This project received funding from the Israel Science Foundation under grant agreement No. 1379/18; the Jabotinsky Scholarship of the Israeli Ministry of Science and Technology for Applied and Engineering Sciences for Direct PhD Students No. 3-15892 for D.S.; and the European Union&#39;s Horizon 2020 research and innovation program under grant agreement No. 858149 (AlternativesToGd).

The joint ethics committee (IACUC) of the Hebrew University and Hadassah Medical Center approved the study protocol for animal welfare (MD-19-15827-1).
1. Krebs-Henseleit buffer preparation
<ol>
	<li>A day before the experiment, prepare a modified version of the Krebs-Henseleit buffer (KHB)26. Initially, dissolve 118 mM NaCl, 4.7 mM KCl, 0.5 mM pyruvate, 1.2 mM MgSO4, 25 mM NaHCO3, and 1.2 mM KH2PO4&#160;in double-distilled H2O.</li>
	<li>Bubble this mixture with 95%/5% O2/CO2&#160;for 20 min, and then add 1.2 mM CaCl2.</li>
	<li>Adjust the pH of the buffer to 7.4 with HCl or NaOH.</li>
	<li>On the day of the experiment, add 10 mM glucose and 72 U/L insulin to the KHB prepared in step 1.2. 
	NOTE: Insulin is added to the perfusion buffer as described in the work of Kolwicz et al.26&#160;and in agreement with previous studies reporting that insulin increases the contractile function27&#160;and the intensity of the hyperpolarized [13C]bicarbonate signal28.</li>
</ol>
2. Perfusion system preparation
<ol>
	<li>Keep a reservoir of 200 mL of KHB in a water bath at 40 &#176;C, and bubble with 95%/5% O2/CO2&#160;at a flow rate of 4 L/min for 1 h prior to cardiac perfusion. Keep the buffer continuously bubbling with this gas mixture throughout the experiment.
	<ol>
		<li>First, set the water bath to 40 &#176;C. Insert the KHB reservoir. Use a peristaltic pump (see Table of Materials) and medical-grade extension tubes to recirculate the KHB between the buffer reservoir and the 10 mm NMR tube at a constant flow rate of 7.5 mL/min.</li>
		<li>Connect three platinum-cured silicone tubes (3 mm i.d.) to the pump (one inflow tube and two outflow tubes for the KH buffer). Insert the outflow and inflow lines into the heated KH buffer. Then, insert the oxygen line into the heated KH buffer.</li>
		<li>Use thin polyether ether ketone (PEEK, see Table of Materials) lines for the buffer and hyperpolarized agent to flow to and from the NMR tube within the spectrometer&#39;s bore.</li>
	</ol>
	</li>
	<li>Ensure that the temperature is maintained at 37-37.5 &#176;C. Follow the steps below.</li>
	<li>Wrap the inflow line (from the buffer reservoir to the NMR tube) with a heating tape set to 42 &#176;C.</li>
	<li>Heat the NMR tube inside the spectrometer with a warm airflow that is regulated by the spectrometer.</li>
	<li>Use an NMR-compatible temperature sensor (see Table of Materials) to measure the temperature inside the NMR tube. The temperature is adjusted to 37-37.5 &#176;C.</li>
</ol>
3. Calibration and preparation of the NMR spectrometer for acquisition
<ol>
	<li>On the day of the experiment, insert a 13C standard sample that contains 1,4-dioxane (Table of Materials) into the spectrometer, and tune and match the NMR probe for 13C. Then, obtain a spectrum showing the thermal equilibrium signal of 1,4-dioxane with a nutation angle of 90&#176;.</li>
	<li>Now exchange the 13C standard sample with a 31P standard sample (Table of Materials), that contains 105 mM of ATP in D2O. Tune and match the NMR probe for 31P. 
	NOTE: A spectrum showing the thermal equilibrium phosphate signals is obtained with a nutation angle of 50&#176;.</li>
	<li>Insert the inflow line, the outflow line, and the temperature probe into a 10 mm NMR tube, and then insert the tube into the magnetic bore. Adjust the heating tape to 42 &#176;C.</li>
	<li>Acquire a&#160;31P NMR spectrum of the circulating KH buffer to be used in that experiment for 30 min, with a nutation angle of 50&#176; and a TR of 1.1 s (1,640 acquisitions).</li>
</ol>
4. Animal preparation, surgical procedure, and perfusion of the heart in the NMR tube
<ol>
	<li>Anesthetize a male HSD:ICR (CD-1) mouse with 3.3% isoflurane in room air (Table of Materials) at 340 mL/min for 5 min using a gas anesthesia system (Table of Materials) in an induction chamber.</li>
