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 of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly—or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. Förster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.
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.
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.
2. Hard- and Software Preparation
3. Preparation of the Heart
4. Electrophysiological Parameters and Arrhythmogenesis
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 µm; 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.
6. Förster Resonance Energy Transfer (FRET)-based Cyclic Adenosine Monophosphate (cAMP) Imaging
NOTE: For FRET-based measurements, harvest hearts from CAG-Epac1-camps transgenic mice16.
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.
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&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).
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. Please click here to view a larger version of this figure.
Figure 2: Cardiac whole mount stainings depicting parts of the autonomic nervous system. A) Depiction of a cardiac H&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 µm). 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ˚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 authors have nothing to disclose.
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 Förderverein des Universitären Herzzentrums Hamburg e.V. and by the DZHK (German Centre for Cardiovascular Research) [FKZ 81Z4710141].
Sodium chloride | Sigma-Aldrich | S3014 | Modified Krebs-Henleit solution |
Sodium hydrogencarbonate | Sigma-Aldrich | 401676 | Modified Krebs-Henleit solution |
Potassium chloride | Sigma-Aldrich | P5405 | Modified Krebs-Henleit solution |
Potassium phosphate monobasic | Sigma-Aldrich | P5655 | Modified Krebs-Henleit solution |
Magnesium sulfate heptahydrate | Sigma-Aldrich | M1880 | Modified Krebs-Henleit solution |
Calcium chloride dihydrate | Sigma-Aldrich | C7902 | Modified Krebs-Henleit solution |
Glucose | Sigma-Aldrich | G5767 | Modified Krebs-Henleit solution |
Sodium pyruvate bioXtra | Sigma-Aldrich | P8574 | Modified Krebs-Henleit solution |
Carbogen (95% O2 / 5% CO2) | SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany | Modified Krebs-Henleit solution | |
Sterile filter steritop-GP 0.22 | EMD Millipore | SCGPT05RE | Modified Krebs-Henleit solution |
Atropine sulfate | Sigma-Aldrich | A0257 | Neuromodulation |
Hexamethonium chloride | Sigma-Aldrich | H2138 | Neuromodulation |
Nicotine free base 98-100% | Sigma-Aldrich | N3876 | Neuromodulation |
Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | Whole mount staining |
Tris(hydroxymethyl)aminomethane | Sigma-Aldrich | 252859 | Whole mount staining |
Methanol | Sigma-Aldrich | 34860 | Whole mount staining |
Hydrogen peroxide solution 30% (w/w) in H2O | Merck, KGA, Darmstadt, Germany | H1009 | Whole mount staining |
Dimethyl sulfoxide | Merck, KGA, Darmstadt, Germany | D8418 | Whole mount staining |
Phosphate-buffered saline tablets | Gibco / Invitrogen | 18912-014 | Whole mount staining |
Triton-x-100 | Sigma-Aldrich | T8787 | Whole mount staining |
Albumin bovine fraction V | Biomol, Hamburg, Germany | 11924.03 | Whole mount staining |
Chicken anti neurofilament | EMD Millipore | AB5539 | Whole mount staining |
Rabbit anti tyrosine hydroxylase | EMD Millipore | AB152 | Whole mount staining |
Goat anti choline acetyltransferase | EMD Millipore | AP144P | Whole mount staining |
Donkey α rabbit IgG Alexa 488 | Thermo Fisher Scientific | A21206 | Whole mount staining |
Donkey α goat IgG Alexa 568 | Thermo Fisher Scientific | A11057 | Whole mount staining |
Donkey α chicken IgY Alexa 647 | Merck, KGA, Darmstadt, Germany | AP194SA6 | Whole mount staining |
Biotin-conjugated donkey α rabbit igG | R&D Systems | AP182B | Whole mount staining |
Biotin-conjugated donkey α goat igG | R&D Systems | AP192P | Whole mount staining |
Biotin-conjugated goat α chicken igY | R&D Systems | BAD010 | Whole mount staining |
Vectashield mounting medium | Vector laboratories, Burlingame, CA, USA | H-1000 | Immunohistochemistry |
Vectastain ABC kit | Vector laboratories, Burlingame, CA, USA | PK-4000 | Immunohistochemistry |
Steady DAB/Plus | Abcam plc, Cambridge, UK | ab103723 | Whole mount staining |
HistoClear | DiaTec, Bamberg, Germany | HS2002 | Immunohistochemistry |
BisBenzimide H33342 trihydrochloride (Hoechst) | Sigma-Aldrich, St. Louis, MO, USA | B2261 | Immunohistochemistry |
Vectashield HardSet mounting medium | Vector laboratories, Burlingame, CA, USA | VEC-H-1400 | Immunohistochemistry |
Perfusion system | HUGO SACHS ELEKTRONIK – HARVARD APPARATUS GmbH, March-Hugstetten, Germany | 73-4343 | Langendorff apparatus |
Data acquisition system and corresponding software for catheter and physiological parameter | Powerlab 8/30 & Labchart, ADInstruments, Dunedin, New Zealand | PL3508 PowerLab 8/35 | Langendorff setup |
Octapolar catheter | CIB’ER Mouse, NuMed Inc., Hopkinton, NY, USA | custom | Langendorff setup |
Stimulus generator | STG4002, Multi Channel Systems, Reutlingen, Germany | STG4002-160µA | Stimulation setup |
Stimulation software | Multi Channel Systems, Reutlingen, Germany | MC_Stimulus II | Stimulation setup |
Data acquisition system and corresponding software for epicardial electrograms | ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany | USB-ME128-System | MEA setup |
Multi-electrode array | MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany | EcoFlexMEA36 | MEA setup |
Multi-electrode array recording software | Multi Channel Systems, Reutlingen, Germany | MC_Rack | MEA setup |
Spring scissors | Fine Science Tools GmbH, Heidelberg, Germany | 15003-08 | Heart Preparation |
Strabismus Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14575-09 | Heart Preparation |
Mayo Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14110-15 | Heart Preparation |
Dumont SS Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11203-25 | Heart Preparation |
London Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11080-02 | Heart Preparation |
Narrow Pattern Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11003-13 | Heart Preparation |
Plastic Wrap | Parafilm M, Bemis NA, based in Neenah, WI, United States | Consumable Materials | |
Stereomicroscope | Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany | FRET | |
LED | CoolLED, Andover, UK | pE-100 | FRET |
DualView | Photometrics, Tucson, AZ, USA | DV2-SYS | FRET |
DualView filter set | Photometrics, Tucson, AZ, USA | 05-EM | FRET |
optiMOS scientific CMOS camera | Qimaging, Surrey, BC, Canada | 01-OPTIMOS-R-M-16-C | FRET |
Imaging software | Micro-Manager; Vale Lab, University of California San Francisco, CA, USA | FRET | |
Analysis Software | Image J software; Public Domain, NIH, USA | FRET |