Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Summary

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (³¹PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Abstract

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (³¹PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo ³¹PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. ³¹PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for ³¹PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Introduction

The goal of this work is to outline a reproducible method to noninvasively measure in vivo skeletal muscle mitochondrial function in individuals possessing a wide range of abilities. Aberrant mitochondrial impairment is a hallmark of a wide range of metabolic syndromes and genetic diseases, from common conditions such as aging and diabetes to rare disorders such as Friedreich's ataxia.

Metabolic Syndrome and Mitochondrial Dysfunction

Metabolic syndrome has been shown to disrupt mitochondrial function, depress skeletal muscle OXPHOS, and lead to ectopic lipid storage in skeletal muscle^1,2. As critical organelles regulating metabolic and energy homeostasis, mitochondria are implicated in the pathophysiology of obesity^3,4, insulin resistance⁵, Type 2 Diabetes Mellitus (T2DM)^6,7, diabetes-related micro-^8,9,10,11 and macrovascular complications^12,13, and non-alcoholic fatty liver disease (NAFLD)^14,15,16, among others.Insulin resistance is characterized by profound changes in skeletal muscle mitochondrial activity, including decreased mitochondrial tricarboxylic acid (TCA) flux rate, ATP synthesis rate, and citrate synthase and NADH:O₂ oxidoreductase activity⁵. One hypothesis is that these alterations could be due to the accumulation of free fatty acid (FFA) metabolites in the muscle, which are markedly augmented during obesity and other obesity-related diseases^2,17. The exposure of muscle to elevated FFAs and lipid intermediates can decrease the expression of genes in the lipid oxidative pathway as well as the TCA cycle and electron-transport chain (ETC)¹⁸. This reduction in mitochondrial skeletal muscle OXPHOS capacity in the setting of a lipid overload is accompanied by a decline in the quantitative (content and biogenesis of mitochondria)¹⁹ and qualitative function of skeletal muscle mitochondria²⁰. Exposing skeletal muscle and myocytes to FFAs leads to severe insulin resistance, and increased FFA uptake in muscle is associated with insulin resistance in both humans and rodents²¹. The lipid intermediates ceramide and diacylglycerol (DAG) have been shown to directly inhibit the insulin signaling pathway by altering the activity of kinases, such as protein kinase C and protein kinase B²¹. Therefore, lipid-derived molecules appear to play a prominent role in the development of skeletal muscle insulin resistance and T2DM. However, it remains unclear whether changes in mitochondrial capacity are a cause or a consequence of insulin resistance²².

Friedrich's Ataxia and Mitochondrial Dysfunction

Decreased OXPHOS can also arise from genetic defects. Friedrich's ataxia (FA), the most common form of hereditary ataxia, is a genetic disorder caused by a mutation in the frataxin (FXN) gene, resulting in intra-mitochondrial iron accumulation, reactive oxygen species production, and abnormalities of oxidative phosphorylation^23,24,25,26. This important discovery has led to the development of targeted therapies, which aim to improve mitochondrial function at the sub-cellular level. Despite this understanding, there has been limited development of in vivo, reproducible biomarkers for FA clinical research. In fact, a critical barrier in the effective evaluation of targeted therapies in FA is the inability to track changes in mitochondrial function. Current functional measures, for example, can identify decreased cardiac output; however, they are incapable of determining the level at which the dysfunction occurs (Figure 1). The development of a reliable marker of mitochondrial function that can be used to identify and evaluate disease progression in Friedrich's ataxia is crucial to gauge the relevant mechanistic impact of targeted therapies.