	<li>Use nasal anesthesia for the maintenance of general anesthesia with 2.9% isoflurane. 
	NOTE: Care is taken to minimize pain and discomfort to the animal.</li>
	<li>Secure the limbs of the animal with tape, ensure a negative pedal pain reflex, and then inject 300 IU of sodium heparin intraperitoneally.</li>
	<li>Wet the mouse&#39;s chest wall and abdomen thoroughly with 70% alcohol to ensure cleanliness and avoid hair contamination or obstruction during the surgical procedure.</li>
	<li>At 1 min after the heparin injection, cut the skin and muscle of the abdominal cavity with small scissors.</li>
	<li>Place the small curved-jaw hemostat-locking mosquito clamp between the xiphoid process and the chest skin to lift the chest and expose the diaphragm. Puncture and cut the right lobe of the diaphragm.</li>
	<li>Cut the chest across the midline, retract to the sides, and then remove.</li>
	<li>Inject the left ventricle of the heart with 200 IU of sodium heparin to prevent blood coagulation. Then, inject 0.1 mL of ice-cold 0.5 mol/L KCl to achieve cardiac arrest, as previously described25. Cardiac arrest is essential to be able to cannulate the heart.</li>
	<li>Identify the thymus, and remove it using scissors to expose the aorta. Remove residual rib cage tissue.</li>
	<li>Identify the aortic arch, and use curved forceps to place a loose knot with a 3-0 silk suture (Table of Materials) around the ascending aorta. Inject 3 mL of KHB into the left ventricle to remove blood clots from the aorta.</li>
	<li>Use curved forceps to retract the heart inferiorly for better visualization of the ascending aorta.</li>
	<li>Perform cannulation in situ with a 22 G intravenous catheter (Table of Materials). Apply cyanoacrylate adhesive in the cannulated region, and then perform double suture tying. Inject additional KH buffer into the heart, and verify that it flows through the cannulation tube.</li>
	<li>Remove the curved forceps. Disconnect the heart from the surrounding viscera, and perfuse it retrogradely with ice-cold KHB (4 &#176;C) through the intravenous catheter.</li>
	<li>Connect the heart to the inflow line of the perfusion system via the intravenous catheter. Upon the initiation of perfusion with warm buffer (37-37.5 &#176;C) at 7.5 mL/min, the heart begins beating spontaneously.</li>
	<li>Fix the NMR tube with the beating heart, outflow lines, and temperature probe, and insert into the bore of the spectrometer, making sure the heart is at the center of the NMR probe.</li>
</ol>
5. Acquiring data for cardiac energetics and pH
<ol>
	<li>Acquire&#160;31P spectra for about 1 h with a flip angle of 50 &#176; and a TR of 1.1 s.</li>
</ol>
6. DNP spin polarization and dissolution
<ol>
	<li>Prepare a 28.5 mg formulation of [1-13C]pyruvate. This formulation consists of 11.1 mM to 14.0 mM OX063 radical in the neat acid.</li>
	<li>Prepare 4 mL of dissolution medium. The dissolution medium consists of TRIS-phosphate buffer, which contains 11.2 mM NaH2PO4, 38.8 mM Na2HPO4, 33 mM TRIS, and 2 mM HCl. This medium composition is adjusted such that upon the addition of 28.5 mg of [1-13C]pyruvic acid formulation to 4 mL of this buffer (in the dissolution phase), the pH of the resulting solution will be 7.4.</li>
	<li>Perform spin polarization and fast dissolution in a dissolution-DNP (dDNP) spin polarization device according to the manufacturer&#39;s instructions (Table of Materials). Apply microwave irradiation at a frequency of 94.110 GHz for the polarization of the [1-13C]pyruvic acid formulation at 1.45 K to 1.55 K for about 1.5 h.</li>
	<li>Quickly mix the 4 mL of hyperpolarized medium from the dDNP device with a well-oxygenated solution that complements the hyperpolarized dissolution medium to obtain a composition closely matching the perfusion medium. 
	NOTE: The final volume of the medium perfusing the heart during the hyperpolarized injections with 14 mM hyperpolarized [1-13C]pyruvate is 26 mL. The final composition of the injected medium (after mixing) contains 4.7 mM KCl, 1.2 mM MgSO4, 70 mM NaCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 10 mM glucose, 1.2 mM CaCl2, and 72 U/L insulin.</li>
