The authors gratefully acknowledge Dr. Matthew Kay for helpful experimental guidance, and Manelle Ramadan and Muhaymin Chowdhury for technical assistance. This work was supported by the National Institutes of Health (R01HL139472 to NGP, R01 HL139712 to NI), Children&#8217;s Research Institute, Children&#8217;s National Heart Institute and Sheikh Zayed Institute for Pediatric Surgical Innovation.

The authors have nothing to disclose.

Although cardiovascular research models range from cellular to in vivo preparations, there is an inherent trade-off between clinical relevance and experimental utility. On this spectrum, the isolated Langendorff-perfused heart remains a useful compromise for studying cardiac physiology48. The whole heart model represents a higher level of functional and structural integration than single cell or tissue monolayers, but also avoids the confounding complexities associated with in vivo models. A major advantage during dual optical mapping experiments is that the epicardial surface of the isolated heart can be observed, and fluorescence imaging of transmembrane potential and calcium handling can be used to monitor cardiac physiology34.
Rodent models are most commonly used for isolated heart preparations as opposed to larger animals, due in part to the associated cost of up-sizing all the elements involved (e.g., solution volume, perfusion circuit, quantity of dyes and mechanical uncouplers) along with greater instability and propensity for arrhythmias in larger animals10,36,49. One advantage to using pig hearts is that they closely resemble the human heart in structure, size and rate of contraction, therefore more accurately modeling hemodynamic parameters like coronary blood flow and cardiac output. Likewise, humans and pigs have similar calcium handling, electrocardiogram intervals37, and action potential morphology including the underlying channels that it represents12,50,51,52. This protocol describes in detail the steps for creating a reproducible large animal model to comprehensively characterize myocardial function. Simultaneous imaging of transmembrane voltage (RH237) and intracellular calcium (Rhod2), used in conjunction with established electrophysiological protocols, provides the opportunity to pinpoint mechanisms that are responsible for altered cardiac function. The described methodology can be used for preclinical safety testing, toxicological screening and the investigation of genetic or other disease pathologies. Moreover, the described methodology can be modified and adapted for use with other cardiac models (e.g., canine, human) depending on the specific research focus53,54,55.
There are a few critical modifications to keep in mind when transitioning from a smaller rodent model to a larger pig model for isolated, whole heart preparations. During preparation and setup, we recommend adding albumin to the perfusate to maintain oncotic pressure and reduce edema (plus antifoam, if needed)56,57,58,59. Moreover, perfusate containing albumin can also aid in metabolic studies that also require fatty acid-supplementation to the media60,61. Unlike rodent hearts, the larger pig heart does not need to be submerged in warm media due to its smaller surface to volume ratio and the increased volume of warmed media flowing through the coronary vessels which better maintains the temperature. As noted earlier, we placed temperature probes inside the right ventricle and on the epicardial surface of both the right and left ventricles, observing only slight temperature fluctuations of 1&#8722;2 &#176;C in all three locations throughout the study. Importantly, such faster flow rates can also increase the likelihood of bubbles and a potential embolism. To circumvent this problem, we recommend using a bubble trap with large bore tubing leading straight down to the aortic cannula. Similarly, we found it most useful to have two individuals working in tandem to cannulate the aorta on a larger (and heavier) heart; one person to hold the aorta open with sturdy hemostats and another to secure the aorta to the canula using umbilical tape. In the described methodology, we found that perfusion with cardioplegia and defibrillation were vital to cardiac recovery, which is contrary to rodent heart preparations. In our experience, only a few excised hearts resumed normal sinus-driven activity without cardioversion.
To improve optical imaging endpoints, a hanging heart preparation limited the effect of glare that can occur with a submerged heart. Moreover, the hanging heart also avoids any compression or compromise of the coronary vessels on the posterior aspect of the heart that can occur when laying the heart down horizontally for vertical imaging. We also found that loading fluorescent dyes after the bubble trap (close to the aortic cannula) greatly improved tissue staining and optical signals. Finally, to improve cardiac electrophysiology endpoints, the use of a larger coaxial stimulation electrode facilitated successful atrial pacing. Although we describe the use of electrocardiograms to identify capture and loss of capture for various EP parameters, intracardiac catheters or bipolar recording electrodes can also be used.
Our study was focused on developing a methodology for dual optical mapping and cardiac electrophysiological assessment in an isolated, intact porcine heart model. Due to similarities with the juvenile human heart, the porcine heart remains a popular model for studies focused on pediatric cardiology or congenital heart defects. Importantly, the described approach can be adapted to use with larger sized adult hearts and/or different species of interest. Indeed, other laboratories may find that the use of canine or human hearts (either donor or diseased) are more applicable for their specific research focus53,54,55. Another potential limitation to this study is the use of a mechanical uncoupler to reduce motion artifact during imaging. Blebbistatin has become the uncoupler of choice in cardiac imaging applications due to its minimal effects on ECG parameters, activation and refractory periods41,62,63. BDM is a less expensive choice, which can be particularly important in large animal studies that require greater volumes of perfusate and mechanical uncoupler, but it is known to have a greater impact on potassium and calcium currents that can alter action potential morphology64,65,66,67. If BDM is used, note that APD shortening increases the hearts vulnerability to shock-induced arrythmias68. Conversely, the main limitation to using blebbistatin is its photosensitivity and phototoxicity, although alternative formulations that have reduced these effects69,70,71. Finally, the described methodology utilizes a single camera system for dual optical mapping experimentation, but it is important to note that research studies focused on ventricular fibrillation and/or tracking of electrical waves across the epicardial surface would need to modify this approach to include three-dimensional panoramic imaging, as described by others15,19,72,73,74,75.

