Measuring perfusion and bioenergetics simultaneously in mouse skeletal muscle: a multiparametric functional-NMR approach.

A totally noninvasive set-up was developed for comprehensive NMR evaluation of mouse skeletal muscle function in vivo. Dynamic pulsed arterial spin labeling-NMRI perfusion and blood oxygenation level-dependent (BOLD) signal measurements were interleaved with (31)P NMRS to measure both vascular response and oxidative capacities during stimulated exercise and subsequent recovery. Force output was recorded with a dedicated ergometer. Twelve exercise bouts were performed. The perfusion, BOLD signal, pH and force-time integral were obtained from mouse legs for each exercise. All reached a steady state after the second exercise, justifying the pointwise summation of the last 10 exercises to compensate for the limited (31)P signal. In this way, a high temporal resolution of 2.5 s was achieved to provide a time constant for phosphocreatine (PCr) recovery (τ(PCr)). The higher signal-to-noise ratio improved the precision of τ(PCr) measurement [coefficient of variation (CV) = 16.5% vs CV = 49.2% for a single exercise at a resolution of 30 s]. Inter-animal summation confirmed that τ(PCr) was stable at steady state, but shorter (89.3 ± 8.6 s) than after the first exercise (148 s, p < 0.05). This novel experimental approach provides an assessment of muscle vascular response simultaneously to energetic function in vivo. Its pertinence was illustrated by observing the establishment of a metabolic steady state. This comprehensive tool offers new perspectives for the study of muscle pathology in mice models.

Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy.

Inhibiting myostatin (mstn) causes spectacular increase in muscle mass, spurring research for therapeutic approaches against neuromuscular disorders. Yet, possible functional deterioration and compromised force production have been reported in isolated muscle of null mstn(-/-) mice. We analyzed vascular and metabolic response to repeated electro-stimulated exercise in vivo in mstn(-/-) mice compared with FVB wild-type controls (WT), using interleaved multi-parametric functional nuclear magnetic resonance (NMR) imaging and spectroscopy. At steady-state exercise, specific force of plantar flexion, phosphocreatine consumption measured by phosphorus spectroscopy and maximum perfusion measured by arterial spin-labeled (ASL) NMR imaging were identical in both groups. After exercise, phosphorus spectroscopy revealed reduced oxidative mitochondrial capacity in mstn(-/-), whereas early recovery perfusion was identical and oxygen extraction, estimated from the blood oxygen level-dependent (BOLD) contrast, was decreased when compared with WT. Hyperemia was prolonged, indicating specific regulation of the perfusional response in mstn(-/-) mice. Histology showed an increased proportion of type IIb fibers in hypertrophied muscles, but the distribution of capillary contacts per fiber between oxidative and glycolytic fibers was unaltered in mstn(-/-) compared with WT. These integrated results formed coherent evidence of a congruous, non-pathologic shift toward a more glycolytic metabolism in this model of mstn(-/-).

Muscle blood flow and oxygenation measured by NMR imaging and spectroscopy.

Tissue perfusion and oxygenation in many organs can be evaluated by various NMR techniques. This review focuses on the specificities, limitations and adaptations of the NMR tools available to investigate perfusion and oxygenation in the skeletal muscle of humans and animal models. A description of how they may be used simultaneously is provided as well. 1H NMR spectroscopy of myoglobin (Mb) monitors intramyocytic oxygenation. It measures the level of deoxy-Mb, from which Mb concentration, Mb desaturation/resaturation rates, muscle oxygenation changes and intracellular partial oxygen pressure (pO2) can be calculated. Positive and negative blood oxygen level-dependent (BOLD) contrasts exist in skeletal muscle. BOLD contrasts primarily reflect changes in capillary-venous oxygenation, but are also directly or indirectly dependent on muscle blood volume, perfusion, vascular network architecture and angulation, relative to the main magnetic field. Arterial spin labelling (ASL) techniques, having high spatial and temporal resolution, are the methods of choice to quantify and map skeletal muscle perfusion non-invasively. Limitations of ASL are poor contrast-to-noise ratio and sensitivity to movement; however, with the introduction of specific adaptations, it has been proven possible to measure skeletal muscle perfusion at both rest and during exercise. The possibility of combining these NMR measurements with others into a single dynamic protocol is most interesting. The 'multiparametric functional (mpf) NMR' concept can be extended to include the evaluation of muscle energy metabolism simultaneously with 31P NMR or with lactate double quantum filtered 1H NMR spectroscopy, an approach which would make NMR an exceptional tool for non-invasive investigations of integrative physiology and biochemistry in skeletal muscle in vivo.