Impaired OXPHOS and Cardiac Dysfunction

Aberrant mitochondrial function, either acquired or genetic, could contribute to the development or progression of cardiac dysfunction. Under the conditions of pressure overload and heart failure, the primary energy substrate preference switches from FFA to glucose. This is associated with decreased ETC activity and oxidative phosphorylation²⁷. The pathophysiology of mitochondrial bioenergetics in cardiac dysfunction can be different depending on the primary origin of the mitochondrial defect. Diabetes and metabolic syndrome results in mitochondrial abnormalities in myocardium, such as impaired biogenesis and fatty acid metabolism, which lead to reduced substrate flexibility, energy efficiency, and eventually, diastolic dysfunction^28,29. In FA, on the other hand, a frataxin deficiency results in significant mitochondrial iron accumulation in cardiomyocytes^30,31. Iron accumulation leads to the production of free radicals via the Fenton reaction³² and increases the chance of free radical-induced cardiomyocyte damage. Intra-mitochondrial iron accumulation is also associated with an increased sensitivity to oxidative stress and a reduced oxidative capacity^30,31. Iron accumulation and subsequent aberrant mitochondrial function, due to frataxin deficiency, may therefore be responsible for the impaired cardiac energetics and cardiomyopathy observed in FA^33,34. It is also interesting to note that the reduced oxidative capacity in skeletal muscle mitochondria parallels the exercise intolerance and reduced metabolic capacity in heart failure (HF)³⁵. Measurement of skeletal muscle OXPHOS capacity, as detailed herein, is readily implementable and robust; coupled with the significance of skeletal muscle OXPHOS in HF, these features make it an appealing biomarker in comprehensive studies of heart disease³⁶.

Impaired OXPHOS and the accompanying cardiac dysfunction is not an inconsequential aspect of metabolic and mitochondrial disease. Subjects with diabetes and metabolic disease are at a higher risk of developing cardiovascular disease and have excess mortality after myocardial infarction (MI)^{37,38,39,40,41}; over half of FA subjects have cardiomyopathy, and many die of cardiac arrhythmia or heart failure⁴². Therefore, quantification of reduced OXPHOS could not only allow for early detection and treatment of cardiac dysfunction, but it could also alleviate a major clinical burden in these diseases.

Targeted therapies to directly increase OXPHOS capacity is a promising area to improve the treatment of subjects, whether the cause of metabolic dysfunction is genetic or acquired. Currently, the development of novel targeted drugs that either alleviate abnormal mitochondrial function⁴³ or correct the primary genetic defect⁴⁴ can improve the deranged bioenergetics characteristic of FA. In the case of acquired mitochondrial dysfunction, increased physical activity can improve mitochondrial function ^45,46,47.

³¹Phosphorous Magnetic Resonance Spectroscopy as a Non-invasive Biomarker of Mitochondrial Function

Regardless of the tested therapy, an integrated in vivo assessment of skeletal muscle bioenergetics is a crucial tool to assess the impact of targeted interventions, especially in subjects with severe exercise intolerance or the inability to undergo conventional metabolic testing. Magnetic resonance spectroscopy tuned to phosphorous (³¹PMRS), an endogenous nucleus found in various high-energy substrates within cells throughout the body, has been used to quantify mitochondrial oxidative capacity using a variety of approaches, including in-magnet exercise-recovery protocols and muscle stimulation protocols⁴⁸. The exercise-recovery protocols rely upon a variety of apparatus ranging in complexity from MRI-compatible ergometers that regulate and measure workload to simple configurations of straps and pads allowing for burst-type resistive and quasi-static exercise. One of the primary goals of any of these protocols is to produce an energy imbalance for which the demand for adenosine triphosphate (ATP) is initially met through the enzymatic breakdown of phosphocreatine (PCr) through the creatine kinase reaction⁴⁹. Upon cessation of exercise, the rate of ATP production is dominated by oxidative phosphorylation and represents the maximum in vivo capacity of the mitochondria⁵⁰. Furthermore, OXPHOS during post-exercise recovery can be described by a first-order rate reaction⁵¹. The post-exercise recovery of PCr can therefore be quantified by the fitting of an exponential time constant (τ_PCr), with smaller values of τ_PCr representing greater capacities for oxidative ATP synthesis. Significant efforts have been made to validate ³¹PMRS against ex vivo and more direct measures of OXPHOS and demonstrate the potential clinical applicability of this technique^52,53,54,55.

Notably, the protocol described in this work can be implemented on clinically-available scanners, and it has been widely validated as a noninvasive biomarker of mitochondrial function⁵⁶. However, an exercise ³¹PMRS protocol optimized for application to individuals with varying severities of neuromuscular impairment or mobility has not been well established⁵⁷. A well-defined, broadly-applicable exercise protocol and ³¹PMRS technique would be particularly useful in the evaluation of diseases with fundamental abnormalities in mitochondrial function.

