The overall goal of this procedure is to simultaneously optically map free intra sarcoplasmic reticulum, calcium and transmembrane potential in the intact isolated LOR perfused rabbit heart. This is accomplished by first excising the rabbit heart and cannulating the aorta for retrograde lanor perfusion. The second step is to eliminate contraction by adding an excitation contraction uncoupling to the perfu eight.
Next, the perfusion is switched to a small volume recirculating loop and a low affinity calcium indicator is added at room temperature. The final steps are to warm the heart to 37 degrees Celsius using the large volume profusion loop and to add the voltage sensitive dye. Thus, during the experiment, changes in calcium and transmembrane potential are measured by exciting the heart with blue light and measuring the emitted light with high speed CM OS cameras.
The main advantage of this technique over existing methods is that it allows investigators to directly probe sarcoplasmic reticulum or SR for short calcium dynamics without interference from transmembrane calcium signals. This method can also help answer key questions related to the role of SR calcium handling in contributing to arrhythmias, because transmembrane potential is recorded simultaneously with SR calcium, Generally individuals new to this method will struggle because optical SR calcium signals are very low amplitude. Therefore, the proper loading of the calcium indicator is essential For this protocol, it is necessary to have freshly made tyro solution on hand.
Four liters will suffice. Don't mix the concentrated stock solutions without dilution. Also have the other solutions prepared in advance as directed in the text protocol bubble the supply of tyros with 95%oxygen and 5%carbon dioxide, and adjust the gas flow to set the pH of the solution very close to 7.4.
The solution should be at room temperature. Prepare the recirculating L andorf perfusion system by placing an inline nylon filter with 11 micron pores to filter the perfu sate. Now prime the main profusion system with about two liters of the oxygenated tyros.
The two liter total volume includes the solution in the reservoir. Next, prepare and prime a short profusion line that bypasses the solution reservoir in order to create a small volume recirculating profusion loop for flu oh five N am. Downloading the total volume in the smaller recirculation system should be about 150 milliliters.
To prepare the pressure monitoring system, connect a pressure transducer to the profusion line and connect the output of the transducer to a trans bridge amplifier. Now turn on the data acquisition system. Set it to continuously monitor the ECG and profusion pressure.
Next, adjust the baseline perfusion pressure to zero millimeters of mercury. Lastly, align the optical mapping camera as described in the text protocol. To begin secure the rabbit and anesthetize it with pentobarbital, along with a 1000 unit bolus of heparin.
Cover the eyes to calm the rabbit. Begin with a midline incision to expose the sternum and ribs using blunt tip scissors. Cut through the sternum between the xiphoid and meibum.
Next, carefully open the sternum without damaging the heart and spread the ribs to expose the heart. Then quickly remove the entire heart lung block by cutting away all the surrounding vessels and tissues. Transfer the heart lung block into about 100 milliliters of ice cold tyro solution.
In the excised tissue block, locate the aorta and cut the ascending aorta just proximal to the three branches of the aortic arch. Then cannulate the aorta with the eight gauge cannula attached to the profusion system. Secure the aorta to the cannula with a piece of USP zero silk suture.
After securing the cannula, carefully dissect away the trachea, lungs, and epicardial fat. Next, identify the mitral valve and carefully use sharp forceps and scissors to damage or remove one leaflet. Thus, solution congestion in the left ventricle cannot occur.
Continue by submerging the heart in the perfusion chamber horizontally with the anterior side up. Place the cannula onto a piece of sard. Secure the cannula with U-shaped pins inserted through the piece of sard and into the silicon bottom of the perfusion dish.
This will prevent the heart from moving. During imaging, a small insect pin can be used to stabilize the apex if needed, measure the amount of tyro solution used during the cannulation and replace it with fresh solution to keep the total circulating solution at two liters. Now fully submerge ECG electrodes into the bath on either side of the heart, but not in contact with the heart.
Then verify that this makes a normal lead one ECG morphology. Meanwhile, maintain the aortic pressure at 60 to 70 millimeters of mercury by adjusting the perfuses flow between 25 and 35 milliliters per minute. Now, turn off the room lights and add 0.3 to 0.6 milliliters of BLEs statin stock solution to the PERFU eight for a final concentration of 10 to 20 micromolar.
After 10 to 15 minutes, heart contractions should stop. Then switch over to the small volume profusion system. One of the keys to successful SR calcium imaging is proper loading of the flow five N am dye.
This requires a small volume perfusion to achieve high dye concentration and room temperature conditions. To avoid D esterification of the D in the cytosol, Now prepare 500 microliters of flu O five N am loading solution and mix it with 500 microliters of road solution. Then add it to the PERFU eight.
Let the downloading proceed for 45 minutes and then turn on the circulating water bath to warm the system to 37 degrees Celsius. After an hour of downloading, switch back to the larger volume of Perfu eight. Next, mix 50 microliters of the voltage sensitive dye RH 2 37 into a milliliter of warm tyros.
Slowly inject this into the system over about five minutes and proceed with the optical mapping of the heart. To begin, focus the optical mapping cameras on the appropriate field to reduce the motion on the PERFU eight surface. Float a cover slip on the solution.
Now adjust the LED beam of light to provide uniform illumination to the heart. For each optical recording, set the desired pacing protocol. Turn on the excitation light and collect one to four seconds of data within one to two hours.
Noise from leaked dye will put an end to the experiment. Then wash the system out with deionized water. Then 70%ethanol and finish with another rinse of deionized water.
Using the described protocol, the spectral separation of the transmembrane potential signal and the sarcoplasmic reticulum or SR calcium signal can be separated. Optical traces during continuous pacing were also recorded. Activation maps were then constructed from the data by plotting the activation time of each pixel.
On an isochronal graph, there is a typical delay of eight to 10 milliseconds between the two signals. It was possible to quantify the relative change in diastolic SR.Calcium load and systolic SR calcium release amplitudes at various pacing cycles. Pathological SR calcium handling was observed following the application of the beta adrenergic receptor agonist isoproterenol.
The red arrows point out a clear calcium leak from the sr. Under these conditions, calcium mediated triggering of action potentials can occur. After watching this video, you should have a good idea of how to perform simultaneous optical mapping of transmembrane potential and SR calcium signals in the intact Lang Endorf Perfused rabbit heart.