Influence of vascular filling and perfusion on BOLD contrast during reactive hyperemia in human skeletal muscle.

Mechanisms generating BOLD contrast are complex and depend on parameters that are prone to large variations, in particular in skeletal muscle. Here, we simultaneously measured perfusion by ASL, and BOLD response in the calf muscle of 6 healthy volunteers during post-ischemic reactive hyperemia. We tested whether the relation between the two was altered for varying degrees of leg vascular replenishment induced by prior positioning of the leg at different heights relative to the heart. We found that the BOLD response depended on perfusion, but also on the degree of repletion of leg blood vessels. We conclude that simultaneous determination of perfusion by ASL is important to identify the mechanisms underlying BOLD contrast in the skeletal muscle.

How to investigate oxygen supply, uptake, and utilization simultaneously by interleaved NMR imaging and spectroscopy of the skeletal muscle.

Human skeletal muscle perfusion, oxygenation, and high-energy phosphate distribution were measured simultaneously by interleaved (1)H and (31)P NMR spectroscopy and (1)H NMR imaging in vivo. From these parameters, arterial oxygen supply (DO(2)), muscle reoxygenation rate, mitochondrial ATP production, and O(2) consumption (VO(2)) were deduced at the recovery phase of a short ischemic exercise bout. In addition, by using a reformulation of the mass conservation law, muscle maximum O(2) extraction was calculated from these parameters.

Metabolic and vascular support for the role of myoglobin in humans: a multiparametric NMR study.

In human muscle the role of myoglobin (Mb) and its relationship to factors such as muscle perfusion and metabolic capacity are not well understood. We utilized nuclear magnetic resonance (NMR) to simultaneously study the Mb concentration ([Mb]), perfusion, and metabolic characteristics in calf muscles of athletes trained long term for either sprint or endurance running after plantar flexion exercise and cuff ischemia. The acquisitions for (1)H assessment of Mb desaturation and concentration, arterial spin labeling measurement of muscle perfusion, and (31)P spectroscopy to monitor high-energy phosphate metabolites were interleaved in a 4-T magnet. The endurance-trained runners had a significantly elevated [Mb] (0.28 +/- 0.06 vs. 0.20 +/- 0.03 mmol/kg). The time constant of creatine rephosphorylation (tauPCr), an indicator of oxidative capacity, was both shorter in the endurance-trained group (34 +/- 6 vs. 64 +/- 20 s) and negatively correlated with [Mb] across all subjects (r = 0.58). The time to reach maximal perfusion after cuff release was also both shorter in the endurance-trained group (306 +/- 74 vs. 560 +/- 240 s) and negatively correlated with [Mb] (r = 0.56). Finally, Mb reoxygenation rate tended to be higher in the endurance-trained group and was positively correlated with tauPCr (r = 0.75). In summary, these NMR data reveal that [Mb] is increased in human muscle with a high oxidative capacity and a highly responsive vasculature, and the rate at which Mb resaturates is well correlated with the rephosphorylation rate of Cr, each of which support a teleological role for Mb in O(2) transport within highly oxidative human skeletal muscle.

[Exploration of exercise intolerance by 31P NMR spectroscopy of calf muscles coupled with MRI and ergometry]. / Exploration de l'intolérance à l'exercice par spectroscopie RMN du phosphore des muscles fléchisseurs plantaires.