Several prior studies have explored the applications of non-invasive techniques to quantify mitochondrial function in subjects. For instance, these techniques have shown impaired OXPHOS in subjects with type 2 diabetes³⁶. Lodi et al. first tested the feasibility of PMRS techniques in subjects with FA and found that 1) the fundamental genetic defect in FA impairs skeletal muscle OXPHOS and 2) the number of GAA triplet repeats is inversely proportional to skeletal muscle OXPHOS³³. More recently, Nachbauer et al. used PMRS as a secondary outcome measure in an FA drug trial with 7 subjects. PCr recovery times were significantly longer in subjects compared to controls, reaffirming Lodi's earlier work and indicating that the effects of aberrant frataxin expression in FA can result in a decline in mitochondrial capacity that is detectable using PMRS techniques⁵⁸.

Reliable methods to adequately define in vivo skeletal muscle function in a feasible, cost-effective, and reproducible manner are critical to improving subject outcomes in a range of diseases that affect mitochondrial function.

This work outlines a robust procedure for obtaining in vivo maximum oxidative capacity of skeletal muscle using ³¹PMRS. The in-magnet exercise protocol is well tolerated by individuals spanning a wide range of physical and functional abilities and affords a simplified subject setup using inexpensive and widely-available equipment.

Protocol

This protocol is approved by and follows the guidelines of the Ohio State University Institutional Review Board for human subjects research. It is critically important that all procedures involving MR equipment are performed by adequately trained personnel adhering to the highest standards of MR safety⁵⁹.

1. Materials and Preparation

1. Ensure that all necessary materials are available prior to the experiment (Figure 2).
2. Plug the ³¹P coil into the in-table coil connector at the end of the exam table closest to the bore. Place a large triangle foam cushion near the head of the MR exam table, but not directly on the ³¹P coil. Place a head pillow at the other end of the MR exam table, farthest from the bore, for subject comfort.

2. Subject Positioning (Figure 3a)

1. Instruct the subject to lie supine, feet first on the MR table. Place a foam cushion under the knees to support the leg in a partially flexed position.
2. Position the subject close to the right side of the table (the subject's right) in order to center the left thigh as closely to the magnet isocenter as possible, thus ensuring optimal B0 homogeneity in the thigh muscle under examination. Provide the subject with ear plugs and/or headphones.
3. Position the ³¹P RF coil on the left quadriceps at approximately the midpoint between the patella and the femoral head, and secure to the leg using straps. Place the coil over the lateral portion of the leg, above the vastus lateralis.
4. Secure the baby oil to the medial aspect of the thigh with the same straps used to secure the coil to the leg. This facilitates scan localization.
5. Bind the subject's legs together with a strap placed below the coil and above the knee. Secure the subject's legs to the MR table with additional straps, one above the knee and one midway between the knee and ankle.
6. Use the laser light guide to delineate the center of the coil and move the table to the magnet isocenter using this centering landmark.

3. Exercise Protocol

1. Explain to the subject that the exercise protocol consists of three phases: an initial, baseline phase; a short, intense exercise phase; and a recovery phase.
2. Instruct the subject to lie still and relax their leg muscles during the baseline and recovery phases of the spectroscopy acquisition in order to minimize motion artifacts.
3. Provide a countdown to the subject indicating the start of exercise. At this point, have the subject initiate knee extension/flexion as forcefully and as rapidly as possible against the resistance of the straps.
   NOTE: The quadriceps muscles are used to move the left lower leg up and down, until instructed to stop.
4. Terminate exercise after a 30% drop in the PCr peak height.
   1. Observe the PCr peak height in the acquisition viewer window, and also view it upon completion of the exercise sequence.
      NOTE: A general guideline is that an approximate 30% drop in PCr peak height corresponds to a Pi peak that is 50% of the height of the PCr peak. If PCr depletion is not occurring rapidly enough to achieve a 30% drop during the exercise phase of the exam, encourage the subject to kick harder or faster while exercising.
      NOTE: Cessation of exercise is determined by monitoring the PCr peak height and duration of exercise. This may result in slightly different durations of exercise in different patients and can be accounted for in the analysis.

