by an MEA is depicted.
Figure 4 shows FRET measurements in a whole heart being retrogradely perfused in the Langendorff apparatus. Different areas of the heart can be analyzed as needed (Figure 4A). A global as well as local topical application of pharmaceuticals is easily possible in this setup (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57617/57617fig1.jpg" /> 
Figure 1: Langendorff setup including multi-electrode arrays (MEAs). The octapolar stimulation and recording catheter is placed close to the area in which the heart will be attached. The lower part of the chamber will be moved upwards (white arrows) after the heart has been attached to the apparatus so that a stable temperature is ensured. <a href="/files/ftp_upload/57617/57617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57617/57617fig2v2.jpg" /> 
Figure 2: Cardiac whole mount stainings depicting parts of the autonomic nervous system.&#160;A) Depiction of a cardiac H&#38;E-stained paraffin section (scale bar 1 mm). B) An exemplary enlargement of one immunohistochemically stained atrial ganglion demonstrates the predominantly parasympathetic cells (red, ChAT-positive) compared to less numerous sympathetic cells (green, TH-positive; scale bar 75 &#181;m). C-E) Representative immunohistochemical stainings of neural (Figure 2C, green, neurofilament, NF) and sympathetic fibers (Figure 2D, red, TH, and their overlay in Figure 2E) traverse from the atria via the coronary sinus (CS) towards the posterior ventricles. Exemplary fibers are marked by arrowheads. Asterisks denote atrial ganglia. Scale bar 1 mm. LA, left atrium; LV, left ventricle; NF, neurofilament; PV, pulmonary veins; RA, right atrium; RAA, right atrial appendage; RV, right ventricle. <a href="/files/ftp_upload/57617/57617fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57617/57617fig3.jpg" /> 
Figure 3: Intra- and epicardial measurements using the Langendorff setup. A. This panel shows an example of a murine heart within the Langendorff system. The intracardiac octapolar catheter, which is inserted in the right atrium and ventricle, and one epicardial multi-electrode array (MEA) are depicted. B. Arrhythmia susceptibility testing using burst stimulation without (control) or with the induction of a self-terminating ventricular tachycardia [after partial atrial denervation (PAD)] are depicted. C. The epicardial MEA is depicted with an enlargement of the schematic electrode layout. D. Wave propagation velocity was analyzed using a custom-made software. The distance between the isochrones is 2 m/s. <a href="/files/ftp_upload/57617/57617fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57617/57617fig4.jpg" /> 
Figure 4: FRET measurements in a Langendorff setup. A. The two differentcAMP biosensor fluorescence channels [yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)] during FRET measurements in a retrograde perfused heart are depicted. If needed, different parts of the heart (e.g., atria and ventricle) can be analyzed (scale bar: 1 mm). B. This panel shows a representative FRET experiment, which measures cAMP levels during a pharmacological stimulation in the atrium and left ventricle. First, the heart was systemically perfused with the adenylyl cyclase activator NKH477, a forskolin analogon, to increase cAMP levels. Then nicotine was topically applied and targeted at the atrial ganglia, which acutely reduced cAMP levels. <a href="/files/ftp_upload/57617/57617fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

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

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.

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 ...

impact-intracardiac-neurons-on-cardiac-electrophysiology

Nanyang Technological University

Research

JoVE Journal

Medicine

10.6K Views.  University Hospital Hamburg-Eppendorf. Hier presenteren we een protocol voor de modulatie van het intracardiac autonome zenuwstelsel en de beoordeling van de invloed daarvan op fundamentele electrofysiologie, arrhythmogenesis en kamp dynamiek met behulp van een ex vivo Langendorff setup.

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

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

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

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.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Bioengineering

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

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.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Biology

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

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

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

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.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

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

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

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.

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

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

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

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.

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small ...

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

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

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.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

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

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can be done using optogenetics through selective expression of channelrhodopsin-2 (ChR2) in cardiac autonomic neurons. Such cardiac applications of optogenetics allow for the study of the intrinsic release of neurotransmitters in a spatiotemporal manner. This method illustrates an ex vivo approach for specific optogenetic stimulation of cardiac neurons in perfused mouse hearts. Transgenic mice were bred to express ChR2 in either choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) neurons throughout the body. A micro-LED (465 nm) encased in a silicone elastomer was prepared for stimulating the neurons of the right atrium that innervate the sinoatrial node. The micro-LED was connected to a function generator set to pulse waves at 10 Hz with a 30 ms pulse width. Hearts with confirmed expression were excised and retrogradely perfused on a Langendorff system circulating Krebs-Henseleit solution. Electrocardiogram (ECG), temperature, and coronary flow rate were recorded using the LabChart software. Once the heart stabilized, the micro-LED was placed on the right atrium and tested for optimal heart rate response. An application of this approach combines the intrinsic release of cholinergic neurotransmitter (acetylcholine) during optogenetic activation of a ChAT-ChR2 mouse heart simultaneously with increasing exogenous catecholaminergic neurotransmitter (norepinephrine) added to the perfusate. The resulting changes in heart rate during the simultaneous cholinergic and catecholaminergic activation are presented. This method describes a valuable experimental approach for investigating the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interactions between cardiac cholinergic and catecholaminergic activity.

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.

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can ...

Comprehensive 3D Analysis of Human Hearts from Micro to Macro Scales

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results&#160;to illustrate progress in the research of human cardiac anatomy.

Author Spotlight: Advancing Human Cardiac Anatomy Through Multi-Scale Analysis of Hearts

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...

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

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