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Fatty infiltration of muscles is a marker of disease progression in many neuromuscular disorders. Muscle MRI is capable of revealing patterns of muscles involvement that are disease specific and facilitates the diagnostic workup of patients. Although routine T1-weighted (T1w) imaging can give an indication of the presence or absence of muscular fat infiltration, it is difficult to extract quantitative data from these images. On the contrary, Dixon methods provide quantitative measure of fat fraction. Usually, whole-body (WB) exams consist in the acquisition of T1w images, followed by Dixon acquisitions on targeted regions to quantitatively assess fat infiltration. With the aim of improving and accelerating the qualitative assessment of neuromuscular disorders, we propose to avoid the T1w acquisition altogether, by allowing to perform the visual diagnosis workup on a WB Dixon imaging. 20 patients underwent WB MRI at 3T. WB T1w images were acquired with a 2D TSE sequence (resolution=1.1×1.1mm 2 , slice thickness=6mm, T acq =5min 40s). WB Dixon acquisition consisted in a 3D VIBE sequence with 3 echoes (resolution=1×1×5mm 3 , T acq =14min 5s). Quantitative fat fraction maps were derived using a standard 3-points Dixon reconstruction method. A customed lookup table was embedded in the DICOM file to provide a colored lecture of fat fraction maps corresponding to the Mercuri's scale. Our results show that the acquisition of a high resolution WB Dixon imaging is possible in less than 15min using an optimized VIBE sequence. This provides quantitative data that are more suitable than T1w images for longitudinal natural history studies, or therapeutic clinical trials. Moreover, the color representation renders the visual grading of the muscles more convenient and less operator dependent as it is based on actual fat fraction measurements. WB Dixon might then overcome the use of WB T1w images for diagnostic of neuromuscular disorders.

We have previously demonstrated that a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping injected in GRMD by locoregional transvenous perfusion of the forelimb, restores up to 80% dystrophin expression and improves histological, Nuclear Magnetic Resonance (NMR) pathological indices and strength in a dose-dependent manner at 3 months post injection (PInj). A recent paper suggested that dystrophin-positive fibers and rAAV genome drastically decrease with time, especially between 3 and 6 months PInj. This may compromise the clinical development of an AAV-mediated gene transfer approach. To assess this point, we realized a 6 to 7 months follow-up in a group of 3 GRMD aged 3–4 months when injected in both forelimbs with 2.5E13 vg/kg of a rAAV8 U7snRNA vector promoting exon skipping. Dogs were injected, followed up and evaluated (NMR, strength assessment, Dystrophin expression, vector biodistribution) in the same way as the previously reported large cohort of dogs that were followed for 3 months. Muscle biopsies were performed at 3 months and complete autopsy at 6 to 7 months PInj. Compared to previously reported dogs injected with the same dose, evaluation of dystrophin expression, histological aspect, AAV genome, NMR parameters and strength showed not differences indicating that the treatment effect was fully maintained over 6 months. No loss in AAV genome quantification and dystrophin expression was observed between the 3 months muscle biopsy specimens and 6 months muscle autopsy in the 3 dogs. A decrement in transgene copy numbers at 6 months PInj was observed in non muscular tissues. These data show, for the first time, that AAV-mediated exon skipping fully maintains dystrophin expression and functional recovery over a six months period. We suggest that long-term rAAV genome stability is obtained in dystrophic muscles when a substantial reversion of the muscular pathological pattern is reached.

Objective: The aim is to search for a pattern of muscle involvement in patients with nemaline myopathy due to mutations in the NEB gene and to correlate MRI findings with clinical severity. Methods: The series consisted of seven patients followed in two different French centers and for whom whole body MRI (WBMRI) was done. T1-TSE sequences in axial and frontal views were performed. Results: the clinical phenotype was determined by the severity of motor and respiratory involvement. Two patients had severe phenotype: wheelchair bound and total dependence on mechanical ventilation. Two cases had moderate phenotype: able to walk but limitation of activity outside due to fatigue, severe restrictive lung disease. Three patients have mild phenotype: complete autonomy, proximal weakness after prolonged activity, and no need for ventilator support. WBMRI showed in all patients marked involvement of tongue and tibialis anterior muscles. Other muscles also altered were lateral pterygoid, soleus and extensor digitorum (severe cases); temporal, abdominal belt and soleus muscles (moderate); extensor digitorum (mild). Discussion: NEB-mutated patients present a wide spectrum of severity. A positive correlation between the clinical severity and the degree of muscle abnormalities is observed. Although there is variability in the distribution of the abnormalities in trunk and proximal limbs, a cranial and distal limb involvement is identified. Conclusion: Selective involvement of the tongue and tibialis anterior muscles in NEB mutated patients makes WBMRI a useful tool for diagnosis of nemaline myopathy.

