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 border="0" cellpadding="0" cellspacing="0" width="956">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S271-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">CaCl2 (2H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">C79-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">KCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S217-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MgCl2 (6H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">M33-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaH2PO4 (H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S369-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaHCO3</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S233-3</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">D-Glucose</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">D16-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">95% O2 5% CO2</td>
			<td style="width:167px;">AirGas</td>
			<td style="width:151px;">carbogen</td>
			<td style="width:423px;">For oxygenation and pH of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Blebbistatin</td>
			<td style="width:167px;">Tocris Bioscience</td>
			<td style="width:151px;">1760</td>
			<td style="width:423px;">Excitation-contraction uncoupler</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">RH237</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">61018</td>
			<td style="width:423px;">Voltage-sensitive dye</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Fluo-5N AM</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:151px;">F-26915</td>
			<td style="width:423px;">Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pluronic F127</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">59004</td>
			<td style="width:423px;">For Ca2+ indicator loading; Warm until the solutiion is clear before use</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td style="width:423px;">For dissolving blebbistatin and dyes</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Filter</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:151px;">NY1104700</td>
			<td style="width:423px;">11um in-line filter</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pressure Transducer</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">BLPR2</td>
			<td style="width:423px;">For measuring perfusion pressure</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Transbridge Transducer Amplifier</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">SYS-TBM4M</td>
			<td style="width:423px;">For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">PowerLab 26T</td>
			<td style="width:167px;">ADInstruments</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">For continuous recording of pressure and ECG signals</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">THT Macroscope</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1x3.1 cm, depth of focus = ~1.5mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MiCam Ultima-L CMOS</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Optical mapping cameras</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Precision LED Spot Light</td>
			<td style="width:167px;">Mightex</td>
			<td style="width:151px;">PLS-0470-030-15-S</td>
			<td style="width:423px;">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...

Watch this Scientific Journal Video about Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart at JoVE.com

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 ...

optical-mapping-intra-sarcoplasmic-reticulum-ca2-transmembrane

Research

JoVE Journal

Biology

9.1K Views.  University of California Davis. 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.