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

Summary

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.

Abstract

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, 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.

Introduction

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 ....

Protocol

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

1. 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 in double-distilled H₂O.
2. Bubble this mixture with 95%/5% O₂/CO₂ for 20 min, and then add 1.2 mM CaCl₂.

Results

The ³¹P spectra recorded from a mouse heart perfused with KHB and from the buffer alone are shown in Figure 1A. The signals of α-, β-, and γ-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. The latter patt.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
HyperSense DNP Polariser	Oxford Instruments	52-ZNP91000	HyperSense, 3.35 T, preclinical dissolution-DNP hyperpolarizer
NMR spectrometer	RS2D		NMR Cube, 5.8 T, equipped with a 10 mm broad-band probe
Peristaltic pump	Cole-Parmer	07554-95
Temperature probe	Osensa	FTX-100-LUX+	NMR compatible temprature probe
Somnosuite low-flow anesthesia system	Kent Scientific
Lines, tubings, suture
Platinum cured silicone tubes	Cole-Parmer	HV-96119-16	L/S 16 I.D. 3.1 mm
Thin polyether ether ketone (PEEK) lines	Upchurch Scientific		id. 0.040”
Intravenous catheter	BD Medical	381323	22 G
Silk suture	Ethicon	W577H	Wire diameter of 3-0
Chemicals and pharmaceuticals
[1-13C]pyruvic acid	Cambridge Isotope Laboratories	CLM-8077-1
Calcium chloride	Sigma-Aldrich	21074	CAS: 10043-52-4
D-(+)-Glucose	Sigma-Aldrich	G7528	CAS: 50-99-77
Heparin sodium	Rotexmedica	HEP5A0130C0160
Hydrochloric acid 37%	Sigma-Aldrich	258148	CAS: 7647-01-0
Insulin aspart (NovoLog)	Novo Nordisk
Isoflurane	Terrel
Magnesium Sulfate	Sigma-Aldrich	793612	CAS: 7487-88-9
Potassium chloride	Sigma-Aldrich	P4504	CAS: 7447-40-7
Potassium phosphate monobasic	Sigma-Aldrich	P9791	CAS: 7778-77-0
Sodium bicarbonate	Gadot Group		CAS: 144-55-8
Sodium chloride	Sigma-Aldrich	S9625	CAS: 7647-14-5
Sodium hydroxide	Sigma-Aldrich	655104	CAS: 1310-73-2
Sodium phosphate dibasic	Sigma-Aldrich	S7907	CAS: 7558-79-4
Sodium phosphate monobasic dihydrate	Merck	6345	CAS: 13472-35-0
TRIS (biotechnology grade)	Amresco	0826	CAS: 77-86-1
Trityl radical OX063	GE Healthcare AS	NC100136	OX063
NMR standards
13C standard sample	Cambridge Isotope Laboratories	DLM-72A	40% p-dioxane in benzene-D6
31P standard sample	Made in house		105 mM ATP and 120 mM phenylphosphonic acid in D2O
Software
Excel 2016	Microsoft
MNova	Mestrelab Research