The SCOPE-DMD project builds on results from the TREAT-NMD and BIO-NMD EU projects to investigate a personalised-medicinal product through to market for the treatment of Duchenne Muscular Dystrophy (DMD). The initial goal of the project is to develop a therapy that can restore the expression of a functional dystrophin protein in targeted DMD patients that would potentially benefit from skipping of exon 45 of the DMD gene. The project uses an innovative clinical study design and novel outcome measures to reduce development timelines. The SCOPE-DMD study is an exploratory, open-label, dose-escalation phase IIb clinical trial that will assess the efficacy, safety, pharmacodynamics and pharmacokinetics of weekly subcutaneous doses of PRO045 in subjects with DMD. The study is of seamless design that is driven by the need to maximise the data from an exceptionally small patient population and to provide continuity of care in this rapidly progressing disease – a result of direct feedback from parents. It is made up of two phases: a dose escalation phase with 15 patients and an extended pivotal phase with an additional 55 patients. The consortium consists of 2 SMEs and 3 academic institutes coordinated by Newcastle University. Based on extensive experience from clinical trials of two other AONs in DMD patients, the consortium has worked on a highly innovative development plan that if successful, could be applied to future clinical trials in DMD and other rare diseases. Also magnetic resonance imaging, novel biomarkers and functional outcome measures including accelerometry, myotools and the Performance of Upper Limb (PUL) module will be incorporated into the trial protocol in addition to more established ones. Both expertise and scientific knowledge come together in this consortium to launch an innovative drug product but also to provide a regulatory and pathway-to-market precedence to benefit future patients with rare disorders getting earlier access to treatment.

The evolution of quantitative NMR imaging (NMRI) and spectroscopy (NMRS) indices over one year is reported in forearms of 21 Duchenne Muscular Dystrophy (DMD) patients amenable to exon 53 skipping therapy. Patients aged 6 to 18 yrs, 12 non-ambulant at inclusion, were followed up to characterize course of natural disease in upper limbs, which are to be targeted in future trials. Boys underwent NMR at 3T, with arm resting along the body. The two arms were examined in separate sessions on the same day, and repeated at a one year interval. Examinations comprised NMRI (measurement of T2 of muscle water and of fat fraction by 3-point Dixon imaging) in flexor and extensor muscles (FLEX, EXT) and non localized NMRS of phosphate metabolites. Each item lasted ∼20 min. Compared to data obtained in arms of 7 control subjects (aged 7–20), the data at inclusion in patients showed muscle water T2, deconvoluted from fat, was above 95% confidence limits in 20% of cases, with excessive T2 heterogeneity in 33%. Percentage fat signal (%F) exceeded 6.6%in 80% of cases and FLEX had higher %F than EXT muscles. Metabolic ratios combining ATP, phosphocreatine, phospho mono- and di-esters and two pools of inorganic phosphate: cytosolic (Pia) and an anomalous alkaline pool present in dystrophic muscle (Pib) were abnormal in 80–100% of cases and correlated strongly to %F. These indices progressed markedly faster after loss of ambulation. After one year, %F increased by 1.0% on average ( p p = 0.02); Pib/Ptot (+10%, p = 0.04), Pia/Pitot (−5%, p = 0.04), were always highest and most significant in non-ambulant patients. NMRI and NMRS markers quantified disease progression in metabolism and fat infiltration in arms of DMD boys over one year. Progression appeared most marked for those muscles that were already severely affected.

To describe 6 patients with asymptomatic Pompe disease (APD). Background. Pompe disease (PD) is a muscle glycogenosis due to acid alpha-glucosidase (GAA) deficiency, potentially leading to limb girdle muscle weakness and respiratory insufficiency in adults. Because of the availability of enzyme replacement therapy (ERT), and the improvement of enzymatic assessment techniques, an increasing number of patients are now diagnosed at an early stage of the disease. We studied 6 patients diagnosed with APD in France in the 1991–2013 interval. PD diagnosis was established on GAA enzyme activity levels and GAA gene analysis. All patients underwent standardized clinical examination, pulmonary function tests (PFT), echocardiography, muscle biopsy, and determination of serum creatine kinase (CK) levels. Whole body muscle MRI was performed in 4 patients. Four females and 2 males with a mean age of 40 years (24–68) and a mean follow-up of 6 years (1–22) were analyzed. PD was investigated because of a positive family history for PD in 3 cases, and because of an isolated hyperCKemia in 3 cases. Clinical examination, PFT and echocardiography were normal in all patients. Muscle pathology was normal in 3 cases, and demonstrated vacuoles in 3 cases. CK levels were normal in 1 patient, and mildly raised in 5 cases. Whole body muscle MRI was normal in 2 cases, and demonstrated variable involvement of tongue, subscapularis and hamstring muscles in 2 cases. GAA deficiency was assessed in blood, and genetic analysis revealed 2 mutations in the GAA gene in all patients. Our study demonstrates that patients with APD present with a heterogeneous combination of biological, pathological and MRI abnormalities. Patients with APD should be closely monitored for muscle disease using clinical examination, PFT and muscle MRI in order to help deciding when to start ERT.