Cardiovascular disease is a leading cause of illness and death worldwide. As such, a primary research focus is to optimize methodologies that can be used to study normal cardiac physiology and underlying mechanisms that can contribute to morbidity and mortality in humans. Basic cardiovascular research has traditionally relied on small animal models, including rodents and rabbits1,2,3, due to the availability of genetically modified species4,5, lower-cost, smaller experimental footprint, and higher throughput. However, the use of a pig model has the potential to provide more clinically relevant data6. Indeed, previous studies have documented similarities in cardiac electrophysiology (EP) between humans and pigs, including similar ion currents7, action potential shape8, and responses to pharmacological testing9. Moreover, the porcine heart has contractile and relaxation kinetics that are more comparable to humans than either rodents or rabbits10. Compared to a canine model, the porcine coronary anatomy more closely resembles a human heart11,12 and is the model of choice for studies focused on heart development, pediatric cardiology and/or congenital heart defects13. Although there are differences between the pig and human heart8, these similarities make the porcine heart a valuable model for cardiovascular research14.
Retrograde perfusion of the heart has become a standard protocol for studying cardiac dynamics ex vivo15 since first established by Oskar Langendorff16. Accordingly, Langendorff-perfusion can be used to support an isolated, intact heart in the absence of autonomic influences. This model is a useful tool for directly comparing cardiac electrophysiology and contractility between healthy and non-healthy hearts. Since cardiac dynamics are both temporally and spatially complex, a slight alteration in one region can dramatically affect the entire heart&#8217;s ability to work as a syncytium17. Therefore, high spatiotemporal imaging of parameter sensitive dyes is a useful tool for monitoring cardiac function across the surface of the heart18,19. Indeed, simultaneous dual imaging of voltage and calcium-sensitive fluorescent probes allows for the assessment of electrical activity, calcium handling and excitation-contraction coupling at the tissue level20,21,22,23,24,25,26,27,28. Langendorff-perfusion and/or optical mapping techniques have previously been used to document the decline in cardiac performance due to aging or genetic mutations, and to assess the safety of pharmacological agents or environmental exposures29,30,31,32,33.
In the clinical setting, an invasive cardiac electrophysiology study is often used to investigate cardiac rhythm disturbances, identify pathologies, and pinpoint possible treatment options. Similarly, we describe an EP protocol that can be used to assess sinus node function, measure atrioventricular conduction, and identify the refractoriness of myocardial tissue. The described EP study can be performed in conjunction with optical mapping, or optocardiography34, to fully characterize cardiac physiology in isolated hearts. In the described protocol, high spatiotemporal resolution fluorescence imaging was performed with a combination of voltage (RH237) and calcium (Rhod-2AM) dyes in a dual emission setup. Additionally, cardiac electrophysiology parameters were monitored under both sinus rhythm and in response to programmed electrical stimulation.