One hundred patients presenting with exercise intolerance or rhabdomyolysis episodes have been examined successively by 31P Nuclear Magnetic Resonance Spectroscopy (MRS) of leg plantar flexor muscles with exercise test. In all cases a muscle biopsy was performed. At the end of investigations, diagnosis of a metabolic myopathy was made in 33 patients: glycogenolysis or glycolysis deficiency in 8 cases, mitochondrial myopathy in 24 cases and CPT II deficiency in one case. Muscular dystrophy or congenital myopathy were diagnosed in 6 cases. No precise etiology could be found in 30 patients with either high CK levels or muscle biopsy abnormalities. Seven patients had rhabdomyolysis related to excessive physical activities. Twenty-four patients had functional symptoms. The principal MRS parameters used for diagnosis were the values of intracellular pH at the end of exercise and the time constant of phosphocreatine resynthesis during recovery. Lack of acidosis after exercise was observed in all patients with blockade of glycogenolysis or glycolysis. A slowing in phosphocreatine resynthesis was found in 66 p.cent of patients with definite mitochondrial myopathy. The specificity of these parameters were respectively 92.4 p.cent and 85.5 p.cent for the two groups. In conclusion (31)P MRS allows the detection of muscular glycogenoses with a sensitivity close to 100 p.cent. However, its sensitivity was lower for the detection of mitochondrial myopathies, as is also known for the other in vivo metabolic investigations, reflecting the heterogeneity of expression of mitochondrial abnormalities in a given muscle. The integration of imaging in the examination protocol may help to orientate towards the diagnostic of a dystrophy in some patients.

A comparison of voluntary and electrically induced contractions by interleaved 1H- and 31P-NMRS in humans.

Skeletal muscle voluntary contractions (VC) and electrical stimulations (ES) were compared in eight healthy men. High-energy phosphates and myoglobin oxygenation were simultaneously monitored in the quadriceps by interleaved (1)H- and (31)P-NMR spectroscopy. For the VC protocol, subjects performed five or six bouts of 5 min with a workload increment of 10% of maximal voluntary torque (MVT) at each step. The ES protocol consisted of a 13-min exercise with a load corresponding to 10% MVT. For both protocols, exercise consisted of 6-s isometric contractions and 6-s rest cycles. For an identical mechanical level (10% MVT), ES induced larger changes than VC in the P(i)-to-phosphocreatine ratio [1.38 +/- 1.14 (ES) vs. 0.13 +/- 0.04 (VC)], pH [6.69 +/- 0.11 (ES) vs. 7.04 +/- 0.07 (VC)] and myoglobin desaturation [43 +/- 15.9 (ES) vs. 6.1 +/- 4.6% (VC)]. ES activated the muscle facing the NMR coil to a greater extent than did VCs when evaluated under identical technical conditions. This metabolic pattern can be interpreted in terms of specific temporal and spatial muscle cell recruitment. Furthermore, at identical levels of energy charge, the muscle was more acidotic and cytoplasm appeared more oxygenated during ES than during VC. These results are in accordance with a preferential recruitment of type II fibers and a relative muscle hyperperfusion during ES.

Determination of skeletal muscle perfusion using arterial spin labeling NMRI: validation by comparison with venous occlusion plethysmography.

