Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-¹³C]Pyruvate and ¹³C/³¹P NMR Spectroscopy

Hi.In this video you will learn how to perform hyperpolarized MR studies of the perfused mouse heart. David Shaul, an MD/PhD student in the lab, will guide you through the various techniques and procedures. Today we learn a protocol for studying hyperpolarized carbon-13 pyruvate metabolism, combined with 31P NMR spectroscopy.

Pyruvate metabolism stands at the crossroad of aerobic and anaerobic metabolism. And as such, it is of great value for studying various conditions of the heart. So, let's get started.

The mouse heart is isolated and perfused with Krebs-Hensleit buffer, inside a 10 millimeter NMR tube. The heart is then inserted to the NMR spectrometer. There, a 31P spectrum is recorded to observe cardiac energetics and pH, by observing the resonances of adenosine triphosphate, phosphocreatine and inorganic phosphate.

Meanwhile, a pyruvate sample that is labeled with carbon-13 in the first position is hyperpolarized. And after dissolution occurs, it is injected to the heart to allow for measuring of lactate, inorganics, and pyruvate the organized activities in real time. A day before the experiment, prepare 400 milliliters of modified Krebs-Henseleit buffer.

As the first step, we solve these ingredients in double distilled water. Then, bubble this solution with this oxygen mixture for 20 minutes, and then add calcium chloride. Adjust the pH to 7.4.

On the day of the experiment, add glucose and insulin. Set the water bath to 40 degrees Celsius. Insert the medium bottle with 200 milliliters of KH buffer to the bath.

Use peristaltic pump to recycle the KH buffer. Connect to it, one inflow line and two outflow lines. Insert the inflow and outflow lines to the heated KH buffer.

Use an oxygen mixer with 95%O2 and 5%CO2. Then, insert the oxygen line to the heated KH buffer. Turn on the peristaltic pump and adjust it to a constant flow weight of 7.5 milliliters per minute.

We want to calibrate the system before introducing the heart. Therefore, insert the inflow and outflow lines to a 10 millimeter NMR tube, and insert optical temperature probe that is NMR compatible. Insert the NMR tube to the magnet bore.

Adjust the heating tank to 42 degrees Celsius and use the NMR heating to adjust the temperature inside the magnet to 37 degrees Celsius. Note that the NMR temperature is actually monitored in the KH buffer that is inside the magnet, using an NMR compatible temperature core. Now, we can use 31P spectroscopy to observe the signal of inorganic phosphate inside the buffer.

This is the equipment that is required for the surgical procedure. Place 100 milliliter of KH buffer in ice and bubble it with the same oxygen mixture. Anesthetize the mouse inside the box chamber with 3.3%isoflurane mixed with room air for induction, at a flow rate of 340 milliliters per minute.

Transfer the mouse to nasal anesthesia. Reduce to 2.9%isoflurane at the same flow rate. Pinch the foot to verify a negative pedal pain reflex, and that the mouse is fully anesthetized.

Inject the mouse with 300 units of sodium heparin, intraperitoneally. Cut the skin two centimeters below the xiphoid process. Cut the abdominal wall to expose the abdominal cavity.

Place scissor clamp between the xiphoid process and the chest skin, and use it to retract the chest wall and exposing the diaphragm. Puncture the right lobe of the diaphragm and then cut the rest of the diaphragm. Cut across the chest wall midline while avoiding contact with deeper organs such as the heart and the vessels.

Inject 200 units of sodium heparin to the left ventricle of the heart. Inject the heart with 0.1 milliliters of 0.5 molar of KCL to achieve cardiac arrest. Retract the thymus tissue anteriorly and cut it from it root to expose the aorta underneath it.

Try to remove as much thymus tissue as you can while avoiding damage to the aorta. Remove residual ribcage tissue to allow for free passage for the intravenous catheter. Place the curved forceps underneath the aorta and use it to place silk suture knot around the aorta.

Place curved forceps at the aortic root and retract the heart inferiorly to expose and stretch the aorta. Inject three milliliters of ice-cold KH buffer to the left ventricle to remove blood clots from the aorta and for preserving the heart viability. Place several drops on the heart surface as well.

Use intravenous catheter needle to puncture the arterial wall of the aorta without damaging the posterior wall. Then, insert the catheter with the needle, about three millimeters. Afterwards, remove the needle and simultaneously insert the catheter tube for additional five millimeters but avoid entering the left ventricle chamber.

Place cyanoacrylate adhesive in the puncture region of the aorta, to prevent from the catheter tube to slide out of the aorta while tightening the suture. Gently double tie a knot between the aorta and the cannulation tube. Inject additional five milliliters of KH buffer to the left ventricle and verify that it flows through the cannulation tube.

