This work was supported in part by the US National Institutes of Health (R01 HL 111600) and the American Heart Association (12SDG9010015).

All procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis, and adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
1. Preparation
<ol>
	<li>Prepare two concentrated (25X) stocks of modified Tyrode&#8217;s solution in advance and store at 4 &#176;C: (1) Stock I (in mM: NaCl 3,205, CaCl2 32.5, KCl 117.5, NaH2PO4 29.75, MgCl2 26.25) and (2) Stock II (in mM: NaHCO3 500).
	<ol>
		<li>Freshly prepare 2 L Tyrode&#39;s solution of the following composition (in mM): NaCl 128.2, CaCl2 1.3, KCl 4.7, MgCl2 1.05, NaH2PO4 1.19, NaHCO3 20 and glucose 11.1 by combining 1840 ml&#160;of deionized (DI) water, 80 ml&#160;Stock I, 80 ml&#160;of Stock II, and 4 g of glucose. Do not directly mix Stock I and Stock II; add them to 1,840 ml&#160;DI to avoid precipitation.</li>
	</ol>
	</li>
	<li>Prepare stock solution of the excitation-contraction uncoupler blebbistatin at 10 mg/ml in anhydrous di-methylsulfoxide (DMSO) in advance and store the stock solution at 4 &#176;C.</li>
	<li>Prepare stock solutions of fluorescent dyes: (1) Voltage-sensitive dye RH237 (1 mg/ml&#160;in DMSO) and (2) Low-affinity calcium indicator Fluo-5N AM (2 mg/ml in DMSO) according to manufacturer&#8217;s recommendations. Use the preparations immediately to avoid decomposition with subsequent loss of loading capacity.</li>
	<li>Prime the recirculating Langendorff perfusion system with oxygenated (95% O2, 5% CO2) Tyrode&#8217;s solution. Adjust the flow of O2/CO2 to keep the pH of the Tyrode&#8217;s solution at 7.4 &#177; 0.05. Use an in-line nylon woven net filter (pore size: 11 &#181;m) to continuously filter the perfusate.
	<ol>
		<li>Prime the perfusion system at RT, as subsequent loading of Fluo-5N AM is performed at RT. For Fluo-5N AM loading, use a small volume of recirculating perfusate (~ 150 ml). After dye loading, use a larger volume of perfusate (1 - 2 L, see Figure 1C).</li>
	</ol>
	</li>
	<li>Turn on data acquisition system and prepare for continuous monitoring of ECG and perfusion pressure. Prepare pressure-monitoring system: Connect a pressure transducer to the perfusion line and monitor the perfusion pressure with a transbridge amplifier. Adjust the baseline perfusion pressure to 0 mmHg when the heart is not attached to the perfusion system.</li>
	<li>Align the optical mapping cameras to ensure spatial alignment between the Vm and SR Ca2+ signals.
	<ol>
		<li>First, focus the cameras by placing a ruler or an object with text into the perfusion dish. Turn the mapping cameras into a live view mode and adjust the focus until the text is crisp and clear. Acquire a single frame image from each camera.</li>
		<li>Overlay these images and adjust the transparency of the top image so that both images are visible. The text in the images should overlap exactly. If not perfectly aligned, adjust the angle of the dichroic mirror or the position of each camera until the two images are aligned.</li>
	</ol>
	</li>
</ol>
2. Harvesting, Perfusion, and Dye-loading of Rabbit Heart
<ol>
	<li>Secure the rabbit in an approved rabbit restrainer. Deeply anesthetize via an intravenous injection (marginal ear vein) of sodium pentobarbital (50 mg/kg) and heparin (1,000 IU).
	<ol>
		<li>When the rabbit displays lack of nociceptive reflexes, make a midline skin incision to expose the sternum and ribs. Cut through the sternum with blunt-tip surgical scissors from the xyphoid to the manubrium. Take care not to damage the heart while opening the sternum. Spread the ribs to expose the heart.</li>
		<li>Quickly excise the entire heart-lung block by rapidly cutting all vessels and connective tissue. Immediately place the heart-lung block into ice-cold Tyrode&#8217;s solution.</li>
	</ol>
	</li>
	<li>Locate the aorta for retrograde perfusion. Cut the ascending aorta just proximal to the three branches of the aortic arch. Cannulate the aorta to an 8 G cannula connected to the perfusion system. Use a piece of USP 0 silk suture to secure the aorta onto the cannula.</li>
	<li>Carefully dissect the trachea, lungs, and epicardial fat from the heart. With a sharp forceps and dissection scissors, locate the mitral valve and carefully damage or remove one leaflet to prevent solution congestion in the left ventricle.</li>
	<li>Submerge the heart in the glass-jacketed perfusion chamber horizontally with the anterior surface of the heart facing up for imaging. Secure the cannula to a piece of Sylgard with U-shaped pins onto the silicon bottom of the dish to prevent movement of the heart during mapping (Figure 1D). If desired, insert a small insect pin at the apex of the heart for stabilization.</li>