T(1)-based determination of perfusion was performed with the high temporal and spatial resolution that monitoring of exercise physiology requires. As no data were available on the validation of this approach in human muscles, T(1)-based NMRI of perfusion was compared to standard strain-gauge venous occlusion plethysmography performed simultaneously within a 4 T magnet. Two different situations were investigated in 21 healthy young volunteers: 1) a 5-min ischemia of the leg, or 2) a 2-3 min ischemic exercise consisting of a plantar flexion on an amagnetic ergometer. Leg perfusion was monitored over 5-15 min of the recovery phase, after the air-cuff arterial occlusion had been released. The interesting features of the sequence were the use of a saturation-recovery module for the introduction of a T(1) modulation and of single-shot spin echo for imaging. Spatial resolution was 1.7 x 2.0 mm and temporal resolution was 2 s. For data analysis, ROIs were traced on different muscles and perfusion was calculated from the differences in muscle signal intensity in successive images. To allow comparison with the global measurement of perfusion by plethysmography, the T(1)-based NMR measurements in exercising muscles were rescaled to the leg cross-section. The perfusion measurements obtained by plethysmography and NMRI were in close agreement with a correlation coefficient between 0.87 and 0.92. This indicates that pulsed arterial techniques provide determination of muscle perfusion not only with superior spatial and temporal resolution but also with exactitude.

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice.

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.

Effect of chronic magnesium supplementation on magnesium distribution in healthy volunteers evaluated by 31P-NMRS and ion selective electrodes.

AIMS: The role of magnesium (Mg) intake in the prevention and treatment of diseases is greatly debated. Mg biodistribution after chronic Mg supplementation was investigated, using state-of-the-art technology to detect changes in free ionized Mg, both at extra- and intracellular levels. METHODS: Thirty young healthy male volunteers participated in a randomised, placebo (P)-controlled, double-blind trial. The treated group (MgS) took 12 mmol magnesium lactate daily for 1 month. Subjects underwent in vivo 31P-NMR spectroscopy and complete clinical and biological examinations, on the first and last day of the trial. Total Mg was measured in plasma, red blood cells and 24 h urine ([Mg]U ). Plasma ionized Mg was measured by ion-selective electrodes. Intracellular free Mg concentrations of skeletal muscle and brain tissues were determined noninvasively by in vivo 31P-NMR at 3T. NMR data were automatically processed with the dedicated software MAGAN. RESULTS: Only [Mg]U changed significantly after treatment (in mmol/24 h, for P, from 4.2+/-1.4 before to 4.1+/-1.3 after and, for MgS, from 3.9+/-1.1 before to 5. 1+/-1.1 after, t=2.15, P=0.04). The two groups did not differ, either before or after the trial, in any other parameter, whether clinical, biological or in relation with the Mg status. CONCLUSIONS: Chronic oral administration of Mg tablets to young healthy male volunteers at usual pharmaceutical doses does not alter Mg biodistribution. This study shows that an adequate and very complete noninvasive methodology is now available and compatible with the organization of clinical protocols which aim at a thorough evaluation of Mg biodistribution.

1H NMR spectroscopy study of the dynamic properties of glycogen in solution by steady-state magnetisation measurement with off-resonance irradiation.

The dynamics of size-selected fractions of glycogen in solution have been investigated by proton NMR spectroscopy, using a recently described relaxation study method which relies on strong offresonance irradiation. The dependence of the steady-state magnetisation on angle and intensity of the effective radio-frequency field was measured and compared to theoretical curves derived from different models of motion. Absence or presence of contributions to relaxation from molecular motions on the microsecond time scale can be tested with this method, without having to resort to models. We found that glycogen dipolar relaxation did not result from isotropic Brownian rotation, and despite some contribution from slow motion (> 1 microsecond) to relaxation in glycogen alpha-particles extracted from rat liver, bulk movement of the molecules did not appear to participate in averaging the dipolar term to zero. Whereas hepatic glycogen rat beta-particles and commercial oyster glycogen displayed very similar relaxation properties, alpha-particles showed significantly different behaviour. However, all results were compatible with a diversity of movements within the molecule, ranging from freely rotating pyranoside rings through collective chain motion and possibly to bulk movement of the beta sub-units within the alpha-particle.

An interleaved heteronuclear NMRI-NMRS approach to non-invasive investigation of exercising human skeletal muscle.

