BackgroundOutcome measures for clinical trials in neuromuscular diseases are typically based on physical assessments which are dependent on patient effort, combine the effort of different muscle groups, and may not be sensitive to progression over short trial periods in slow-progressing diseases. We hypothesised that quantitative fat imaging by MRI (Dixon technique) could provide more discriminating quantitative, patient-independent measurements of the progress of muscle fat replacement within individual muscle groups.ObjectiveTo determine whether quantitative fat imaging could measure disease progression in a cohort of limb-girdle muscular dystrophy 2I (LGMD2I) patients over a 12 month period.Methods32 adult patients (17 male;15 female) from 4 European tertiary referral centres with the homozygous c.826C>A mutation in the fukutin-related protein gene (FKRP) completed baseline and follow up measurements 12 months later. Quantitative fat imaging was performed and muscle fat fraction change was compared with (i) muscle strength and function assessed using standardized physical tests and (ii) standard T1-weighted MRI graded on a 6 point scale.ResultsThere was a significant increase in muscle fat fraction in 9 of the 14 muscles analyzed using the quantitative MRI technique from baseline to 12 months follow up. Changes were not seen in the conventional longitudinal physical assessments or in qualitative scoring of the T1w images.ConclusionsQuantitative muscle MRI, using the Dixon technique, could be used as an important longitudinal outcome measure to assess muscle pathology and monitor therapeutic efficacy in patients with LGMD2I.

Intracellular oxygen (O 2 ) availability and the impact of ambient hypoxia have far reaching ramifications in terms of cell signalling and homeostasis; however, in vivo cellular oxygenation has been an elusive variable to assess. Within skeletal muscle the extent to which myoglobin desaturates (deoxy-Mb) and the extent of this desaturation in relation to O 2 availability provide an endogenous probe for intracellular O 2 partial pressure (P iO 2 ). By combining proton nuclear magnetic resonance spectroscopy ( 1 H NMRS) at a high field strength (4 T), assessing a large muscle volume in a highly efficient coil, and extended signal averaging (30 min) we assessed the level of skeletal muscle deoxy-Mb in 10 healthy men (30 ± 4 years) at rest in both normoxia and hypoxia (10% O 2 ). In normoxia there was an average deoxy-Mb signal of 9 ± 1%, which, when converted to P iO 2 using an O 2 /Mb half-saturation (P 50 ) of 3.2 mmHg, revealed an P iO 2 of 34 ± 6 mmHg. In ambient hypoxia the deoxy-Mb signal rose to 13 ± 3% (P iO 2 = 23 ± 6 mmHg). However, intersubject variation in the defence of arterial oxygenation (S aO 2 ) in hypoxia (S aO 2 range: 86-67%) revealed a significant relationship between the changes in S aO 2 and P iO 2 (r 2 = 0.5). These data are the first to document resting intracellular oxygenation in human skeletal muscle, highlighting the relatively high P iO 2 values that contrast markedly with those previously recorded during exercise (∼2-5 mmHg). Additionally, the impact of ambient hypoxia on P iO 2 and the relationship between changes in S aO 2 and P iO 2 stress the importance of the O 2 cascade from air to cell that ultimately effects O 2 availability and O 2 sensing at the cellular level.

The tri-exponential model is an accurate tool to monitor muscle tissue disease activity devoid of bias introduced by fat infiltration.

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 ؋ 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.

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.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients.

Recent years have seen tremendous progress towards therapy of many previously incurable neuromuscular diseases. This new context has acted as a driving force for the development of novel non-invasive outcome measures. These can be organized in three main categories: functional tools, fluid biomarkers and imagery. In the latest category, nuclear magnetic resonance imaging (NMRI) offers a considerable range of possibilities for the characterization of skeletal muscle composition, function and metabolism. Nowadays, three NMR outcome measures are frequently integrated in clinical research protocols. They are: 1/ the muscle cross sectional area or volume, 2/ the percentage of intramuscular fat and 3/ the muscle water T2, which quantity muscle trophicity, chronic fatty degenerative changes and oedema (or more broadly, “disease activity”), respectively. A fourth biomarker, the contractile tissue volume is easily derived from the first two ones. The fat fraction maps most often acquired with Dixon sequences have proven their capability to detect small changes in muscle composition and have repeatedly shown superior sensitivity over standard functional evaluation. This outcome measure will more than likely be the first of the series to be validated as an endpoint by regulatory agencies. The versatility of contrast generated by NMR has opened many additional possibilities for characterization of the skeletal muscle and will result in the proposal of more NMR biomarkers. Ultra-short TE (UTE) sequences, late gadolinium enhancement and NMR elastography are being investigated as candidates to evaluate skeletal muscle interstitial fibrosis. Many options exist to measure muscle perfusion and oxygenation by NMR. Diffusion NMR as well as texture analysis algorithms could generate complementary information on muscle organization at microscopic and mesoscopic scales, respectively. 31P NMR spectroscopy is the reference technique to assess muscle energetics non-invasively during and after exercise. In dystrophic muscle, 31P NMR spectrum at rest is profoundly perturbed, and several resonances inform on cell membrane integrity. Considerable efforts are being directed towards acceleration of image acquisitions using a variety of approaches, from the extraction of fat content and water T2 maps from one single acquisition to partial matrices acquisition schemes. Spectacular decreases in examination time are expected in the near future. They will reinforce the attractiveness of NMR outcome measures and will further facilitate their integration in clinical research trials.

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.