Name: phosphorus-31 magnetic resonance spectroscopy: a tool for measuring in vivo mitochondrial oxidative phosphorylation capacity in human skeletal muscle
Uploaded: 2017-01-19T15:00:00
Description: Watch this Scientific Journal Video about Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle at JoVE.

This work was supported in part by a Davis Heart and Lung Research Institute Trifit Award, as well as by the Intramural Research Program of the NIH National Institute on Aging.

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
<ol>
	<li>Ensure that all necessary materials are available prior to the experiment (Figure 2).</li>
	<li>Plug ...

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...

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&#39;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 muscle1,2. As critical organelles regulating metabolic and energy homeostasis, mitochondria are implicated in the pathophysiology of obesity3,4, insulin resistance5, Type 2 Diabetes Mellitus (T2DM)6,7, diabetes-related micro-8,9,10,11 and macrovascular complications12,13, 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 diseases2,17. 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&#39;s Ataxia and Mitochondrial Dysfunction
Decreased OXPHOS can also arise from genetic defects. Friedrich&#39;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 phosphorylation23,24,25,26. 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&#39;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 dysfunction28,29. In FA, on the other hand, a frataxin deficiency results in significant mitochondrial iron accumulation in cardiomyocytes30,31. 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 capacity30,31. Iron accumulation and subsequent aberrant mitochondrial function, due to frataxin deficiency, may therefore be responsible for the impaired cardiac energetics and cardiomyopathy observed in FA33,34. 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 (&#964;PCr), with smaller values of &#964;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 technique52,53,54,55.
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&#39;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.

<table border="0" cellpadding="0" cellspacing="0" width="883">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1.5 T MR Scanner</td>
			<td style="width:165px;">Siemens</td>
			<td style="width:136px;"></td>
			<td style="width:251px;">manufacturer will not affect results</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">10 cm 31P transmit-receive coil, 1.5T compatible</td>
			<td style="width:165px;">PulseTeq</td>
			<td style="width:136px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">3 fl oz Baby Oil</td>
			<td style="width:165px;">Johnson &#38; Johnson</td>
			<td style="width:136px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Foam triangle cushion (Knee)</td>
			<td style="width:165px;">Siemens</td>
			<td style="width:136px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">(3) plastic buckle resistive straps; table to table</td>
			<td style="width:165px;">Siemens</td>
			<td style="width:136px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">(1) plastic buckle resistive strap; self-connecting</td>
			<td style="width:165px;">Siemens</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

phosphorus-31 magnetic resonance spectroscopy: a tool for measuring in vivo mitochondrial oxidative phosphorylation capacity in human skeletal muscle

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.

Reproducibility Study
Six volunteers (4 men and 2 women; mean age: 24.5 &#177; 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...

Watch this Scientific Journal Video about Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle at JoVE.

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

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.

