The overall goal of the following experiment is to observe cardiac function and energetics in an isolated perfused mouse heart. This is achieved by perfusing a mouse heart in LOR mode. With Krebs hand select buffer enriched with glucose and pyruvate as a second step, a water-filled balloon is inserted into the left ventricle or lv, which allows for the constant monitoring of cardiac function during the experiment.
Next, the heart is placed into a 10 millimeter NMR tube and inserted into the magnet in order to assess cardiac energetics via observing the resonances of phospho creatine phosphates of a TP and the inorganic phosphate results were obtained that show cardiac function and energetics based on the LV pressure waves and P 31 and a mass spectroscopy p. Hi, I am Steve Kitz from the MIT Cadre Metabolism Center in the Department of Anesthesiology at the University of Washington. Today I'll show you a procedure for isolated heart perfusion combined with 31 PNMR spectroscopy.
We use this procedure in our laboratory to assess cardiac function and energetics in our bioengineered mouse models. So let's get started. To begin this protocol, prepare one liter of kres, hence light or KH buffer.
Introduce 5%carbon dioxide and 95%oxygen bubbles into the mixture for 10 to 15 minutes. Then add two millimolar calcium chloride, followed by substrates, 10 millimolar glucose and 0.5 millimolar pyruvate. For these experiments, two separate systems are utilized simultaneously per acquisition of P 31 Spectra.
A BRUER 14 T magnet is interfaced with the advanced three console and a computer equipped with top spin V 2.1 software for assessment of cardiac function. A custom built heart perfusion system is interfaced with the power lab. Four 30 data requisition.
Equipped with the lab chart PRO six software for data analysis. The heart perfusion apparatus includes a mini pulse three peristaltic pump controlled by an STH pump controller used to perfuse the KH buffer into the heart temperature regulation during the NMR experiment is critical. Heated circulators are used to maintain the temperature between 37.0 to 37.5 degrees Celsius while the heart is inside the magnet and the temperature is monitored for the duration of the experiment using a fiber optic temperature probe.
Throughout the experiment, the LV pressure and perfusion pressure are monitored via pressure transducers that are attached to a data acquisition system and displayed using the included software. A physiological transducer is used to monitor LV pressure and a disposable blood pressure transducer is used to monitor perfusion pressure. Finally, an umbilical cord is used to supply the heated oxygenated buffer to the heart.
Once inside the magnet, prior to the start of the experiment, calibrate the pressure transducers with the standards fig moment manometer first place, no pressure into the transducer, to zero the calibration. Then inflate this fig mode manometer to a higher pressure. Enter this value into the computer program.
Then confirm that the pressure transducers have been adequately calibrated by inflating this fig moment manometer to known pressures and verifying them on the computer screen. Once the pressure transducers have been calibrated, calibrate the NMR probe with a 10 millimeter NMR tube containing a standard sample of 150 millimolar sodium phosphate. Perform the calibration using the top spin software program.
First, tune the sample to set the frequency of the phosphorus resonance. Then shim the sample to create a homogenous magnetic field. This initial calibration facilitates the signal and decreases the time necessary to begin the acquisition period.
Once the heart is positioned within the probe to start the mouse heart extraction, inject the mouse with 200 units of heparin intraperitoneal to reduce coagulation. After five minutes, administer 175 milligrams per kilogram of sodium Penta barbital intraperitoneal. Once the mouse is anesthetized, place it on its back and tape the arms and legs to expose the chest area.
Perform a toe pinch to ensure that the mouse is fully anesthetized.Cut. Open the peritoneal cavity and chest with scissors and use forceps to peel back the rib cage and expose a thoracic cavity with forceps. Hold onto the lung tissue and gently lift the heart with curved scissors.
Cut the great vessels behind the heart and continue slowly towards the head of the mouse until the heart is free. Immediately arrest the heart in ice cold KH buffer. Keep the organs on ice and quickly remove the lungs.