	<li>Position electrocardiogram (ECG) electrodes in the bath on the right and left side of the heart. Fully submerge the electrodes in the bath and ensure they are not in contact with the heart surface to provide a volume-conducted ECG analogous to a Lead I configuration. Verify a normal Lead I ECG morphology. Adjust the flow (25 - 35 ml/min) to ensure that the aortic pressure is maintained at 60 - 70 mmHg.</li>
	<li>Turn off the room lights and add 0.3 - 0.6 ml&#160;of blebbistatin stock solution (step 1.2) to the perfusate (final concentration 10 - 20 &#181;M).
	<ol>
		<li>Turn off room lights to avoid photoinactivation of blebbistatin. If necessary, a small spotlight or headlamp can be used to provide task lighting. 
		Note: Blebbistatin is a myosin ATPase inhibitor and an excitation-contraction uncoupler. Because Fluo-5N loading requires extended (60 min) RT (hypothermia) conditions (see steps 2.8 &#8211; 2.10), cardiac energy production may be compromised. Therefore, addition of blebbistatin into the perfusate prior to dye loading may reduce the energy demand16 and eliminate motion artifacts during subsequent optical recordings17.</li>
	</ol>
	</li>
	<li>When contraction of the heart has ceased (10 - 15 min, verified on the aortic pressure recording), switch to the small-volume recirculating perfusion system (see step 1.4.1).</li>
	<li>Prepare and load Fluo-5N AM loading solution: Add 0.25 ml&#160;of Fluo-5N AM stock solution (step 1.3) to 0.25 ml&#160;warm 20% Pluronic F127. Add 0.5 ml&#160;warm Tyrode&#8217;s solution to the mixture, mix well, and add to the small-volume recirculating perfusion system.</li>
	<li>Continuously monitor perfusion pressure and ECG and adjust the flow if necessary. Dye loading takes approximately 1 hr at RT. 45 min after loading has begun, turn on the circulating water bath to begin warming the perfusion system and perfusate to 37 &#176;C.</li>
	<li>After 60 min of Fluo-5N AM loading, switch the perfusion to the larger volume recirculating perfusion system (see step 1.4).</li>
	<li>Dilute 50 &#181;l&#160;of voltage sensitive dye RH237 stock solution (step 1.3) in 1 ml&#160;of warm Tyrode&#39;s solution and add slowly (over ~ 5 min) into an injection port proximal to the cannula.</li>
</ol>
3. Optical Mapping
<ol>
	<li>During the final moments of dye loading, position the heart and focus the optical mapping cameras to ensure the appropriate field of view for the experiment.</li>
	<li>If desired, place a bipolar pacing electrode on the epicardial surface of the heart for pacing.</li>
	<li>Place a plastic or glass cover slip on the surface of the perfusion chamber to reduce motion artifact on the liquid surface that may be present due to recirculation of the perfusate. 
	Note: As an alternative to a cover slip, use a clean plastic 50 mm Petri dish cover.</li>
	<li>Focus the light guides to uniformly illuminate the surface of the heart with excitation light. Use blue light emitting diode (LED) light sources and further filter the light with a 475 - 495 nm bandpass filter (Figure 1A).</li>
	<li>Collect emitted fluorescence with a macroscope and two complementary metal-oxide-semiconductor (CMOS) optical mapping cameras. Split the emitted light with a dichroic mirror at 545 nm.
	<ol>
		<li>Long-pass filter the longer wavelength moiety, containing the Vm signal, at 700 nm. Band-pass filter the shorter wavelength moiety, containing the SR Ca2+ signal, from 502 - 534 nm (Figure 1A). Set the frequency of optical data acquisition to 0.5 - 1 kHz.</li>
	</ol>
	</li>
	<li>For each optical recording, first initiate the desired pacing protocol, turn on the excitation light, and collect 1 - 4 sec of data. If desired, synchronize the pacing protocols and data acquisition. 
	Note: It is not possible to re-load the Fluo-5N AM, therefore the signal-to-noise ratio (SNR) will decrease throughout the experiment due to dye leakage from the SR. Limit experimental protocol to 1 - 2 hr&#160;to ensure high SNR values.</li>
	<li>Following the experiment, wash the perfusion system in the sequence of DI water, 70% reagent alcohol, and again with DI water.</li>
	<li>Filter each dataset with a spatial Gaussian filter (radius 3 pixels) using commercially available software packages according to the manufacturer&#8217;s protocol.</li>
	<li>If necessary, spatially align the Vm and SR Ca2+ data sets. As in Step 1.6, overlay images from each camera and verify that the anatomical structures of the heart (i.e., coronary vasculature, edges of the atria or ventricles) or other items in the field of view (i.e., cannula, pacing electrode) exactly overlap. If not, spatial alignment of the datasets is necessary.</li>
	<li>Shift the top transparent image in the x- and y-direction until exact overlap is achieved, making note of the number of pixels the image must be shifted in each direction. The entire dataset corresponding to the top image (either Vm or SR Ca2+) must then be shifted by the determined number of pixels in each direction to assure precise alignment between the Vm and SR Ca2+ datasets.</li>
</ol>