<table><tbody><tr><td>(-)-Blebbistatin</td><td>Sigma-Aldrich</td><td>B0560-5MG</td><td>Mechanical Uncoupler</td></tr><tr><td>2,3-Butanedione monoxime (BDM)</td><td>Sigma-Aldrich</td><td>B0753-100G</td><td>Mechanical Uncoupler</td></tr><tr><td>Albumin</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A9418</td><td></td></tr><tr><td>Analog signal interface</td><td>emka Technologies</td><td>itf16USB</td><td></td></tr><tr><td>Antifoam</td><td>Sigma-Aldrich</td><td>A5758-250ML</td><td></td></tr><tr><td>Antifoam Y-30 Emulsion</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A5758</td><td></td></tr><tr><td>Aortic cannula, 5/16&amp;rdquo;</td><td>Cole-Parmer</td><td>45509-60</td><td></td></tr><tr><td>Bubble trap</td><td>Sigma-Aldrich</td><td>CLS430641U-100EA</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>C77-500</td><td></td></tr><tr><td>Camera, sCMOS</td><td>Andor Technology</td><td>Zyla 4.2 PLUS</td><td></td></tr><tr><td>Coaxial stimulation electrode (atria)</td><td>Harvard Apparatus</td><td>73-0219</td><td></td></tr><tr><td>Defibrillator</td><td>Zoll</td><td>M Series</td><td></td></tr><tr><td>Dichroic mirror</td><td>Chroma Technology</td><td>T660lpxrxt-UF2</td><td></td></tr><tr><td>Differential amplifier</td><td>Warner Instruments</td><td>DP-304A</td><td></td></tr><tr><td>Emission filter, calcium</td><td>Chroma Technology</td><td>ET585/40m</td><td></td></tr><tr><td>Emission filter, voltage</td><td>Chroma Technology</td><td>ET710lp</td><td></td></tr><tr><td>EP stimulator (Bloom)</td><td>Fisher Medical</td><td>DTU-215B</td><td></td></tr><tr><td>Excitation filter</td><td>Chroma Technology</td><td>CT510/60bp</td><td></td></tr><tr><td>Excitation lights</td><td>Thorlabs</td><td>SOLIS-525C</td><td></td></tr><tr><td>Filter</td><td>McMaster-Carr</td><td>8147K52</td><td></td></tr><tr><td>Filter cartridge, polypropylene</td><td>Pentair</td><td>PD-5-934</td><td></td></tr><tr><td>Filter housing</td><td>McMaster-Carr</td><td>9979T21</td><td></td></tr><tr><td>Flow transducer</td><td>Transonic</td><td>ME6PXN</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, St. Louis, MO</td><td>158968</td><td></td></tr><tr><td>Heating coil</td><td>Radnoti</td><td>158821</td><td></td></tr><tr><td>Hemofilter</td><td>Hemocor</td><td>HPH 400</td><td></td></tr><tr><td>Hemostatic Forceps</td><td>World Precision Instruments</td><td>501326</td><td></td></tr><tr><td>Image Splitter</td><td>Cairn Research</td><td>OptoSplit II</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louis, MO</td><td>P3911</td><td></td></tr><tr><td>KH2PO4</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>423-316</td><td></td></tr><tr><td>Large-bore tubing, I.D. 3/8&amp;rdquo;</td><td>Fisher Scientific</td><td>14-169-7H</td><td></td></tr><tr><td>Lens 50 mm, 0.95 f-stop</td><td>Navitar</td><td>DO-5095</td><td></td></tr><tr><td>Metamorph</td><td>Molecular Devices</td><td></td><td>Image Alignment</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>M-7506</td><td></td></tr><tr><td>Mucasol detergent</td><td>Sigma-Aldrich</td><td>Z637181-2L</td><td></td></tr><tr><td>Na Pyruvate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>P2256</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louis, MO</td><td>S-3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>S-233</td><td></td></tr><tr><td>Needle Electrodes 29 G, 2 mm</td><td>AD Instruments Inc.</td><td>MLA1204</td><td></td></tr><tr><td>Noise eliminator</td><td>Quest Scientific</td><td>Humbug</td><td></td></tr><tr><td>Perfusion pump</td><td>PolyStan</td><td>A/S 1481</td><td></td></tr><tr><td>Pressure transducer</td><td>World Precision Instruments</td><td>BLPR2</td><td></td></tr><tr><td>Reservoir, 2 L</td><td>Cole-Parmer</td><td>UX-34541-07</td><td></td></tr><tr><td>RH237</td><td>AAT Bioquest Inc.