Excessive accumulation of fibrous connective tissue in the SKM extracellular matrix is one of the main long term consequences of the chronic inflammatory/necrotic/degenerative insults in muscle disorders. Quantitation and distribution of intramuscular connective tissue (IMCT) will constitute an important and new biomarker of disease progression and of response to treatments. Although UTE sequences were shown to be able to image and characterize connective tissue structures such as tendons and cartilage no parametric image of the short T2 component has ever been reported in the SKM. Our goal was to develop an ultra-short TE (UTE)-based NMR tool for imaging short T2 components (∼1ms) in the skeletal muscle (SKM) tissue, expected to reveal IMCT content. Fat-fraction- and T2 ∗ -maps were extracted from six 2D-UTE image series of the same axial slice acquired in the calf of 8 healthy volunteers at echo-times (TE) of 2.5,3,4,6,12.5 and 26.5ms. A short T2-fraction-map, Sf, was calculated by the equation Sf=(∣ S (t0)∣e t0/T2∗ −∣ S (t1)∣e t1/T2∗ − F ∣ S (t0)∣(f0−f1))/( S (t0) e t0/T2∗ ) S (ti) is the signal of the image acquired at TE=ti, t0/t1=0.2/2.5ms, F is the relative signal contribution from lipids and flΞ∣ ∑ k pke iwk·tl ∣; pk and wk are the relative fraction and frequency of each peak in the frequency spectrum of lipids. A significant relative fraction of signal from "water" protons with short T2 was systematically observed in all muscles. The highest values were found within fascia. No short T2 was observed in the subcutaneous fat and spongy bone, confirming that there were no contributions from lipids. The average short T2 fraction observed in SKM was 4.2(0.7)%. Very short T2 components were imaged for the first time in SKM tissue. Although the characterization of a short T2 component in SKM has been previously reported, suggesting that it might represent IMCT, healthy and diseased tissues must be further investigated in order to ensure the origin of this fast relaxing signal.

A conventional T1-weighted spin-echo (SE) magnetic resonance (MR) imaging sequence was compared with breath-hold and non–breath-hold half- Fourier single-shot fast SE MR sequences with black-blood preparation and high spatial resolution for imaging of various cardiac diseases. The optimized single-shot fast SE sequence provided better or equal image quality in less time. Breath-hold and non–breath-hold single-shot fast SE sequences may replace the conventional T1-weighted SE sequence for first-line cardiac MR imaging.

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.

To elucidate the low proton nuclear magnetic resonance (NMR) visibility of muscle lactate previously demonstrated in excised rat muscle, lactate transverse relaxation was investigated in the same model using double quantum editing sequences with effective echo times ranging from 55 to 475 msec. On this time scale, muscle lactate clearly exhibits a bi-exponential transverse relaxation with a short T 2 of 33 ± 5 msec (mean ± SE, n = 3) and a long T 2 of 230 ± 10 msec. The relative populations (84 ± 4% vs. 16 ± 4%, respectively) of these two lactate pools are compatible with compartmentation between intra- and extracellular muscle lactate.

We have developed a magnetic resonance imaging (MRI) technique based on a multiple gradient-echo sequence designed to probe perfusion and oxygenation simultaneously within skeletal muscle. Processing of the images acquired at successive echo times (TEs) generates two functional maps: one of the signal intensity (SI) extrapolated to zero echo time, which is sensitive to perfusion; and a second one of R2*, which reflects oxygenation. An advantage of the processing procedure lies in the selection of tissue of interest through the profile of T2* decay, leading to automatic rejection of pixels containing small vessels. This allows a more specific assessment of tissue perfusion and oxygenation. This technique was demonstrated successfully during post-ischemic reactive hyperemia in human calf. A perfusion peak of 123 mL x 100 g(-)1 x min(-1) was measured immediately after ischemia, whereas R2* value showed an 11.5% decrease at the same time, essentially reflecting blood oxygenation changes. Differences in the time courses of reperfusion and re-oxygenation were observed, oxygenation presenting a slower recovery. The mechanisms responsible for such a differential dynamic response are discussed.

Among the ultrafast MRI techniques, the single-shot fast spin-echo sequence offers a robust alternative to echo planar imaging, essentially because of a much reduced sensitivity to B0 inhomogeneity. This property is particularly appealing in situations in which B0 inhomogeneities can be severe and difficult to correct, such as in cardiac imaging. With single-shot cardiac imaging, however, achieving high resolution over the necessarily large field of views without introducing back-folding artifacts is problematic. One option is to use multishot sequences. However, then issues related to cardiac gating arise. Another solution is to use, optimized presaturation slabs with quadratic phase pulses generated by the Shinnar-LeRoux algorithm. These can be set to reduce the field of view in the phase-encoding direction, resulting in a reduction in the number of phase-encoding steps. For instance, for a 1 x 2-mm spatial resolution, over a rectangular, 250 x 125-mm field of view, and using a half Fourier acquisition, an echo-train length of only 40 is required. With a 4.5-msec echo spacing, the total imaging time is approximately 180 msec. The efficacy of this solution on phantoms and volunteers is demonstrated. Multislice short-axis examinations of the whole heart, realized within a single short breath-hold of approximately 10 seconds, are shown. The possibility of investigating not only cardiac anatomy but also both contractility and myocardial perfusion is discussed.

Mutations in the LAMA2 gene underlie a severe congenital type of muscular dystrophy (MDC1A). We report the clinical, histological and genotypic features of 2 patients aged respectively 35 and 31 years-old, presenting unusual phenotypes. The first patient, aged 36, exhibit a late-onset muscle weakness predominantly in the limb girdle. Contractures of the elbow, wriest, fingers and Achilles tendons were the first symptoms with an onset at 15 years of age. Pelvic muscle weakness was observed at 25 years of age. The CK was elevated (2000 UI/L). In addition, he presented at age 30 a dilated cardiomyopathy with ventricular arrhythmias, requiring the implantation of a cardioverter-defibrillator. The second patient, aged 31 years, presented axial weakness at age 1, followed by the occurrence of rigid spine around 10 years of age associated to contractures at elbow, fingers, hips and Achilles tendons. At age 30, he had predominantly axial and limb girdle muscle weakness associated with upward gaze deficit. He has also generalized and partial complex seizures, beginning at age 6. The brain MRI disclosed bilateral occipital polymicrogyria and lissencephaly. Muscle histology showed dystrophic features, rimmed vacuoles, and partial loss of laminin a immunoreactivity. Nerve conduction study was normal in both patients. High-throughput analyses using “NMD-Chips” DNA capture and next generation sequencing identified novel nonsense mutations of LAMA2 gene at homozygous stage in the two patients. Previous sequencing of LMNA, EMD, DES, FHL1, MYH7, FKRP, FKTN, COL6A1,2,3, SEPN1 genes disclosed no mutation. In summary, these 2 patients disclosed a mild limb-girdle-type pattern of weakness and contractures associated respectively in each patient with dilated cardiomyopathy and cortical dysplasia. Work partly supported by the NMD-CHIP Consortium, an European Commission FP7 HEALTH project (HEALTH-F5-2008-223026).