Novel tools are presented that aim at more comprehensive NMR investigations of human skeletal muscle metabolism, in particular during exercise protocols. They integrate imaging (NMRI) and spectroscopy (NMRS) experiments in a single dynamic examination. The first sequence that we propose combine NMR-plethysmography, 1H-NMRS of deoxymyoglobin and 31P-NMRS. This allows simultaneous determination of skeletal muscle perfusion, oxygenation and high-energy phosphates status. It is very well suited to the study of interplay between blood supply and energy metabolism during the recovery period from aerobic or anaerobic exercise. In a second sequence, the same spectroscopic measurements are associated to a 1H double quantum coherence (DQC) edition of lactate. It is, this time, possible to estimate muscle lactate production concurrently with oxygen content, high-energy phosphates distribution and intracellular pH. This sequence is intended mainly for metabolic investigations of ischemic bouts. Examples are given of the use of these sequences in normal adult volunteers. They demonstrate the technical feasibility of these new approaches and illustrate their potential for future applications, particularly non-invasive of regulatory mechanisms of muscle metabolism in situ.

Evidence for 100% 13C NMR visibility of glucose in human skeletal muscle.

The accuracy of the measurement of total muscle glucose by in vivo 13C NMR spectroscopy was tested in five normal volunteers during a euglycemic [1-13C]glucose infusion. The NMR visible concentration calibrated using an external reference was compared with that calculated from plasma glucose concentration, assuming that glucose remained extracellular. The NMR measurement always provided higher values than the calculation from plasma glucose: 0.51 +/- 0.035 (mean +/- SE) versus 0.38 +/- 0.005 mmol/liter of muscle on average. This systematic difference was interpreted as reflecting the presence of muscle glucose-6-phosphate, co-resonating with free glucose. Thus, glucose appeared to be virtually 100% NMR visible in human skeletal muscle.

Simultaneous determination of muscle perfusion and oxygenation by interleaved NMR plethysmography and deoxymyoglobin spectroscopy.

A novel approach is presented that combines NMR-plethysmography and NMRS of deoxymyoglobin in real-time, using line-by-line interleaved acquisitions of both gradient echo images during venous occlusion and of the N-delta proton signal of myoglobin's proximal F8 histidine. This method allowed simultaneous measurement of peripheral regional perfusion and skeletal muscle oxygen content. During reactive hyperaemia, using our combined NMRI-NMRS protocol, we explored the relationship between muscle reoxygenation (myoglobin resaturation half-time, y in s) and reperfusion (x in ml/100 g tissue/min) and found it to be highly significant (y = 70.83x-0.94; r2 = 0.70; F = 64.40; p = 9.73 x 10(-9). We also demonstrated that at low flow, muscle perfusion was a rate-limiting factor to reoxygenation. Making certain hypotheses, muscle oxygen extraction was derived from perfusion and myoglobin resaturation rate. Muscle oxygen extraction during early post-ischemic recovery (0.78 +/- 0.11, 0.79 +/- 0.09 and 0.72 +/- 0.05 at 0, 60 and 100 Torr counter-pressure, respectively) was shown to be independent of perfusion and maximum at each step of the protocol in most volunteers but also to display significant variability among subjects in this supposedly normal population sample.

C13 NMR spectroscopy of lipids: a simple method for absolute quantitation.

There is both epidemiological and experimental evidence of the effect of fatty acid molecular structure, particularly the degree of saturation in fatty acyl chains, on the growth and regulation of certain tumours. In vivo carbon nuclear magnetic resonance spectroscopy has previously been shown to offer a non invasive technique for the evaluation of proportions of monounsaturated, polyunsaturated and saturated fatty acids in human adipose tissue. We present a simple method, which uses both endogenous water and fat as reference, to quantify in molar terms these lipid sub-categories for tissues other than pure fat. This could provide additional information in the debate on the protective effect in cancer of high polyunsaturated fatty acid (PUFA) diet. The method was validated by characterization of a lipid emulsion of known composition in various experimental set-ups and was applied to measure the lipid composition of the calves of two volunteers. Limitations and perspectives of the method are discussed.