Phosphorus-31 Magnetic Resonance Spectroscopy for Serial Measurements of Cardiac Energetics During Temperature-Controlled Heart Preservation

Summary

In this article, we have described a reproducible, temperature-controlled protocol for ex vivo Phosphorus-31 Magnetic Resonance Spectroscopy, enabling serial, quantitative assessment of myocardial energetics during organ preservation.

Abstract

Reliable organ preservation is critical to increasing donor availability and improving transplant outcomes. While static storage remains the standard for donor hearts, there is a need for precise methods to monitor myocardial energetics throughout preservation.

Phosphorus-31 Magnetic Resonance Spectroscopy (³¹P MRS) enables direct, dynamic measurement of key energy metabolites, including Phosphocreatine (PCr), Adenosine TriPhosphate (ATP), inorganic phosphate (Pi), and intracellular pH. This technique allows for longitudinal, quantitative, and precise evaluation of myocardial metabolic status during ex vivo storage. Advantages of MRS include dynamic assessment of energy status and a global assessment of ventricular energy.

The protocol begins with swine donor recovery, cardioplegia administration, and cardiectomy. The aortic arch is cut proximal to the brachiocephalic trunk to maximize the length of the ascending aorta. A ribbed cannula is secured into the distal ascending aorta, the left atrium is marked, and the heart is placed in a cylindrical, insulated container pre-filled with temperature-specific cardioplegia. The lid connects to the cannula, suspending the heart inside, and the container is sealed, maintaining a temperature within 1 °C for 1 h.

The container is placed in a tailored Styrofoam box with an opening for a phosphorus tuned coil, aligning the coil center with the center of the left ventricle (LV). Scout images confirm LV placement near the ³¹P coil. B₀ shimming ensures uniform excitation, followed by acquisition of a non-localized ³¹P MR spectrum. Post-processing identifies spectral peaks for calculating PCr/ATP, PCr/Pi ratios, and intracellular pH.

In our protocol, ³¹P MRS is performed at serial timepoints between h 0 to 8. Between timepoints, the heart is stored in a standard storage device at the desired temperature.

³¹P MRS has allowed us to study time-dependent changes to energy depletion and the role of storage temperature in mitigating energy depletion over time.

Introduction

Heart transplantation remains the definitive treatment for select patients with end-stage heart failure¹. However, a persistent shortage of suitable donor organs and the limitations of the current preservation strategies are major barriers to optimal outcomes and expanded transplant availability^2,3. Traditionally, donor hearts are preserved using cold static storage, which is straightforward and cost-effective, but is susceptible to subclinical energetic depletion during prolonged transport^2,4. As programs increasingly accept hi....

Protocol

This protocol follows institutional animal care guidelines and was approved by the Johns Hopkins Animal Care and Use Committee (Protocol number SW24M319) for all procedures involving live animals up to and including surgical harvest. All subsequent ex vivo experiments were performed using tissues obtained after euthanasia.

1. Pre-Harvest procedure

1. Set the incubator to a temperature that is 2 °C below the target preservation temperature. Allow the incubator to stabilize for at least 2 h.
2. Place 6-7 ice packs in the incubator to pre-chill them to the set temperature. Ensure ice packs are sufficie....

Results

The localized 2^nd order shimming¹⁶ resulted in a more homogenous B₀ magnetic field across the region of interest, as indicated by a reduction in the standard deviation at each time point (0 h: 14.0 to 4.0 Hz; 4 h: 17.5 to 3.2 Hz; 8 h: 16.0 to 3.2 Hz). This ensured that excellent spectral quality was achieved at each time point (Figure 7). The myocardial metabolic changes during ex vivo cold storage indicate a rapid depletion of high-ener.......

Discussion

This manuscript describes a surgical preparation and a ³¹P MRS acquisition and analysis protocol for the serial assessment of myocardial energetics during heart preservation. ³¹P MRS allows for serial, non-destructive assessment of key cardiac metabolites at different cold storage temperatures, which provides novel insights into the metabolic viability of the myocardial tissue. This ³¹P MRS protocol is relatively fast, requiring minimal scanner time, which allows for the assessment of car.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Incubator	Benchmark Scientific	H2265-HC	MyTemp 65HC Heating Cooling Incubator
Insulating cooler	Gatorade	UPC 052000047097	Gatorade Insulated Sport Beverage Cooler
jMRUI version 7.0	Java	www.jmrui.eu	Graphical user interface for data analysis
Myocardial temperature probe	DeRoyal	81-030415	DeRoyal 400 series myocardial temperature probe (15mm)
Magnetic Resonance Scanner	Philips	https://www.philips.com/healthcare/diagnostic-imaging/magnetic-resonance-imaging	Achieva 3T, Philips Healthcare
Single loop phosphorus coil	Philips Healthcare	https://www.philips.com/healthcare/diagnostic-imaging/magnetic-resonance-imaging	P140, Philips Healthcare