	<li>Administer the hyperpolarized [1-13C]pyruvate-containing medium to the isolated heart using a continuous flow setup29. 
	NOTE: This is done to ensure that the heart perfusion is not disturbed at any point during the experiment and that the hyperpolarized medium is administered at a known rate and for a known duration.</li>
</ol>
7. Hyperpolarized 13C spectroscopy
<ol>
	<li>Acquire hyperpolarized 13C data using product-selective saturating-excitation pulses15&#160;by applying 2.5 ms cardinal sine (Sinc) pulses, as previously described15,16. Selectively excite [1-13C]lactate and [13C]bicarbonate consecutively at 6 s intervals to obtain a 12 s interval for each metabolite.</li>
	<li>For [1-13C]lactate detection, center the selective Sinc pulse at the [1-13C]pyruvate hydrate frequency (179.4 ppm), which results in signal intensity ratio (lac) of 0.113 for the C1&#160;signals of [1-13C]pyruvate to [1-13C]lactate.</li>
	<li>For [13C]bicarbonate detection, center the selective Sinc pulse at 157.7 ppm, which is 214 Hz down-field of the [13C]bicarbonate signal (161.1 ppm); this results in a signal intensity ratio (bic) of 0.139 for the C1signal of [1-13C]pyruvate to [13C]bicarbonate.</li>
</ol>
8. Determination of the tissue wet weight and volume
<ol>
	<li>At the end of the experiment, detach the heart from the perfusion system, and gently dry it with tissue paper. Subsequently, weigh the heart to obtain the tissue wet weight.</li>
	<li>Determine the volume of the heart using a density factor of 1.05 g/cm3,&#160;as determined previously for the mouse heart30.</li>
</ol>
9. ATP content quantification
<ol>
	<li>Integrate the &#947;-ATP signal of a single acquisition of the ATP standard sample and a 30 min acquisition (TR of 1.1 s and 1,640 acquisitions) of the isolated heart.</li>
	<li>Quantify the ATP content of the heart by comparing the integral of the heart&#39;s &#947;-ATP signal to that of the standard (Table of Materials), where the latter has a known concentration (105 mM), and correct for the number of acquisitions and relaxation effects.</li>
</ol>
10. Resolving the Pi signal of the heart
NOTE: In order to evaluate the tissue pH, it is first necessary to deconvolve the heart&#39;s Pi signal from that of the total Pi signal (Pit). This is done by omitting the signal of the KHB Pi (PiKH) from that of the Pit.
<ol>
	<li>In a 31P spectrum of KHB that shows a single Pi signal (PiKH, Figure 1A), fit the PiKH signal to a Lorentzian function using Excel (Table of Materials).</li>
	<li>The volume visible to the NMR probe (Vp, 1.375 mL), contains more KHB when the sample tube does not contain the heart. To correct for this filling effect, calculate the attenuated buffer signal (Pib) using Eq. 1A. 
	<img alt="Equation 1" src="/files/ftp_upload/63188/63188eq01.jpg" style="margin: auto;" />&#160; &#160;Eq. 1A 
	where Vh is the heart&#39;s volume, as determined in step 8.</li>
	<li>Subtract this signal from the Pit according to Eq. 1B to obtain the Pi signal arising solely from the perfused heart (Pih, Figure 1B). 
	<img alt="Equation 2" src="/files/ftp_upload/63188/63188eq02.jpg" style="margin: auto;" />&#160; &#160;Eq. 1B</li>
</ol>
11. Multi-parametric pH analysis
<ol>
	<li>Perform the conversion of the Pih signal chemical shift distribution to the pH with reference to the chemical shift of PCr using Eq. 231. 
	<img alt="Equation 3" src="/files/ftp_upload/63188/63188eq03.jpg" style="margin: auto;" />&#160; &#160;Eq. 2 
	where &#916;&#948; is the chemical shift difference, pKa is 6.72, &#948; free base is 5.69, and &#948; free acid is 3.27, as previously described31.</li>
	<li>Correct the resulting pH distribution curve for the non-linearity between the Pih chemical shift scale and the pH scale according to Lutz et al.32. A typical pH distribution resulting from this calculation is presented in Figure 1C.</li>
	<li>Analyze the tissue pH distribution with a multi-parametric approach using seven statistical parameters following the work of Lutz et al.32. Four of these parameters are presented here as they appear to be the most descriptive of tissue pH: 1) global maximum pH; 2) weighted mean pH; 3) weighted median pH; and 4) skewness of the pH plot (Figure 1C).</li>