This marks that the cannulation was successful. Remove the curved forceps from the aortic root. Disconnect the heart from the surrounding viscera while avoiding contact with the cannulation agent and with the cardiac tissue.

Immediately connect the cannula to a flowing ice-cold KH buffer syringe. It is important to avoid introduction of air bubbles into the heart. Remove non-cardiac tissue.

At this stage, you should observe the heart resume beating. Cut the remaining silk suture edges. In the NMR room, disconnect the cannula from the syringe and connect it to the 37 degrees KH buffer of the perfusion system.

Then, insert the heart to the 10 millimeter NMR tube. Place the heart at the center of the probe and then insert the NMR tube with the heart to the bore of the NMR spectrometer. Perform shim"using the water signal on the 1H channel until line width of 10 to 20 hertz is achieved.

Then, acquire 31P spectra of the heart by using flip angle of 50 degrees and TR of 1.1 second at steady state. These conditions favor the signal of ATP, over the signals of PCr and inorganic phosphate. Observing the signals of ATP and PCr, means that the tissue is viable inside the NMR spectrometer.

Use a dedicated processing program to analyze the spectra. Perform exponential uperization of seven hertz. Use baseline correction, and then, assign the phosphocreatine signal to minus 2.5 ppm.

Observe the signals of inorganic phosphate, phosphocreatine, and ATP. Polarize the sample of pyruvate, that is labeled in the first position with carbon 13, for 80 minutes. After 80 minutes of polarization, the sample is ready for the solution.

This is how the dissolution occurs. Inject the dissolution content to the heart using the continuous perfusion approach. This approach was designed to deliver the pyruvate without any interruption to the tissue perfusion and oxygenation level.

The solution media that contains the hyperpolarized pyruvate is injected to a conical tube, then injected manually to a bypass, and then, we direct the perfusion through the bypass, and the bypass content is flowing through the heart continuously, and eventually washes out. We use a perfusion rate of 7.5 milliliters per minute and bypass volume of 22 milliliters. So, that the hyperpolarized media is flowing through the heart for about three minutes.

In this time window, we use carbon 13 spectroscopy to measure pyruvate, lactate, and bicarbonate signals. We use an excitation scheme that alternates between lactate excitation and bicarbonate excitation, with intervals of six seconds. We used saturating selective excitation to acquire the carbon 13 signal.

In this approach, the substrate pyruvate is minimally excited, while the metabolites, lactate and bicarbonate, are fully excited. Observe and record carbon 13 pyruvate metabolism for about three minutes. The metabolic investigation is finished after the pyruvate signal decays.

This figure shows 31P spectra recorded from a mouse heart perfused with KH buffer. The spectrum acquired from the heart, shows the signals of alpha, beta, and gamma ATP, PCr, and Pi.The Pi signal is composed of two main components, The component in the left, which appears in a higher field, represents Pi signal that is mostly due to KH buffer at a pH of 7.4. The border and less homogenous component in the right, which is in a lower field, shows the Pi signal that is in a more acidic environment.

This component arises from the cardiac tissue. Next, the tissue's Pi signal was obtained by subtracting the buffer Pi signal from the entire Pi signal. It was then converted from a ppm scale to a pH scale.

The pH is investigated using a multiparametric analysis of the tissue Pi signal by calculating the weighted mean, weighted median, global maximum, and skewness. This figure shows typical carbon 13 NMR spectrum, obtained using the hyperpolarized product selective saturating-excitation approach, during hyperpolarized carbon 13 pyruvate injection to the perfused mouse heart. Notice lactate pyruvate and bicarbonate signals.

Pyruvate signal is truncated here for displayed purposes. The changes in signal intensities of the substrate and the metabolites are due to T1 relaxation, and the frequency of excitation, and flow characteristics. Next, the integrated intensities of these signals are plotted.

In addition, we plotted in black circles, the pyruvate integrated intensity that was collected for T1 decay, and for the effect of 4D frequency excitations, using an effective decay time constant of 32 seconds. This correction was found to yield the expected flow dynamics for the substrate. Wash-in, plateau, and wash-out.

Using this corrected signal time course, we selected for further analysis, the time window that is highlighted in light blue, in which the pyruvate concentration in the NMR tube, was constant and maximal. The rates of LDH and PDH were calculated for each of the selected time points and then averaged. The main values for this injection are provided in units of nanomole per second.

In summary, we have shown the experiment system for performing hyperpolarized pyruvate metabolic studies in the perfused mouse heart. We hope that this information was useful.