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

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

The overall goal of this In Vivo magnetic resonance technique and exercise protocol is to establish a robust and standardized method for the noninvasive measurement of mitochondrial oxidative phosphorylation capacity. Skeletal muscle mitochondrial oxidative phosphorylation capacity can be measured noninvasively with In Vivo phosphorous 31 magnetic resonance spectroscopy. This standardized technique will facilitate the development of targeted therapies to improve oxidative phosphorylation capacity.The main advantages of this technique are that it is noninvasive allowing for serial investigation, and that it is well tolerated by individuals spanning a wide range of functional capabilities. Generally, individuals new to this method will struggle because the equipment set up and subject positioning are technically intensive and crucially important to obtain good results. Prior to imaging, first obtain written and informed consent from the subject and thoroughly screen them for MRI safety.Then, set up for the experiment. This protocol is demonstrated using a Siemens Avanto 1.5 Tesla system. Begin prepping for imaging by first plugging the phosphorous 31 coil into the table coil connector closest to the bore.Then, place a large triangle foam cushion near the other end of the scanner exam table, but not directly on the coil. Also, place a head pillow at the of the MR exam table farthest from the bore, then instruct the subject to lie supine with their feet first on the MR table. Place a foam cushion under their knees to support the leg in a partially flexed position.Be sure to position the subject close to the right side of the table in order to center the left thigh as closely to the magnet isocenter as possible to ensure optimal B0 homogeneity. Then, provide the subject with ear plugs or headphones for hearing protection. Position the phosphorous 31 coil on the subject's left quadriceps at approximately the mid point between the patella and the femoral head.Then, secure it to the leg using straps. The coil should be over the lateral portion of the leg, above the vastus lateralis. Secure a baby oil bottle to the medial aspect of the thigh with the same straps used to secure the coil to the leg, then 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 the ankle. Allow the subject to practice the isometric exercise to ensure that the straps provide sufficient resistance. Then, once the subject is comfortable, use the laser light guide to mark the center of the coil and move the table to the magnet isocenter using the centering landmark.For the baseline phase of the spectroscopy acquisition, instruct the subject to lie still and relax their leg muscles to minimize motion artifacts. Begin by acquiring a triplane localizer to verify positioning and identify the location of the coil. Also, acquire a second localizer, but open the slice view on the first triplane localizer.Next, set up the phosphorous 31 spectroscopy sequence with non-localized pulse acquired sequence parameters as seen here. Center and rotate the slice orientation by left clicking and holding the slice group, and match the final orientation of the slices to the position of the oil bottle. Now, place the shim box by dragging the second triplane localizer into the viewing window, then drag the spectroscopy sequence into the protocol window and double click to open.Use the position tool bar to visualize the shim voxel which will appear on the localizer images. The voxel can be moved by left clicking and holding it in the center of the box, and it's size and rotation can be changed by left clicking and holding it at the corner of the box. Place the shim box directly below the coil, and parallel to the plane of the quadriceps to ensure B0 field homogeneity.Use the localizer images to identify the sensitive region of the coil, and adjust the shim box to encompass this region within the muscle. Open the acquisition viewer window and select the head icon in the tool bar to allow viewing of the spectroscopy acquisition in real time, then run the sequence to obtain a single test spectrum. Next, observe the resulting spectrum and examine the quality of the B0 shimming.Look for a prominent phosphocreatine peak centered at zero parts per million without significant noise. If the spectrum appears noisy, reposition the shim box and reacquire. Copy the test spectroscopy sequence with the best spectral quality and use it for all subsequent measurements.Increase the number of measurements from one to 10, then acquire while the subject is at rest. Next, for exercise acquisition, apply the shim settings from the previous scan and set the sequence to acquire 20 measurements. Instruct the subject to first remain at rest for two measurements, then provide the subject with a count down to indicate when to start the exercise.At this point, the subject should initiate knee extension flexion as forcefully and as rapidly as possible against the resistance of the straps for approximately 30 seconds. After a 30%drop in the PCR peak height is observed, instruct the subject to terminate the exercise and rest. Then, acquire 20 additional post exercise measurements immediately without pause or shimming.After the acquisitions are complete, ensure exercise quality by comparing the phosphocreatine peak heights at the beginning and end of exercise. This graph shows the recovery of the phosphocreatine concentration after it's depletion with the rapid quasi-static knee extension exercise. The line represent the fit of the exponential recovery function.A Bland-Altman analysis of phosphocreatine recovery time demonstrates a mean difference and standard deviation of 1.03 plus or minus 4.83 seconds, and a between trials coefficient of variation of 4.66. Shown here is a representative phosphocreatine recovery curve from the examination of a nonambulatory subject. Note that a phosphocreatine depletion of 64%was attained with this exercise protocol.Finally, this comparison of phosphocreatine recovery times demonstrates sequentially poorer mitochondrial oxidative capacity in control, non-diabetic, and diabetic subjects. Once mastered, this technique can be done in less than 20 minutes if it's performed properly. While attempting this procedure, it's important to remember to both verify that the spectrum has sufficient signal prior to exercise and to ensure that exercise results in a phosphocreatine depletion of at least 30%Following this procedure, other methods like fat muscle quantification imaging can be performed in order to calculate muscle mass, and fat content, which are also important to overall skeletal muscle function.This technique can be used to investigate many diseases including Friedreich's ataxia, where it allowed researchers to correlate genetic markers of disease severity in mitochondrial oxidative capacity. After watching this video you should have a good understanding of how to perform an In Vivo phosphorous 31 magnetic resonance spectroscopy scan and exercise protocol. Don't forget that working with magnetic resonance scanners requires stringent safety measures, and that all MR experiments should be performed by adequately trained personnel.

Research

JoVE Journal

Medicine

In Vivo Canine Muscle Function Assay

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...