</td><td>21480</td><td></td></tr><tr><td>Rhod2-AM</td><td>AAT Bioquest Inc.</td><td>21062</td><td></td></tr><tr><td>Stimulation electrode (ventricle)</td><td>Harvard Apparatus</td><td>73-0160</td><td></td></tr><tr><td>Surgical Suture</td><td>McKesson Medical-Surgical</td><td>890186</td><td></td></tr><tr><td>Transducer amplifier</td><td>World Precision Instruments</td><td>TBM4M</td><td></td></tr><tr><td>Tubing flow console</td><td>Transonic</td><td>TS410</td><td></td></tr><tr><td>Umbilical tape</td><td>Jorvet</td><td>J0025UA</td><td></td></tr><tr><td>Water bath/circulator</td><td>VWR</td><td>89400-970</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bandage shears</td><td>Harvard Apparatus</td><td>72-8448</td><td>Lister Bandage Scissors, Angled, Blunt/Blunt, 42.0 mm blade length, 17.0 cm</td></tr><tr><td>Electrocautery</td><td>Dalwha Corp. Ltd.</td><td>BA2ALD001</td><td>Model: 200 Basic</td></tr><tr><td>Hemostat</td><td>Roboz</td><td>RS-7476</td><td>St Vincent Tube Occluding Forceps</td></tr><tr><td>Hemostatic forceps</td><td>Harvard Apparatus</td><td>72-8960</td><td>Hartmann Hemostatic Forceps, Curved, Serrated 2.2 mm tip width, 9.5 cm</td></tr><tr><td>Hemostats</td><td>Harvard Apparatus</td><td>72-8985</td><td>Halstead-Mosquito Hemostatic Forceps Curved, Serrated, 2 mm tip 14 cm</td></tr><tr><td>Mayo scissors</td><td>WPI</td><td>501749</td><td>14.5 cm, Straight</td></tr><tr><td>Metzenbaum scissors</td><td>WPI</td><td>501747</td><td>11.5 cm, Straight</td></tr><tr><td>Mosquito forceps</td><td>Harvard Apparatus</td><td>72-8980</td><td>Halstead-Mosquito Hemostatic Forceps Straight, Smooth, 2 mm tip width 12 cm</td></tr><tr><td>Needle holder</td><td>Harvard Apparatus</td><td>72-8828</td><td>Webster Needle Holders, Straight, Smooth,13.0 cm overall length</td></tr><tr><td>Pediatric cross clamp</td><td>Roboz</td><td>RS-7660</td><td>Cooley-Derra Clamp 6.25&quot; 5 mm Calibrations</td></tr><tr><td>Right angle forceps</td><td>WPI</td><td>501240</td><td>Baby Mixter Hemostatic Forceps, 14 cm, Right Angle</td></tr><tr><td>Scalpel</td><td>Ted Pella</td><td>549-4</td><td>Scalpel Handle No. 4, 13.7 cm Stainless Steel and 10 No. 22 Blades</td></tr><tr><td>Scissors</td><td>Harvard Apparatus</td><td>72-8380</td><td>Operating Scissors, Straight, Blunt/Blunt, 42 mm blade,12 cm</td></tr><tr><td>Straight Serrated forceps</td><td>WPI</td><td>500363</td><td>Dressing Forceps 15.5 cm</td></tr><tr><td>Towel clamp</td><td>WPI</td><td>501700</td><td>Backhaus Towel Clamp, 13 cm, Curved, Locking handle, SS</td></tr><tr><td>Weitlaner retractor</td><td>WPI</td><td>501314</td><td>Weitlaner Retractor, Self-Retaining, 10.2 cm, 2x 3 Sharp Prongs</td></tr><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3-0 prolene suture</td><td>Various vendors</td><td>Various vendors</td><td></td></tr><tr><td>Vessel loop</td><td>Aspen surgical</td><td>011001PBX</td><td>Sterion&amp;reg; Vessel Loop, 0.8 mm x 406 mm</td></tr><tr><td>Cardioplegia (Plegisol)</td><td>Pfizer</td><td>00409-7969-05</td><td>Plegisol; St Thomas crystalloid cardioplegia solution 20 mL/kg</td></tr><tr><td>Heparin</td><td>Various vendors</td><td>Various vendors</td><td>300 U/kg</td></tr><tr><td>Syringe and Needle</td><td>Various vendors</td><td>Various vendors</td><td>60 mL &amp;amp; 18 G respectively</td></tr><tr><td>Umbilical tape</td><td>Ethicon</td><td>U12T</td><td></td></tr></tbody></table>