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The authors declare that they have no competing financial interests.

The keys to successful Fluo-5N dye loading are the small-volume recirculating perfusion setup, which allows for a high Fluo-5N concentration without the need for large amounts of dye, the length of loading time (1 hr), and performing the loading at RT. If loading is performed at physiological temperatures, cellular enzymatic activity quickly cleaves the &#8211;AM tag when the dye crosses the cell membrane, trapping the dye molecules in the cytosol and not allowing them to cross the SR membrane. At RT, however, enzymatic ...

Dual optical mapping of intracellular Ca2+ and transmembrane potential (Vm) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between Vm and intracellular Ca2+. However, optical mapping of intracellular Ca2+ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca2+ and is unable to distinguish whether these changes are due to transmembrane Ca2+ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca2+ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca2+ concentration5.
Each action potential triggers a rise in intracellular Ca2+, known as the intracellular Ca2+ transient (CaT). In the mammalian heart, approximately 70 &#8211; 90% of the total CaT is due to release of Ca2+ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)6. Within the SR, approximately half of the total Ca2+ is bound to calsequestrin (CSQ) and other intra-SR buffers7, which play an important role in SR Ca2+ homeostasis8,9. The amount of free SR Ca2+ dictates the driving force for SR Ca2+ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca2+ release or reuptake can, in turn, impact Vm via the electrogenic Na+-Ca2+ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca2+ can provide important insights into contractile and electrophysiological dysfunction.
Over the past several years, investigators have made significant advances in the monitoring of SR Ca2+ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca2+ content is then inferred or calculated from the immediate rise in intracellular Ca2+10. Another intriguing approach uses low-affinity Ca2+ indicators, such as Fluo-5N11 or Mag-Fluo412, which bind to free SR Ca2+. These indicators have dissociation constants (Kd) in the range of 10 &#8211; 400 &#956;M and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ concentration ([Ca2+]SR) is in the millimolar range. Using low-affinity Ca2+ indicators, several aspects of SR Ca2+ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca2+ release and the mechanisms of Ca2+ alternans13,14. However, in order to fully understand the heterogeneous nature of SR Ca2+ cycling in the intact heart and the role of SR Ca2+ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca2+ across the epicardial surface of the intact heart are required15.
This article describes methodology for dual optical mapping of free SR Ca2+ and Vm in the intact Langendorff-perfused rabbit heart with the low-affinity Ca2+ indicator Fluo-5N. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+.