The mdx is the mouse model for Duchenne muscular dystrophy (MD), but with a benign phenotype. The large-myd mouse (Lg) has a mutation in the glycosyltransferase like Large gene and models the Congenital MD 1D, with a severe phenotype. The double mutant mdx/Lg, developed in our lab (1), has a more severe phenotype than both parental lineages. Dystrophic muscle alterations have been described by nuclear magnetic resonance (NMR) imaging in several mouse models. This NMR study aims to evaluate yet unexplored mouse MD strains. Eight double mutant mdx/Lg, 11 mdx and 10 Lg mice, aged between 2 and 4 months, and 13 C57Bl normal mice (WT) were imaged at 2T. To calculate T2 maps, two images were acquired at 12 and 40 ms echo times (TR = 1500 ms, 4 slices, 1.5 mm slice thickness, 4 mm interslice distance). T1 weighted (T1w) images with and without fat saturation were also acquired. Four regions were evaluated: lower leg anterior and posterior segments, thigh lateral and medial segments. Muscle T2 was similarly elevated in all muscle groups of dystrophic mice (33.1–38.8 ms) as compared to WT mice (26.0–30.3 ms, p

Non-invasive measurements and quantitative evaluation of the natural course of disease need to be established in anticipation of replacement therapy in Duchenne muscular dystrophy (DMD). A cohort of 23 DMD boys (6–18years, 14 non-ambulant), were enrolled specifically to characterize disease progression of upper limbs, to be targeted in future therapeutic trials. Prior studies in the GRMD canine model showed that Nuclear Magnetic Resonance imaging (NMRI) and phosphorous spectroscopy (NMRS) could provide quantitative indices which graded with therapeutic dose. A similar approach was implemented in patients, who were examined in a 3T-60cm Siemens Magnetom TRIO system, with the arm along the body. The two arms were examined in separate sessions, to be repeated yearly, comprising NMRI (measurement of T2 of muscle water, and of fat fraction by 3-point Dixon imaging) and NMRS of phosphate metabolites. Each item lasted ∼20min. Twelve indices were analysed: –2 in NMRI: %fat signal (%F), muscle water T2 (deconvoluted from fat) –10 in NMRS: 8 ratios of metabolites combining: ATP, phosphocreatine, phospho mono- and di-esters and two pools of inorganic phosphate: cytosolic (Pia) and an anomalous alkaline pool present in dystrophic muscle (Pib), and 2 indices for respective pH values. T2 was above laboratory norms in 15% of cases, % F exceeded 10% of signal in 70% of cases and correlated to patient age ( R =.58, P R =.38 to .46, P F , ( R =.46 to .77, P Quantitative NMRI and NMRS provided a variety of indices which were abnormal in forearms of DMD patients, covering a broad range of fat infiltration, and metabolic abnormalities which graded together and with patient age and could provide biomarkers of therapeutic efficacy.

Skeletal muscle NMR imaging has two distinct roles: 1/ to diagnose disorders and 2/ to monitor disease progression. Both have been played to the benefit of type II glycogenosis patients (GSDII). In this study, we quantified muscle water T2, a marker of disease activity, in thigh and leg muscles of GSDII. We also investigated the relationship between T2 and the extension and progression of fatty degenerative changes in skeletal muscle. The degree of fatty infiltration in 11 thigh and 8 leg muscle heads was calculated for both lower limbs from a 3-point 3D Dixon acquisition. Since quantitative imaging has become standard in our department for the routine follow-up of GSDII in 2009, 36 patients have been examined, 17 were rescanned after 1 yr and 6 rescanned after 2 yrs. Twenty-one patients were treated by enzyme substitution, 2 stopped treatment during the observation. Quite a large number of muscles had abnormally elevated muscle water T2 (>39 ms): 84/296 in the thighs and 62/253 in the legs. Abnormal muscle water T2s were associated with higher percentages of fatty infiltration at 1 yr follow up: in the thighs: 11.6% vs 5.8% in normal T2 muscles, p