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.

Figure 1A shows a diagram of the isolated heart perfusion system, which includes the tubing circuit, pump, filter, oxygenator, reservoirs and heating elements. Placement of the ECG (lead II configuration) and pacing electrodes is shown in Figure 1B, and the imaging setup is depicted in Figure 1C. A schematic of the optical components and light paths are shown in Figure 1D.
Experimental studies were performed on intact, whole hearts isolated from juvenile Yorkshire pigs (14&#8722;42 days, n = 18) that ranged in size from 2.5&#8722;10.5 kg body weight and 18&#8722;137 g heart weight (Figure 2A). After transferring the isolated heart to a Langendorff system (37 &#176;C), the heart rate stabilized to 70 &#177; 4.5 bpm (mean &#177; SEM) within ~10 min of defibrillation and remained constant throughout the duration of study (Figure 2B). An average flow rate of 184 &#177; 17 mL/min (mean &#177; SEM) was measured, which slowed to 70 &#177; 7.5 mL/min after perfusing with warmed media containing a mechanical uncoupler (Figure 2C).
Lead II ECGs were recorded throughout the duration of the study during sinus rhythm (Figure 3A) or in response to external pacing (Figure 3B-E) to quantify electrophysiological parameters. For EP assessment, dynamic pacing (S1&#8722;S1) was applied to the right atrium to pinpoint the WBCL and SNRT (recovery time after S1&#8722;S1 commences, Figure 3C), wherein WBCL was denoted as the shortest PCL that initiated atrial to ventricular conduction. An S1&#8722;S2 pacing protocol was implemented using a bipolar stimulus electrode on the left ventricle in order to identify the shortest coupling interval that initiated ventricular depolarization, thereby pinpointing VERP (Figure 3D). Alternatively, an S1&#8722;S2 atrial pacing protocol is applied to pinpoint AVNERP (S1&#8722;S2), as shown in Figure 3E. Representative examples of pig heart electrophysiology parameters align closely with those previously published37.
Optical mapping experiments were performed during sinus rhythm, spontaneous ventricular fibrillation (Figure 4), or during dynamic pacing (S1&#8722;S1) of the left ventricle (LV) to generate electrical and calcium restitution curves depicted in Figure 5. Representative images of a dye-loaded piglet heart are shown in Figure 4 with corresponding optical action potentials (Vm) and calcium (Ca) transients collected from two regions of interest on the epicardial surface (right ventricle [RV] = blue, LV = red). Unprocessed signals are displayed during sinus rhythm and during ventricular fibrillation. As previously mentioned, dynamic epicardial pacing (S1&#8722;S1) was also used during optical mapping experiments to normalize any slight difference in the intrinsic heart rate (Figure 5A-E). Raw signals are displayed (RV = blue, LV = red), which were used to depict the action potential &#8212; calcium transient coupling time (Figure 5C), activation and duration time (Figure 5D), electrical and calcium restitution (Figure 5E). For thick myocardial preparations, spatial filtering with kernel size ~3 mm x 3 mm is appropriate for epicardial action potential or calcium transient analysis19,47. Accordingly, high spatial resolution images (in the described setup 1240 x 1024 total, or 620 x 512 per channel, 6.5 &#181;m pixel size) are often spatially binned during or post-acquisition (Figure 5C). Image processing can be performed to generate activation and repolarization maps using custom algorithms23,33,43,45 (Figure 3D), with the activation time of each pixel on the heart was defined as the maximum derivative of the action potential or calcium transient upstroke.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60472/60472fig01.jpg" /> 
Figure 1: Experimental setup.&#160;(A) Diagram of the isolated heart perfusion system; arrows denote the direction of flow. (B) A cannulated heart is shown with electrode placement. RA = right atria, RV = right ventricle, LV = left ventricle, ECG = lead II electrocardiogram. (C) The imaging platform in close proximity to the heart tissue. (D) Emission of each complementary probe (voltage, calcium) is separated by wavelength using an image splitting device with appropriate emission filters and dichroic mirror. <a href="https://www.jove.com/files/ftp_upload/60472/60472fig01large.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/60472/60472fig02.jpg" /> 