</ol>
12. Calculation of the LDH and PDH activities
NOTE: The production rates of the hyperpolarized metabolites [1-13C]lactate and [13C]bicarbonate are used to calculate the LDH and PDH activities, respectively. In the product selective saturating-excitation approach15, only newly synthesized hyperpolarized metabolites are detected by each selective excitation.
<ol>
	<li>Use the hyperpolarized [1-13C]pyruvate signal as a reference to determine the corresponding metabolite production level.
	<ol>
		<li>During the perfusion with the hyperpolarized medium,&#160;the [1-13C]pyruvate concentration in the NMR tube increases (wash-in), then plateaus (at a maximal concentration of 14 mM), and then decreases (wash-out).</li>
		<li>To identify the time points at which the pyruvate concentration reached a constant level (plateau), correct the [1-13C]pyruvate signal for signal decay resulting from T1&#160;relaxation and RF pulsation using the effective relaxation constant, Teff.</li>
		<li>For each injection, define the Teff&#160;based on its ability to correct the [1-13C]pyruvate decay curve to show this flow dynamics (Eq.3). 
		<img alt="Equation 4" src="/files/ftp_upload/63188/63188eq04.jpg" style="margin: auto;" />&#160; &#160;Eq. 3 
		where <img alt="Equation 5" src="/files/ftp_upload/63188/63188eq05.jpg" style="margin: auto;" /> is the [1-13C]pyruvate signal that was acquired during the [13C]bicarbonate acquisition. The average Teff in the experiments described herein was found to be 35.8 s &#177; 2.3 s (n = 5 hearts).</li>
		<li>Select the data points in which the concentration is within 10% of the maximal corrected [1-13C]pyruvate signal for further analysis.</li>
	</ol>
	</li>
	<li>Use the corresponding data of [1-13C]lactate and [13C]bicarbonate production for the time points selected in step 12.1 for the calculation of the metabolite production rates using Eq. 4A and Eq. 4B, provided that the SNR of the metabolite signal is larger than 2 (threshold for analysis). A typical example of such a selection of time points is shown in Figure 2B (highlighted temporal window).</li>
	<li>Calculate the production rates of each of the selected data points using Eq. 4A and Eq. 4B: 
	<img alt="Equation 17" src="/files/ftp_upload/63188/63188eq17.jpg" style="margin: auto;" />&#160; &#160;Eq. 4A 
	<img alt="Equation 6" src="/files/ftp_upload/63188/63188eq06.jpg" style="margin: auto;" />&#160; &#160;Eq. 4B 
	where <img alt="Equation 7" src="/files/ftp_upload/63188/63188eq07.jpg" style="margin: auto;" /> and <img alt="Equation 8" src="/files/ftp_upload/63188/63188eq08.jpg" style="margin: auto;" /> are the production rates of [1-13C]lactate or [13C]bicarbonate at each time point, respectively. <img alt="Equation 9" src="/files/ftp_upload/63188/63188eq09.jpg" style="margin: auto;" /> and <img alt="Equation 10" src="/files/ftp_upload/63188/63188eq10.jpg" style="margin: auto;" /> are factors that represent the relative excitation of [1-13C]pyruvate and the products [1-13C]lactate or [13C]bicarbonate, respectively. These factors were determined previously to be 0.113 and 0.139, respectively29. Vp is the volume that is detected by the NMR probe (1.375 mL), TR denotes the time interval between two consecutive product selective saturating excitations (12 s for each product), <img alt="Equation 11" src="/files/ftp_upload/63188/63188eq11.jpg" style="margin: auto;" /> and <img alt="Equation 12" src="/files/ftp_upload/63188/63188eq12.jpg" style="margin: auto;" /> are the signals of [1-13C]lactate and [13C]bicarbonate, respectively, and <img alt="Equation 13" src="/files/ftp_upload/63188/63188eq13.jpg" style="margin: auto;" /> and <img alt="Equation 14" src="/files/ftp_upload/63188/63188eq14.jpg" style="margin: auto;" /> are the signals of [1-13C]pyruvate that were acquired during the [1-13C]lactate and [13C]bicarbonate excitations, respectively. [Pyr] is the [1-13C]pyruvate concentration, which was 14 mM during the plateau phase.
	<ol>
		<li>Determine the rate for each point and then average per hyperpolarized injection.</li>
	</ol>
	</li>
</ol>

