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.
The overall goal of this ex vivo Langendorff setup is to analyze and modulate the intracardiac autonomic nervous system and assess its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics in decentralized hearts. The main advantage of this technique is that it allows researchers to analyze and modulate the intracardiac nervous system in decentralized hearts. Though this method can provide insight into healthy hearts, we got initial evidence that this approach can also be used to study various forms of structural heart disease.
Begin by starting the water bath and placing the perfusion solution including a mixture of 95%oxygen, 5%carbon dioxide in it. Then, adjust the pump rate before attaching the heart, so that no air bubbles are left in the cannula when mounting it to the apparatus. Set the targeted perfusion pressure to 80 millimeters mercury in the general settings, and start recording.
Use an electrophysiology catheter with platinum electrodes, an electrode surface of 0.5 by 0.5 millimeters, with an electrode spacing of 0.5 millimeters for the data recording and stimulation with a designated digital stimulus generator. Then, place the catheter close to the area where the heart will be positioned after the attachment to the apparatus. Next, attach the cannula quickly to the Langendorff apparatus, and ensure that there are no bubbles left in the cannula.
Switch the perfusion pressure to 80 millimeters mercury, allowing a constant pressure perfusion. 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. Then, start the stimulation with the prepared cycle length of 100 milliseconds for an initial 20-minute equilibration period.
Finally, close the chamber to allow a stable temperature. Begin by applying 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. Then, perform a programmed extrastimulation or burst pacing protocols in line with the Lambeth Conventions to evaluate the ventricular arrhythmogenesis.
Next, place the multi-electrode array, MEA, in the designated area of the heart, and add the grounding to another part of the heart. Place an epicardial stimulation catheter close to the MEA, and start a constant stimulation. Finally, start recording after the confirmation of good contact of the electrodes by checking the signal quality and amplitude.
Using a self-built imaging system around a stereomicroscope, begin by placing the stereomicroscope in front of the heart and adjust it for acuity. Excite the cAMP sensor with a light source, and split the emission light into donor and acceptor channels using a beam-splitter. Take images by using a scientific complementary metal-oxide-semiconductor, sCMOS, camera.
Next, start the image acquisition by pushing the Multi-D Acq. Button and set up a time-lapse, which acquires an image every 10 seconds with the appropriate exposure time, depending on the strength of the fluorescent signal. Then, use the previously described and available FRET Online and FRET Online 2 plugins to split the image into two channels, select the regions of interest, and monitor the ratio tracing.
During the acquisition, perfuse the heart with modified Krebs-Henseleit solution containing different substances. Then, at the end of the experiment, switch off the acquisition by pushing the Stop button and save the stack of images. Finally, 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.
On these whole-mount stainings, parts of the autonomic nervous system are depicted. An exemplary enlargement of one immunohistochemically stained atrial ganglion demonstrates the predominantly parasympathetic cells in red, compared to less numerous sympathetic cells, which are shown in green. In representative immunohistochemical stainings, neural fibers traverse from the atria via the coronary sinus towards the posterior ventricles with sympathetic predominance.
Exemplary fibers are marked by arrowheads. Here, ventricular arrhythmia susceptibility testing via the electrodes in the RV is presented. The induction of a ventricular tachycardia in hearts occurred more frequently after partial atrial denervation.
Further, a global as well as local topical application of pharmaceuticals is easily possible in this experimental setup. After perfusion of the adenylyl cyclase activator to increase cAMP levels, nicotine was applied to acutely reduce cAMP levels. While attempting this procedure, it's important to keep procedure time to a minimum and at a comparable length between experiments.
After watching this video, you should have a good understanding of how to investigate the impact of the intracardiac autonomic nervous system on cardiac electrophysiology, arrhythmogenesis, and cAMP dynamics in an ex vivo Langendorff setup.