Figure 2: Heart weight, rate and flow measurements.&#160;(A) Heart weight to body weight ratio for each piglet used in the study (n = 18). (B) Heart rate measured ~10 min after defibrillation and again at the end of study (approximately 1 h). (C) Coronary flow drops precipitously after perfusion with a mechanical uncoupler (+BDM) due to reduced oxygen demand. Scale bars represent mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/60472/60472fig02large.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/60472/60472fig03.jpg" /> 
Figure 3: Representative examples of lead II electrocardiogram recordings collected during sinus rhythm or in response to external pacing.&#160;(A) Normal sinus rhythm. (B) Example of epicardial pacing at cycle length of 400 ms (S1&#8722;S1), which was used for imaging experiments. (C) Top: Atrial pacing to identify WBCL; successful capture is observed at S1 = 250 ms wherein atrial to ventricular conduction is observed. Note that atrial pacing can be used to determine SNRT (time to sinus node discharge, after commencing external pacing). Bottom: As the S1 cycle length is decreased to 205 ms, the conduction to the ventricle fails. (D) Top: Epicardial pacing (S1&#8722;S2) to identify VERP; successful capture is observed at S1 = 450 ms, S2 = 300 ms. Bottom: As the S2 cycle length is decreased to 250 ms, the ventricular tissue fails to capture. (E) Atrial pacing (S1&#8722;S2) to identify AVNERP. Top: Successful capture is observed at S1 = 450 ms, S2 = 200 ms. Bottom: As the S2 cycle length is decreased to 199 ms, conduction to the ventricle fails. Blue arrows denote pacing spikes, red arrows denote capture (&#8216;C&#8217;) or no capture (&#8216;NC&#8217;). S1&#8722;S1 = dynamic pacing, S1&#8722;S2 = extrastimulus pacing. <a href="https://www.jove.com/files/ftp_upload/60472/60472fig03large.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/60472/60472fig04.jpg" /> 
Figure 4: Optical data during sinus rhythm and ventricular fibrillation.&#160;Left: Representative images of a dye-loaded pig heart (Vm = voltage, RH237; Ca = calcium, Rhod2), anterior view. Spatially filtered transmembrane voltage and intracellular calcium fluorescent signals from a pig heart during sinus rhythm (Center). Voltage and calcium signals during ventricular fibrillation (Right). Signal region sizes (15 x 15 pixels = 2.4 x 2.4 mm2, 30 x 30 = 4.8 x 4.8 mm2 kernel size) represented as red and blue squares. Units = &#916;F/F. <a href="https://www.jove.com/files/ftp_upload/60472/60472fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60472/60472fig05.jpg" /> 
Figure 5: Optical data from Langendorff-perfused pig hearts.&#160;Unprocessed, spatially filtered (A) transmembrane voltage and (B) intracellular calcium fluorescence signals from the right and left ventricles during electrical pacing at the apex. Unfiltered, spatially averaged signals depict optical action potentials and calcium transients from regions of interest (signal units are &#916;F/F). (C) An overlay of normalized transients illustrates action potential-calcium transient coupling time (low-pass filtered at 75 Hz). (D) Processing signals across the epicardial surface to generate isochronal maps of temporal parameters, including activation time (tact) and 80% repolarization time. (E) Electrical and calcium transient restitution curves generated at multiple frequencies (left) with statistical analysis (right) to illustrate longer repolarization time at slower pacing cycle lengths. Scale bars = mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/60472/60472fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Formula</td>
			<td>Molecular weight</td>
			<td>g/L</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>NaCl</td>
			<td>58.44</td>
			<td>5.26</td>
		</tr>
		<tr>
			<td>Sodium gluconate</td>
			<td>C6H11NaO7</td>
			<td>218.14</td>
			<td>5.02</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>C2H3NaO2&#8226;3H2O</td>
			<td>136.08</td>
			<td>3.68</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>KCl</td>
			<td>74.55</td>
			<td>0.63</td>
		</tr>
		<tr>
			<td>Magnesium chloride (anhydrous)</td>
			<td>MgCl2</td>
			<td>95.21</td>
			<td>0.1405</td>
		</tr>
		<tr>
			<td>8.4% Sodium bicarbonate</td>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>13</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>C6H14O6</td>
			<td>182.17</td>
			<td>16.3</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>MgSO4</td>
			<td>120.37</td>
			<td>4</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmolarity (mOsmol/L)</td>
			<td>294</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Modified del Nido&#8217;s cardioplegia recipe.

