Name: investigating cardiac metabolism in the isolated perfused mouse heart with hyperpolarized [1-13c]pyruvate and 13c/31p nmr spectroscopy
Uploaded: 2023-04-21T12:00:00
Description: 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

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 ...

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.

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	<tbody>
		<tr>
			<td>Equipment</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&#160;</td>
			<td>RS2D</td>
			<td></td>
			<td>NMR Cube, 5.8 T, equiped with a 10 mm broad-band probe</td>
		</tr>
		<tr>
			<td>Peristaltic pump&#160;</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>Lines, tubings, suture</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&#160;</td>
		</tr>
		<tr>
			<td>Thin polyether ether ketone (PEEK) lines</td>
			<td>Upchurch Scientific</td>
			<td></td>
			<td>id. 0.040&#8221;</td>
		</tr>
		<tr>
			<td>Intravenous catheter&#160;</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>Chemicals and pharmaceuticals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[1-13C]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 monbasic 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>NMR standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>13C standard sample</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-72A</td>
			<td>40% p-dioxane in benzene-D6</td>
		</tr>
		<tr>
			<td>31P standard sample</td>
			<td>Made in house</td>
			<td></td>
			<td>105 mM ATP and 120 mM phenylphosphonic acid in D2O</td>
		</tr>
		<tr>
			<td>Software</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>
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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 ...

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