There are no disclosures.

We demonstrate an experimental setup that is designed to investigate hyperpolarized [1-13C]pyruvate metabolism, tissue energetics, and pH in an isolated mouse heart model.
The critical steps within the protocol are as follows: 1) ensuring that the pH of the buffer is 7.4; 2) ensuring that all components of the buffer are included; 3) avoiding blood clotting in the cardiac vessels by heparin injections; 4) avoiding ischemic damage to the heart by reducing the metabolic activity (KCl ...

Alterations in cardiac metabolism are associated with a variety of cardiomyopathies and often form the basis of the underlying pathophysiological mechanisms1. However, there are numerous obstacles to studying metabolism in living tissues, as most biochemical assays require the homogenization of the tissue and cell lysis and/or radioactive tracing. Therefore, there is a pressing need for new tools to investigate myocardial metabolism in living tissues. Magnetic resonance (MR) of hyperpolarized 13C-labeled substrates allows for real-time measurements of metabolism in living tissues2, without the use of ionizing radiation, by increasing the MR signal-to-noise (SNR) ratio of the labeled site(s) by several orders of magnitude3. Here, we describe an experimental setup, an acquisition approach, and an analytical approach for studying the rapid metabolism in the isolated mouse heart and, in parallel, present indicators of general tissue energetics and acidity. The cardiac pH is a valuable indicator, as the acid-base balance is disrupted in the early stages of cardiac diseases and conditions such as myocardial ischemia, maladaptive hypertrophy, and heart failure6.
Hyperpolarized [1-13C]lactate and [13C]bicarbonate production from hyperpolarized [1-13C]pyruvate helps in determining the production rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH). Most of the previous studies performed using hyperpolarized substrates in the isolated rodent heart either used complex kinetic models to derive the enzymatic activity of LDH and PDH, or reported the signal intensity ratios of the hyperpolarized product to a substrate without calculating the actual enzyme activity rates2,4,5,6,7,8,9,10,11,12,13,14. Here, we used the product selective saturating-excitations approach15, which allows for the monitoring of the enzyme activity in a model-free manner15,16. In this way, the absolute enzymatic rates (i.e., the number of moles of product produced per unit of time) were determined. 31P spectroscopy was utilized to observe the signals of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (ATP). A multi-parametric analysis was used to characterize the pH distribution of the heart, as demonstrated by the heterogeneous chemical shift in the Pi signal of the tissue.
The retrogradely perfused mouse heart (Langendorff heart)17,18,19&#160;is an ex vivo model for the intact beating heart. In this model, the heart viability and pH are preserved for at least 80 min20, and it has shown potential for recovery following a prolonged ischemic injury21,22. Nevertheless, inadvertent variability during micro-surgery may lead to variability in the tissue viability across hearts. Previous studies have reported on the deterioration of this heart over time19; for example, a reduction in contractile function of 5%-10% per hour has been observed18. The adenosine triphosphate (ATP) signal has previously been shown to report on the myocardial energetic status and viability23. Here, we noted that the perfused heart may occasionally show unintentional variability in viability levels, as demonstrated by the ATP content, despite the fact that we had an uninterrupted perfusion and oxygen supply. We demonstrate here that normalizing the LDH and PDH rates to the ATP content of the heart reduces the inter-heart variability in these rates.
In the following protocol, we describe the surgical procedure used for heart cannulation, isolation, and consequent perfusion in the NMR spectrometer. Of note, other surgical approaches aimed at isolating and perfusing the mouse heart have been described before24,25.
The methodologies used for acquiring data related to enzymatic rates in the beating heart (using 13C spectroscopy and hyperpolarized [1-13C]pyruvate) and the heart&#39;s viability and acidity (using 31P NMR spectroscopy) are described as well. Finally, the analytical methodologies for determining metabolic enzyme activities and tissue viability and acidity are explained.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HyperSense DNP Polariser</td><td>Oxford Instruments</td><td>52-ZNP91000</td><td>HyperSense, 3.35 T, preclinical dissolution-DNP hyperpolarizer</td></tr><tr><td>NMR spectrometer&amp;nbsp;</td><td>RS2D</td><td></td><td>NMR Cube, 5.8 T, equipped with a 10 mm broad-band probe</td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>Cole-Parmer</td><td>07554-95</td><td></td></tr><tr><td>Temperature probe</td><td>Osensa</td><td>FTX-100-LUX+</td><td>NMR compatible temprature probe</td></tr><tr><td>Somnosuite low-flow anesthesia system</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Lines, tubings, suture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Platinum cured silicone tubes</td><td>Cole-Parmer</td><td>HV-96119-16</td><td>L/S 16 I.D. 3.1 mm&amp;nbsp;</td></tr><tr><td>Thin polyether ether ketone (PEEK) lines</td><td>Upchurch Scientific</td><td></td><td>id. 0.040&amp;rdquo;</td></tr><tr><td>Intravenous catheter&amp;nbsp;</td><td>BD Medical</td><td>381323</td><td>22 G</td></tr><tr><td>Silk suture</td><td>Ethicon</td><td>W577H</td><td>Wire diameter of 3-0</td></tr><tr><td>&lt;strong&gt;Chemicals and pharmaceuticals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>[1-&lt;sup&gt;13&lt;/sup&gt;C]pyruvic acid</td><td>Cambridge Isotope Laboratories</td><td>CLM-8077-1</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>21074</td><td>CAS: 10043-52-4</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>CAS: 50-99-77</td></tr><tr><td>Heparin sodium</td><td>Rotexmedica</td><td>HEP5A0130C0160</td><td></td></tr><tr><td>Hydrochloric acid 37%</td><td>Sigma-Aldrich</td><td>258148</td><td>CAS: 7647-01-0</td></tr><tr><td>Insulin aspart (NovoLog)</td><td>Novo Nordisk</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Terrel</td><td></td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>793612</td><td>CAS: 7487-88-9</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P4504</td><td>CAS: 7447-40-7</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td>CAS: 7778-77-0</td></tr><tr><td>Sodium bicarbonate</td><td>Gadot Group</td><td></td><td>CAS: 144-55-8</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9625</td><td>CAS: 7647-14-5</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>655104</td><td>CAS: 1310-73-2</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S7907</td><td>CAS: 7558-79-4</td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Merck</td><td>6345</td><td>CAS: 13472-35-0</td></tr><tr><td>TRIS (biotechnology grade)</td><td>Amresco</td><td>0826</td><td>CAS: 77-86-1</td></tr><tr><td>Trityl radical OX063</td><td>GE Healthcare AS</td><td>NC100136</td><td>OX063</td></tr><tr><td>&lt;strong&gt;NMR standards&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C standard sample</td><td>Cambridge Isotope Laboratories</td><td>DLM-72A</td><td>40% p-dioxane in benzene-D6</td></tr><tr><td>&lt;sup&gt;31&lt;/sup&gt;P standard sample</td><td>Made in house</td><td></td><td>105 mM ATP and 120 mM phenylphosphonic acid in D&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Excel 2016</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MNova</td><td>Mestrelab Research</td><td></td><td></td></tr></tbody></table>