4. Scan Protocol

1. Acquire a tri-plane localizer to verify proper subject positioning and identify the location of the ³¹P coil.
   NOTE: The localizer sequence begins automatically and centers at the indicated position using the laser light guide (step 2.9)
2. Acquire a second tri-plane localizer.
   1. Open the slice view on the first tri-plane localizer images.
      NOTE: This process may be different for different software and hardware systems.
   2. Center and rotate the slice orientation by left-clicking and holding on the slice group. Rotate the slice group. Ensure that the final orientation of slices matches with the position of the baby oil.
   3. In the sequence routine window, increase the number of slices to cover the entire leg in the axial and sagittal images (Figure 3b).
3. ³¹P spectroscopy sequence:
   1. Use the following non-localized pulse-acquire sequence parameters: TR: 1,000 msec; TE: 0.34 msec; spectral width: 2,000 Hz; flip angle: 90 degrees; acquired data points: 1,024; 4 averages resulting in a time resolution of 1 spectrum every 6 sec.
4. ³¹P shim box placement:
   1. Using a mouse, drag the second triplane localizer images into the viewing window at the top of the screen. Drag the spectroscopy sequence into the protocol window and double-click to open.
   2. Use the position tool bar to visualize the shim voxel (select the black rectangle with horizontal lines). After selecting this option, observe a green box on the localizer images.
      NOTE: This is the shim voxel.
   3. Move the voxel by left-clicking and holding the voxel in the center. Change the size and rotate the orientation of the voxel by left-clicking and holding the voxel at the corner of the box. Place the shim box so as to ensure B0 field homogeneity directly below the coil and parallel to the plane of the quadriceps.
      NOTE: This is to ensure proper shimming within the sensitive region under the coil, which is the volume of tissue directly below the center of the coil.
   4. Use the tri-plane localizer images to identify the sensitive region of the coil and adjust the shim box to encompass this region within the quadriceps muscle.
      NOTE: The shim box can be larger than the true coverage of the surface coil in order to ensure B0 homogeneity within the data acquisition voxel (Figure 3c).
   5. ³¹P test acquisition:
      1. Open the acquisition viewer window and select the head icon in the acquisition tool bar. This will allow for viewing of the spectroscopy acquisition in real time.
      2. After placement of the 31P shim voxel, run the sequence to obtain a single spectrum by clicking the "run" button at the top of the protocol window.
      3. Examine the quality of B0 shimming. Observe the resulting spectrum in the acquisition window. Observe a prominent PCr peak centered at 0 ppm and no significant noise (Figure 4a, left).
         NOTE: Troubleshooting: If the spectrum appears noisy, ensure that the shim box is placed within muscle. Adjust the size and position of the shim box to improve the signal-to-noise ratio. Repeat the test acquisition as needed.
      4. In order to see the PCr peak height, open the spectrum in the spectroscopy tool ("Applications" → "Spectroscopy"). Open the patient's folder (folder tree icon), select the appropriate scan, and double-click to load the spectrum.
5. Pre-exercise T1 image:
   1. Obtain a single-slice axial T1-weighted image at the center of a coil.
6. ³¹P pre-exercise acquisition:
   1. Copy the sequence from step 4.4 (that produced the best spectral quality) by left-clicking and dragging the sequence in the protocol window. Use this sequence for all subsequent measurements.
   2. In the sequence routine window, increase the number of measurements from 1 to 10. Select run to acquire 10 measurements while the subject is at rest.
7. ³¹P exercise acquisition:
   NOTE: Make careful note of the start and end exercise times, as this will be important for analysis.
   1. Rest: Apply the shim settings from the previous scan and set the sequence to acquire 20 measurements. Instruct the subject to begin kicking after a countdown. Instruct the subject to remain at rest for 2 measurements.
   2. Exercise: Ask the subject to perform the knee extension exercise for ~ 30 sec (or the time required to achieve a 30% decrease in the PCr peak amplitude). After the subject achieves sufficient PCr depletion, ask them to rest.
8. ³¹P post-exercise acquisitions:
   1. Acquire an additional 20 measurements at rest. Ensure that the post-exercise acquisitions begin immediately following the exercise sequence, without pause or shimming (Figure 4a, right).
      NOTE: The subdivision of this recovery period into two separate acquisitions permits the analysis of the initial 20 dynamic spectra during the acquisition of the second 20 dynamic spectra, allowing the operator to avoid acquisition of the full recovery period if the exercise needs to be repeated.
9. Ensuring exercise quality:
   1. Compare the PCr peak heights at the beginning and end of exercise. High-quality exercise sessions result in a ~ 30% decrease in the PCr concentration.
   2. Verify that the PCr peak height is the same at beginning of rest and at the end of recovery (typically, < 10% difference is desired). This ensures that there was negligible loss of field homogeneity during acquisition.
      NOTE: If the PCr breakdown is insufficient, or if there has been a loss of field homogeneity, then repeat the exercise/recovery portion of the exam (taking care to avoid fatigue), ensure that the coil and straps are securely attached, and extend the duration of exercise and/or encourage more vigorous exercise (Figure 4b).
      NOTE: A comparison of the images obtained in steps 6 and 11 permits an additional quality control step to visualize any displacement of the thigh and coil due to the exercise, thus ensuring that minimal motion occurred during the protocol, which could significantly affect the acquired data.
10. Following post-exercise T1 imaging, repeat the pre-exercise axial T1 imaging (step 4.5) using the same acquisition parameters.
    1. In addition to sufficient depletion of PCr, measure the end exercise pH to ensure that the exercise did not induce acidosis of the muscle.
    2. Perform this by measuring the chemical shift between Pi and PCr (δP_i) and using the following equation⁶⁰:
       pH = 6.77 + log[(δP_i-3.29)/(5.68-δP_i)]
       NOTE: The pH should remain greater than 6.8⁶¹. If the PCr breakdown is sufficient but the pH is too low, repeat the exercise bout for a shorter duration and/or with a decreased intensity.
11. Saving Data:
    1. Save all acquired spectra as DICOM files and export them for processing using JMRUI.
    2. If using a scanner, select all spectroscopy acquisitions in the "Navigator" window.
    3. Under "Applications," select "Dicom Tools" → "Export MR Spectroscopy," and save the DICOM (*.dcm) files to C:/User/MedCom/temp/CDROFFLINE
       (the tool automatically chooses this location).
    4. Under "Transfer," select "Export to Offline." Save to the desired location.