Identify the lobes of the thymus and gently peel them back to expose the aorta. Then remove the thymus. Finally, isolate the aorta by carefully removing any surrounding tissue.
Using micro suturing forceps, generally hold both walls of the aorta to expose the lumen. Then carefully place the aorta onto the cannula. Made from 0.965 millimeter outer diameter polyethylene tubing.
Hold the aorta in place with a micro vessels clamp and quickly tie sutures around the aorta. Once the sutures are tied, remove the clamp. Use the forceps to carefully check that the cannula is above the aortic root.
Add any additional ties that are necessary to hold the heart in place. Remove any extra tissue using the forceps and micro scissors. Then make a small incision into the left oracle.
Gently hold the heart and carefully insert 0.61 millimeter od polyethylene tubing through the left atrium, left ventricular cavity, and out through the apex. Trim the excess tubing. Next, insert a deflated water filled balloon through the atrium into the left ventricle.
Hold the balloon in place using adhesive tape. Then gradually increase the peristaltic pump speed to provide sufficient flow to the heart. Continue to perfuse the heart with KH buffer using a constant flow equivalent to approximately two milliliters per minute until the heart is placed into the NMR probe.
Inflate the LV balloon with a small volume using a micrometer syringe to verify that the LV pressure transducer is functioning for NMR spectroscopy. First, carefully insert the heart into a 10 millimeter NMR tube. A wide boar spinner is placed around the NMR tube to help guide the tube into the proper position.
Within the probe, firmly attach the entire apparatus to the umbilical cord with adhesive tape. Next, slowly lower the umbilical cord into the upper bore of the magnet until the NMR tube containing the heart is inside the coil of the 10 millimeter NMR probe. Once the heart is in the proper position within the probe, adjust the parasitic pump flow in order to achieve a perfusion pressure of 80 millimeters of mercury.
Maintain the perfusion pressure by enabling the hold mechanism on the pump controller. Allow a 15 to 20 minute e equilibration period for the heart. During the equilibrium period, adjust the volume of the LV balloon to achieve an end diastolic pressure of eight to 10 millimeters of mercury.
Then optimize the spectrometer parameters in order to obtain the best possible phosphorous signal. Tune the NMR instrument by setting the radio pulse, so the frequency at which the phosphorus nucleus resonates. Then shim the sample to make the magnetic field homogeneous after the equilibration period.
Begin multiple P 31 NMR Spectra acquisition. The acquisition period for each spectrum is dependent on the field strength of the magnet, the size of the sample, and the signal to noise ratio required for a specific experiment. Here spectra are obtained using a 14 Tesla magnet by averaging the signal obtained from 256 radio frequency pulses of 20 microseconds with a 60 degree flip angle and delays of 2.0 seconds.
This experiment will require approximately 10 minutes. A typical measure of cardiac function, the LV developed pressure is obtained by subtracting the end diastolic pressure or EDP from the systolic pressure. In a normal C 57 black six mouse, heart LV developed pressure is typically between 100 to 110 millimeters of mercury at a fixed end diastolic pressure of eight to 10 millimeters of mercury.
Representative styling curves are shown from control and aortic banded mice. Systolic function is represented by LV developed pressure over increasing LV volumes as determined by the volume of the LV balloon. Diastolic function is represented by EDP over increasing LV volumes.
P 31 NMR spectra of an isolated perfused mouse heart provides signals of PHOSPHOCREATINE or PCR, the three phosphates from a TP and inorganic phosphate or pi. Here, the PI peak is relatively small. This is typical of an aerobically profused heart supplied with pyruvate or fatty acids.
In addition to glucose during periods of ischemia, this peak increases while the PCR peak decreases. I've just shown you how to prepare a mouse heart for perfusion so that both cardiac function and energetics can be measured simultaneously during the experiment. It's important to make sure that the heart is adequately perfused.
Otherwise, your NMR data will not be reliable. So that's it. Thank you and good luck with your experiments.