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there are no strongly effective treatments but new gene-based therapies are currently being developed with particular advances in using exon skipping and other RNA-based approaches, conventional gene replacement strategies, and cell-based gene therapy. In the case of DMD, a number of groups are testing gene therapy with adeno-associated virus vectors expressing engineered microdystrophins (AAV-MDs). In our hands, highly sequence optimised AAV-MDs are available for use in mouse, dog and humans, expressed using a strong skeletal and cardiac muscle specific synthetic promoter, have been tested in detail in mdx mice, and in the GRMD dog. Here we report the outcome of a study of AAV-canine MD delivery (5× E12 vg/kg) by isolated limb perfusion in GRMD dogs from ages 3 months to 6 months. No immunosuppression was applied. In skeletal muscles of the treated limb at 3-months after vector administration we report widespread vector biodistribution, and sustained high level MD expression (up to 95% of fibres). In addition the treated limb exhibited very significantly improved parameters of muscle degeneration, fibrosis, 1H-MRI and 31P-NMR spectroscopy, and muscle strength. In the context of immune parameters, AAV administration elicited anti-AAV2/8 antibodies and a transient elevation of serum cytokines (wk5–10), but no evidence of cellular immunity to microdystrophin or to the AAV vector was observed. This study strongly supports the hypothesis that the current optimised microdystrophin is highly functional, not only in mice, but also in a large animal model, and that AAV2/8 vector delivery results in sustained expression of microdystrophin without adverse immune responses. The current optimised AAV-microdystrophin configuration thus lays a sound basis for a translation programme towards clinical trials in DMD patients.

Autosomal dominant centronuclear myopathy (CNM) is characterized by slowly progressive muscular weakness and wasting, and high frequency of centrally located nuclei in muscle fibers. The disorder involves mainly limb girdle, trunk and neck muscles but may also affect distal muscles. Ptosis and limitation of eye movements are frequent. Molecular analysis has identified recurrent mutations in exons 8 and 11 and rare heterozygous missense mutations in the dynamin-2 (DNM2) gene, which encodes a protein involved in endocytosis and membrane trafficking, actin assembly, and centrosome cohesion. We recently identified an isolated case of a 34 years old male patient, complaining of gait difficulties since the age of 14 years. Two years ago, the condition evolved to limb muscle weakness due to a predominantly distal severe lower limbs muscle atrophy associated to pes cavus. He also presented mild facial weakness with a discrete ptosis without ophtalmoparesis. By sequencing DNM2 exons, we found the rare heterozygous missense mutation c.1948G > A (p.E650K) in the DNM2 gene. This mutation was only described in one Chilean family. T1-weighted whole-body NMR imaging revealed fatty degenerative changes predominantly affecting the legs (score of 3 in a scale of 4), the face and the thighs. Thigh muscle displayed the particular reticular-punctuated pattern of fatty infiltration seen in CNM patients. The pattern of lesion distribution and the particular muscle texture observed in this patient are highly evocative of the disease. Therefore, NMR findings are showing comparable alterations in DNM2-related CNM patients, independently of the pathogenic mutation. FAPESP-CEPID, CNPQ-INCT, FINEP, CAPES-COFECUB.

Skeletal muscle diagnostic imaging is classically based on visual inspection of T1-weighted NMR images and interpretation of patterns of fatty infiltration. The existence of oedematous/inflammatory/necrotic lesions is subjectively appreciated on T2-weighted images, typically acquired with the STIR sequence to minimize fat confounding effects. Because it relies on identification of hyperintensities between and within muscles, the interpretation risk with T2-weighted images is to miss global, homogenous increases in T2. In this report, we demonstrate this concern to be more than theoretical. Three patients aged 6, 7 and 12 were referred for further investigation of progressive muscle wasting (P1) or clinical suspicion of dermatomyositis (P2, P3). All of them had normal CK values. Whole-body T1w showed no fatty infiltration in muscles. Standard T2w imaging was normal in P1, showed discrete differences in signal intensity, particularly between anterior and posterior leg compartments in P2 and was moderately positive with hyperintense streaks in P3. Quantitative T2 maps were obtained from multi TE spin echo data after extraction of the water component by tri-exponential fitting. In the 3 patients, muscle water T2s were abnormally elevated in the scapular and pelvic girdles, the thighs and the legs: 45/51 muscles of P1, 46/60 of P2 and 47/51 of P3 had T2s higher than 39 ms, the upper normal value. Mean T2s were 42.3, 42.3 and 45.9 ms, with highest T2s measured at 50.0, 51.9 and 53.3 ms in P1, P2 and P3 respectively. Juvenile dermatomyositis was histologically confirmed in all patients. After 3 months on steroid, P1 was rescanned. In the previously inflamed muscles, T2 had on average decreased by 3.4 ms ( p

ObjectivesCyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD).MethodsMuscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy.ResultscDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients.ConclusionsIn most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease.

Mutations in COL6A1, COL6A2, and COL6A3 genes encoding collagen VI (ColVI) are responsible for a wide spectrum of muscle diseases, also termed collagen VI myopathies. At one end of the spectrum, Bethlem myopathy (BM) represent a mild proximal myopathy beginning within the first or second decade of life and characterized by joint contractures, a hallmark of this condition. We report the clinical presentation, the course and the genetic analysis of 35 BM patients followed at the Institute of Myology. Onset of the disease was noticed before the age of 10 (80%). Early onset was not correlated with a severe clinical course. The majority of patients presented a retractile phenotype. However, three patients showed atypical phenotype: a LGMD muscle weakness pattern without contractures was observed in two patients and a third one had a diagnosis of minicore myopathy. Finger hyperlaxity and scoliosis were observed in 23% and 26% of patients, respectively. The vast majority of patients had a slow disease progression, with the exception of eight patients who used clippers or rolling chair. On long-term follow-up, only four patients presented a vital capacity (VC) lower than 70% of the theoretical value (record age of 18–50 years) which was not correlated with age disease onset. The pattern of involved muscles on muscle imaging was characteristic of ColVI-myopathies. Pathogenic mutations, including missense or exon skipping mutations affecting the triple helical domains, were identified in the three COL6A genes. The majority of the patients (91%) harbored autosomal dominant mutations (14% with de novo mutations), while three patients presented a recessive inheritance. In conclusion, we presented a series of 35 BM patients, three of them presenting an atypical phenotype namely without retractions misdiagnosed as LGMD or minicore myopathy. However, in these patients, muscle imaging was in accordance with the pattern of COLVI-related myopathies orienting towards molecular study of COL6 genes.