5. Data Processing and Analysis⁶²

1. Analyze the MR spectra with freely-available JMRUI software (version 5.2; http://www.jmrui.eu/).
2. Apodize and phase shift the spectra to ensure uniformity over all acquired time points (Figure 5). The PCr peak will be centered at 0 ppm in the spectra.
3. Use the built-in AMARES algorithm to quantify the amplitude of the PCr peak in each acquired spectrum. The peak amplitude represents the concentration of PCr within the sensitive region of the surface coil at that particular time point.
4. In the computational software, plot the PCr concentrations as a function of acquisition time. Using the built-in computational software curve-fit tool, fit the PCr recovery period data to the following equation^52,63:
   
5. Record the values of the baseline PCr (), the lowest PCr (), and the recovery time(.
6. Ensure that the appropriate conditions are met during the exercise session by calculating the PCr depletion, the percent difference between the baseline PCr and the lowest PCr. Ideal exercise sessions result in a 20 - 50% depletion.
   NOTE: The quality of the curve fitting can be ensured by verifying that the R² value is greater than 0.75. R² values are automatically calculated by the fitting software.

Results

Reproducibility Study

Six volunteers (4 men and 2 women; mean age: 24.5 ± 6.2 years) with no self-reported heart, metabolic, or mitochondrial disease underwent sessions of the described ³¹PMRS exercise and imaging technique on 2 different days within 1 week to evaluate technique reproducibility (Figure 6a). The studies performed on normal volunteers confirm the reproducibility of...

Discussion

This paper describes a standard protocol for ³¹PMRS examination that affords serial and noninvasive in vivo measurement of skeletal muscle mitochondrial function. The protocol holds considerable appeal when considering the breadth of investigations targeting the growing burden of metabolic syndrome and its resulting morbidity and mortality. This ³¹PMRS protocol requires a minimal amount of scanner time and can be incorporated into comprehensive metabolic investigations in subjects at any ce...

Materials

Name	Company	Catalog Number	Comments
1.5 T MR Scanner	Siemens		manufacturer will not affect results
10 cm 31P transmit-receive coil, 1.5T compatible	PulseTeq		manufacturer will not affect results
3 fl oz Baby Oil	Johnson & Johnson		manufacturer will not affect results
Foam triangle cushion (Knee)	Siemens		manufacturer will not affect results
(3) plastic buckle resistive straps; table to table	Siemens		manufacturer will not affect results
(1) plastic buckle resistive strap; self-connecting	Siemens