<table><tbody><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>C79-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M33-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S369-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>AirGas</td><td>carbogen</td><td>For oxygenation and pH of Tyrode&#039;s solution</td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td>Excitation-contraction uncoupler</td></tr><tr><td>RH237</td><td>Biotium</td><td>61018</td><td>Voltage-sensitive dye</td></tr><tr><td>Fluo-5N AM</td><td>Invitrogen</td><td>F-26915</td><td>Low-affinity Ca&lt;sup&gt;2+&lt;/sup&gt; indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td></tr><tr><td>Pluronic F127</td><td>Biotium</td><td>59004</td><td>For Ca&lt;sup&gt;2+&lt;/sup&gt; indicator loading; Warm until the solution is clear before use</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td>For dissolving blebbistatin and dyes</td></tr><tr><td>Filter</td><td>EMD Millipore</td><td>NY1104700</td><td>11 &amp;#956;m in-line filter</td></tr><tr><td>Pressure Transducer</td><td>WPI</td><td>BLPR2</td><td>For measuring perfusion pressure</td></tr><tr><td>Transbridge Transducer Amplifier</td><td>WPI</td><td>SYS-TBM4M</td><td>For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td></tr><tr><td>PowerLab 26T</td><td>ADInstruments</td><td></td><td>For continuous recording of pressure and ECG signals</td></tr><tr><td>THT Macroscope</td><td>SciMedia</td><td></td><td>Macroscopic optical setup. Details: 0.63X objective (NA = 0.31), 2X condensing objective, resultant field of view = 3.1 cm x 3.1 cm, depth of focus = ~ 1.5 mm</td></tr><tr><td>MiCam Ultima-L CMOS</td><td>SciMedia</td><td></td><td>Optical mapping cameras</td></tr><tr><td>Precision LED Spot Light</td><td>Mightex</td><td>PLS-0470-030-15-S</td><td>LED light source</td></tr></tbody></table>

optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Figure 1A shows a schematic diagram of the optical configuration for dual Vm and SR Ca2+ mapping. With this setup, there is complete spectral separation of the Vm and SR Ca2+ signals (Figure 1B). A diagram of the dual-loop perfusion system used for Fluo-5N dye loading is illustrated in Figure 1C. Figure 1D shows the horizontal orientation of the heart in the perfusion dish. Representative Vm and SR C...

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Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

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Research

JoVE Journal

Biology

9.2K Views.  University of California Davis.  Dit artikel beschrijft de gedetailleerde protocol en de apparatuur die nodig is voor dual optische kaart brengen van transmembraan potentiaal (V m) en gratis intra-sarcoplasmatisch reticulum (SR) Ca 2+ in de Langendorff-doorbloed konijn hart. Deze methode maakt directe observatie en kwantificering van Vm en SR Ca 2+ dynamiek in het intacte hart. 

Video: Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

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 ...

Medicine

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 ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

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 ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

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

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

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

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

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

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

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

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca²⁺ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

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