Replacement therapy of dystrophinopathies has begun with promising results both in animals and in patients. Non-invasive quantitative tools are needed to evaluate its potential benefits or side effects but also to determine the optimal protocol. Fifteen GRMD dogs were treated by unilateral high-pressure high-volume injection into the cephalic vein with a rAAV8-U7-ESE6-ESE8 solution. They were divided into 6 groups according to the dose of viral particles and the volume injected. The lowest dose-lower volume combination was skipped. Two dogs were injected with saline only. After high dose injection, some muscles displayed up to 80% of dystrophin positive fibers. Three months after injection, we evaluated both forearms with spectroscopy at 4T and NMR imaging at 3T. Comparisons between injected and non-injected arm were blinded. All indices of mock dogs were within the reference range of the GRMD population. Both spectroscopy and imaging indices showed differences between the two arms at the highest dose irrespective of volume injected and at the intermediate dose-high volume combination. The putative identification of the treated arm proved systematically correct. Among the spectroscopy indices, Pi/PCr, PCr/ATP and PDE/(Pi + PCr) were the most sensitive and 31P NMR showed changes proportional to the number of AAV particles injected. With regard to imaging, 31 indices were evaluated in the extensor carpi radialis and flexor carpi ulnaris. The most relevant were: muscle heterogeneity in T2w images, the T1w/T2w signal ratio (SR), the T2w/PDw SR and the maximal relative signal enhancement after injection of 0.5 mmol/kg gadoteric acid. When high or intermediate doses were injected, indices actually decreased towards the normal range in both arms, with a reduction that was more substantial in the muscles of the injected side. This study demonstrated NMR ability to detect changes in dystrophic muscle structure and metabolism in response to exon-skipping therapy.

In NMR imaging of neuromuscular disorders, and particularly in muscle dystrophy, a prolonged T2 of muscle water can be used as a marker of disease activity. However, when fat contributes to the NMR signal, as in non-fat-suppressed images of infiltrated muscles, then the conventional mono-exponential data fit will result in prolonged T2 values that principally reflect the degree of fat infiltration. Fat-suppression schemes at acquisition are an obvious option but fail over large volumes. Here, we propose and validate a solution to measure muscle water T2 on non-fat-suppressed NMR images, based on analysing the multiple echo series by a tri-exponential model. Thigh images of 10 patients with a variety of neuromuscular diseases were retrospectively analyzed. Muscle fatty infiltration was quantified using a 3D 3-point Dixon sequence. For muscle T2 determination, a standard multi-slice multi spin echo (17 echos with TEs ranging from 9.5 ms to 161 ms) sequence was used, in which muscle signal decay was considered as the combination of two lipid components and one water component. In order to compare the sensitivity to fat infiltration of the mono- and tri- exponential approaches, we determined the T2 values in groups of pixels belonging to the same muscle but with different ranges of fat infiltration, [0–20%] and [20–50%]. The mono-exponential model yielded T2 values that were abnormally high (>70 ms) and heavily dependent on the degree of fat infiltration ( p r 2 = 0.87), the T2s obtained with the tri-exponential method were not ( r 2 = 0.12). Normal muscle water T2 distribution was narrow: 35 ± 2 ms, which permitted detection of T2 alterations of small amplitude, putatively reflecting mild muscle damage, even in heavily fat infiltrated muscles.

Objective To test the hypothesis that brain energy metabolism is abnormal in patients at an early stage of Huntington disease (HD). Background Energy metabolism has been a major focus of HD research for many years due to several observations in both patients and models of the disease. However, there are currently no in vivo biomarkers of brain energy metabolism in HD. Methods We coupled noninvasive 31P-NMR spectroscopy with activation of the occipital cortex in order to measure the levels of ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) before, during and after a visual stimulus. We studied 15 HD patients at an early stage of the disease (mean motor UHDRS =18±9) and 15 age- and sex-matched controls. Results In controls, we observed an 11% increase in Pi/PCr ratio (p=0.024) and a 13% increase in Pi/ATP ratio (p=0.016) during brain activation, reflecting increased ATP synthesis and ADP levels. Subsequently, controls had a return to baseline levels during recovery (p=0.012 et 0.022 respectively). In HD patients, both Pi/PCr and Pi/ATP ratios were unchanged during and after visual stimulation, reflecting altered mitochondrial bioenergetics. In addition, in HD patients the ratio of Pi/ATP correlated with the UHDRS score during the activation (p=0.014) and recovery periods (p=0.009), while Pi/PCr ratio correlated with the UHDRS score during recovery (p=0.016), reflecting a correlation between brain energy metabolism and disease severity in HD. Conclusions 31Phosphorus nuclear magnetic resonance spectroscopy could provide functional biomarkers of brain energy deficit to monitor therapeutic efficacy in Huntington disease.

