Name: optocardiography and electrophysiology studies of ex vivo langendorff-perfused hearts
Uploaded: 2019-11-07T14:00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts at JoVE.com

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&#8221;</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>CaCl2</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&#8221;</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>MgSO4</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>NaHCO3</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Electrodes 29 gauge, 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 liter</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>Surgical Tools</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, 42.0mm 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, 2mm tip 14cm</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, 2mm tip width 12cm</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&#34; 5mm Calibrations</td>
		</tr>
		<tr>
			<td>Right angle forceps</td>
			<td>WPI</td>
			<td>501240</td>
			<td>Baby Mixter Hemostatic Forceps, 14cm, Right Angle</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Ted Pella</td>
			<td>549-4</td>
			<td>Scalpel Handle No. 4, 13.7cm 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, 42mm blade,12cm</td>
		</tr>
		<tr>
			<td>Straight Serrated forceps</td>
			<td>WPI</td>
			<td>500363</td>
			<td>Dressing Forceps 15.5cm</td>
		</tr>
		<tr>
			<td>Towel clamp</td>
			<td>WPI</td>
			<td>501700</td>
			<td>Backhaus Towel Clamp, 13cm, Curved, Locking handle, SS</td>
		</tr>
		<tr>
			<td>Weitlaner retractor</td>
			<td>WPI</td>
			<td>501314</td>
			<td>Weitlaner Retractor, Self-Retaining, 10.2cm, 2x3 Sharp Prongs</td>
		</tr>
		<tr>
			<td>Disposables</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&#174; Vessel Loop, 0.8 x 406mm</td>
		</tr>
		<tr>
			<td>Cardioplegia (Plegisol)</td>
			<td>Pfizer</td>
			<td>00409-7969-05</td>
			<td>Plegisol; St Thomas crystalloid cardioplegia solution 20ml/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>60mL &#38; 18G 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.

Watch this Scientific Journal Video about Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts at JoVE.com

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