investigating cardiac metabolism in the isolated perfused mouse heart with hyperpolarized [1-13c]pyruvate and 13c/31p nmr spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

The 31P spectra recorded from a mouse heart perfused with KHB and from the buffer alone are shown in Figure 1A. The signals of &#945;-, &#946;-, and &#947;-ATP, PCr, and Pi were observed in the heart. The Pi signal was composed of two main components: in the higher field (left side of the signal), the Pi signal was mostly due to the KHB at a pH of 7.4; in the lower field (right side of the signal), the Pi signal was broader and less homogeneous due to the more acidic environment. ...

Watch this Scientific Journal Video about Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy at JoVE.com

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

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.

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

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

investigating-cardiac-metabolism-isolated-perfused-mouse-heart-with

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JoVE Journal

Biology

1.4K Views.  Hebrew University of Jerusalem. Hi.In deze video leert u hoe u hyperpolarized MR-studies van het doordrenkte muizenhart kunt uitvoeren. David Shaul, een MD / PhD-student in het lab, zal je door de verschillende technieken en procedures leiden. Vandaag leren we een protocol voor het bestuderen van hyperpolarized koolstof-13 pyruvaatmetabolisme, gecombineerd met 31P NMR-spectroscopie.Pyruvaat metabolisme staat op het kruispunt van aeroob en anaeroob metabolisme. En als zodanig is het van grote waarde voor het bestudere...

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

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Medicine

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

Cancer Research

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Engineering

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Bioengineering

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

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

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