Objective: To test the hypothesis that brain energy metabolism is abnormal in patients at an early stage of Huntington disease (HD). Background HD is an autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the HTT gene. Energy metabolism has been a major focus of HD research for many years due to several observations in both patients and models of the disease. However, there are currently no in vivo biomarkers of brain energy metabolism in HD. Design/Methods: We coupled noninvasive 31P-nuclear magnetic resonance spectroscopy (NMRS) with activation of the occipital cortex in order to measure the levels of ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) before, during and after a visual stimulus. We studied 15 HD patients at an early stage of the disease (mean motor UHDRS= 18 ±9) and 15 age- and sex-matched controls. Results: In controls, we observed an 11% increase in Pi/PCr ratio (p=0.012) and a 13% increase in Pi/ATP ratio (p=0.008) during brain activation, reflecting increased ATP synthesis and ADP levels. Subsequently, controls had a return to baseline levels during recovery (p=0.006 et 0.011 respectively). In HD patients, both Pi/PCr and Pi/ATP ratios were unchanged during and after visual stimulation, reflecting altered mitochondrial bioenergetics. In addition, in HD patients the ratio of Pi/ATP correlated with the UHDRS score during the activation (p=0.014) and recovery periods (p=0.009), while Pi/PCr ratio correlated with the UHDRS score during recovery (p=0.016), reflecting a correlation between brain energy metabolism and disease severity in HD. Conclusions: We have identified noninvasive functional biomarkers of brain energy deficit to assess therapeutic efficacy in HD. We are now extending our 31P-NMRS studies to presymptomatic individuals. Disclosure: Dr. Mochel has nothing to disclose. Dr. Nguyen has nothing to disclose. Dr. Deelchand has nothing to disclose. Dr. Rinaldi has nothing to disclose. Dr. Valabregue has nothing to disclose. Dr. Wary has nothing to disclose. Dr. Carlier has nothing to disclose. Dr. Durr has nothing to disclose. Dr. Henry has nothing to disclose.

To identify possible alterations in the skeletal muscle high-energy phosphate metabolism at the early phase of endotoxic shock in rats.A prospective, randomized study of skeletal muscle energetics in endotoxemic and in control rats, by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy at rest, under regional ischemia, and during reperfusion.Biochemical NMR laboratory equipped for surgery and hemodynamic monitoring.Wistar rats were randomized to different groups. Eight rats were injected with Escherichia coli endotoxin (15 mg/kg, survival time 19 +/- 4 hrs) intraperitoneally. Seven other rats served as controls. The additional nine rats were studied for the saturation recovery pulse sequence.In the treatment group, endotoxin was injected 8 hrs before NMR spectroscopy. The right hind limbs were studied under anaesthesia using a surface coil NMR probe. Their high-energy phosphate contents and intracellular pH were determined by 31P NMR spectroscopy. After baseline measurements, an ischemia-reperfusion challenge was imposed on the muscle by transient clamping of the abdominal aorta. Contralateral femoral artery pressure was constantly monitored.During the baseline period, the endotoxin-treated muscles did not show any difference in the distribution of the high-energy phosphate compounds or in intracellular pH, as compared with the controls. Ischemia resulted in a significantly faster decline of the inorganic phosphate/creatine phosphate ratio in the endotoxin-treated rats (1.35 +/- 0.17 vs. 0.51 +/- 0.06 at the end of the 38-min ischemic period). Skeletal muscle acidosis developed earlier and was deeper in the endotoxemic animals (pH: 6.94 +/- 0.02 vs. 7.02 +/- 0.03 at the end of ischemia). During reperfusion, the calculated time constants of recovery of inorganic phosphate to phosphocreatine ratios were identical between groups.Resting nonischemic muscles of endotoxin-treated rats show no evidence of alterations in high-energy phosphate metabolism. However, under ischemic conditions, high-energy phosphate metabolism deteriorates faster in the skeletal muscles of treated animals. These data support the hypothesis of a greater mismatch during perfusion at very low pressure between residual oxygen availability and oxygen needs in the endotoxin-treated muscle cell.

Using 31P nuclear magnetic resonance spectroscopy, we compared the state of the high-energy phosphates in rabbit kidneys stored at 4 degrees C for 24 hr with 3 different solutions: Ringer (Rg), University of Wisconsin (UW), and Euro-Collins (EC) solutions. We found the highest phosphomonoester/inorganic phosphate (MP:Pi) ratio in the group of kidneys stored in the Rg solution (Rg, 0.93 +/- 0.04; UW, 0.36 +/- 0.02; EC, 0.28 +/- 0.02). This medium has been demonstrated in previous physiological studies to give poor results in terms of organ preservation compared to the solutions that mimic the "intracellular" fluid, such as the EC and UW solutions. Because the commonly used cold storage solutions contain phosphates, which superimpose on the intracellular Pi and, thus, can distort the results, we attempted to eliminate the contaminating solution around the kidney and in the vasculature by flushing the kidney with a phosphate-free solution (Rg). The MP:Pi ratio increased in the UW and EC groups (UW, 0.82 +/- 0.04; EC, 0.64 +/- 0.04) in identical proportion in the 2 groups. It remained highest in the Rg group (1.02 +/- 0.03). Comparisons of data before and after flush showed that external phosphate contamination was not predominant. There was no equilibrium in phosphate distribution between intra- and extracellular spaces at 24 hr of storage. We conclude that the validity of the MP:Pi ratio, as a viability index of renal transplant, might have to be restricted to comparisons of kidneys preserved in the same storage conditions. Therefore, it would be necessary to establish normal and pathological values of this ratio for each cold storage solution.