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Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

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 ...

optocardiography-electrophysiology-studies-ex-vivo-langendorff

Research

JoVE Journal

Medicine

12.9K Views.  Children's National Hospital. Cardiovasculaire storing is de belangrijkste doodsoorzaak wereldwijd. Het geïsoleerde hartmodel dat we hier presenteren, kan dienen als een experimenteel venster in de menselijke hartdynamiek. Deze aanpak combineert een klassieke elektrofysiologiestudie met de gelijktijdige optische mapping van transmembranespanning en intercellulair calcium om de toestand van het hart te evalueren.Deze methodologie kan worden toegepast op ziektemodellering, farmacologie en toxicologiestudies. Bijvoor...

Video: Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

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 ...

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.

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.

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 ...

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 ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

Coronary Artery Assessment During Ex-Situ Heart Perfusion

Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the current organ shortage. Coronary angiography is recommended in ECD at risk for coronary artery disease but is not systematically performed. These hearts are, therefore, either declined for transplant or procured without screening for coronary artery disease. Coronary angiography during normothermic ex-situ heart perfusion (NESP) could be an interesting approach to enhance the rate of ECD procurement and to reduce the risk of primary graft failure in the absence of coronary angiography in ECD. The present protocol aims to provide material details along with optimal imaging views for coronary angiography during NESP. Reproducible angiographic views were observed, including one dedicated to the right coronary artery, two for the left anterior descending artery, two for the circumflex artery, and a spider view. Continuous lactate extraction was observed in all procedures with a final median concentration of 1.10 mmol/L (0.61-1.75 mmol/L) two hours after coronary angiography, consistent with myocardial viability. The median contrast agent volume used for ex-situ imaging of the isolated perfused heart was 48 mL (38-108 mL). This protocol was reproducible for coronary artery imaging and did not impair myocardial viability during NESP.

Author Spotlight: Innovative Technique for Coronary Angiography in Marginal Donors

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the ...

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 ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

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