Deletion of the myostatin gene (mstn-/-) results in spectacular increase in muscle mass, and opened the path to therapeutic approaches. Yet improvement in strength does not necessarily match the observed increase in mass. If function is to be preserved in hypertrophic muscle, adequate oxygen supply and substrate utilization should also be maintained. Multi-parametric functional (mpf) NMR can explore these aspects in vivo and non-invasively. In this work, we simultaneously assessed muscle perfusion, capillary oxygenation level (BOLD), energetic phosphate metabolism and isometric force in response to electrical stimulation in mstn-/- mice. Mstn-/- (n=6) and wild-type (n=10) FVB mice were repeatedly subjected to a “exercise(2min)- recovery(10min)” protocol inside a 4 Tesla NMR imager-spectrometer, using custom-built coils and ergometer. Perfusion/BOLD and phosphocreatine (PCr) were monitored with a time resolution of 10s and 2.5s. Acquisitions were triggered to the electro-stimulation to allow perfusion measurement even during exercise. Phosphorus data were summed across exercises to compensate for the intrinsically low signal without degrading time resolution. A metabolic steady state was reached after the 2nd exercise for force, perfusion, and BOLD. Mstn-/- force was not different from WT when normalized for the 36% increase in muscle cross sectional area measured by NMR imaging. Maximal perfusion at exercise was the same in both groups, but hyperemic profile was prolonged in mstn-/-, and PCr recovery significantly delayed (P

BACKGROUND AND METHODS We studied the effect of volume resuscitation on lung fluid balance and systemic oxygen extraction during septic shock in eight anesthetized dogs. Sepsis was induced using a 2-hr continuous infusion of Escherichia coli endotoxin at 0.25 micrograms/min.kg. Relationships between oxygen uptake (VO2) and oxygen supply (DO2) were performed acutely during stepwise controlled decrements in cardiac output by progressive inflation of an intracardiac balloon. At each stage, DO2 and corresponding VO2 were measured independently and the individual critical DO2 level was referred to as the point below which the relationship held. The slope of such a constructed relationship was defined as the maximal oxygen extraction ratio. Lung fluid balance was assessed by measurements of extravascular lung water. All values were studied at baseline, after endotoxin insult, and after reversing hypotension by a 10% dextran infusion. RESULTS Endotoxin infusion led to a shock state that associated hypotension (from 135 to 63 mm Hg) with increases in blood lactate (from 0.53 to 3.9 mmol/L). The mean critical DO2 and maximal oxygen extraction ratio were significantly altered from 7.9 to 17.8 mL/min.kg and from 0.81 to 0.38, respectively. After reversing hypotension by 28 mL/kg colloid infusion, the critical DO2 (11.4 mL/min.kg) and maximal oxygen extraction ratio (0.48) were significantly improved. However, restoration of normal values required a state of fluid overload by further dextran infusion (8 mL/kg). At the end of the fluid challenge, extravascular lung water significantly increased from 6.4 to 17.4 mL/kg. CONCLUSIONS These data suggest that volume loading may reverse endotoxin-induced peripheral perfusion abnormalities. However, substantial pulmonary edema may occur, possibly jeopardizing the beneficial effects of fluid expansion.

Lipid linewidths were measured in the plasma proton spectra of young women. The individuals taking a contraceptive pill associating ethinyloestradiol and levonorgestrel or norethisterone were found with an average linewidth 2.5 Hz narrower than controls. As this drug regimen systematically induces an increase in plasma VLDL and a decrease in HDL/LDL ratio, our observation supports the theory that the Fossel test reflects plasma lipids abnormalities in cancer patients.Physicians enrolled at least 140 female soldiers and military employees in a screening program to detect breast and gynecologic cancers at the outpatient clinic of the military hospital in Liege, Belgium using the Fossel test, a magnetic resonance (NMR) spectroscopy of plasma. They also underwent a clinical examination, including a pap smear. Only 1 patient had a grade I cervical lesion and the remaining women had no disease. The plasma lipid average linewidths were distributed normally. The values ranged from 20-48.5Hz with a mean of 33.4Hz + or - .5Hz. 26.9Hz represented the 10th percentile and 41Hz the 90th percentile. The only correlation between test results and clinical parameters was chronic intake of sex steroids (p=.03). A significant association between chronic intake of sex steroids and a decrease in plasma lipids proton NMR linewidths existed (p=.04). Specifically, the association was only significant with oral contraceptives (OCs) containing levonorgestrel or norethisterone (p=.02) which produce androgenic activity. Moreover those who used the higher dose estrogen OCs (50ug ethinyl estradiol) had lower results than the lower dose estrogen OCs or the controls (p=.06). In fact, the OCs with ethinyl estradiol combined to levonorgestrel or norethisterone narrowed plasma lipids linewidths on average 2.5Hz. Such Ocs increase very low density lipids (VLDL) and the low density level-cholesterol to high density level-cholesterol ratio. Continuous administration of sex steroid hormones to cancer patients also increases lipid metabolism and narrows the plasma methyl and methylene resonances. In conclusion, the Fossel test does detect abnormal distribution of plasma lipoproteins in cancer patients, particularly in the proportion of VLDL.