Impaired oxygen demand during exercise is related to oxidative stress and muscle function in Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) causes progressive muscle weakness and loss. This study aims to compare changes in quadriceps oxygenation and hemodynamics during maximal voluntary quadriceps isometric contraction (MVCQ) and to determine the relationships between these parameters and systemic oxidative stress markers and muscle structural parameters and muscle volume in patients with FSHD and healthy controls.


Introduction
Facioscapulohumeral muscular dystrophy (FSHD), one of the most prevalent forms of muscular dystrophy (about 4 individuals every 100,000 people) [1], is an autosomal dominant disorder that causes progressive muscle weakness and skeletal muscle loss. Muscle strength reduction contributes to chronic fatigue through a decreased level of physical activity [2]. Despite major progress in understanding FSHD genetic basis, the specific pathogenic mechanisms of the disorder remain unclear and no curative treatment is available. In previous studies, we demonstrated that the reduced physical performance of patients with FSHD is associated with mitochondrial dysfunction and oxidative stress imbalance [3] and can be improved by antioxidant supplementation [4].
Recently, it has been reported that near infrared spectroscopy (NIRS) can be used to precisely assess muscle mitochondrial respiratory capacity in humans [5], thus allowing the non-invasive, real-time monitoring of muscle oxygenation at rest and during exercise (for a review, see [6,7]). In the field of neuromuscular diseases, NIRS has been used mainly to explore oxygen (O 2 ) uptake impairment in patients with mitochondrial diseases [8][9][10]. To the best of our knowledge, there are only few reports on muscle oxygenation in patients with muscular dystrophy [11][12][13]. In these studies, NIRS was used to monitor muscle oxygenation during isokinetic contractions, treadmill exercise or continuous isometric contractions at low intensity. Different results concerning the level of muscle oxygenation during contraction were obtained in function of the dystrophy type and exercise workload [11,12,14].
As patients with FSHD present peripheral skeletal muscle dysfunction [3,4] and reduced cytochrome c oxidase activity and ATP synthesis [3], we hypothesized that NIRS signal during maximal muscle contraction (MVC) is altered in these patients compared with age-matched healthy sedentary controls. We also hypothesized that in patients with FSHD, variations in muscle mitochondrial respiratory capacity determined by NIRS could be related to the level of systemic oxidative stress markers, muscle volume parameters and muscle typology.

Study design
This study was not randomized. This was a transversal comparative study of a sub-group of patients recruited from both arms of a double-blind, placebocontrolled, randomized trial in patients with FSHD entitled "Effects Antioxidants Supplementation on Muscular Function Patients Facioscapulohumeral Dystrophy (FSHD)" at the Clinical Physiology Department, Montpellier University Hospital (France). Recruitment to the main study "Effects Antioxidants Supplementation on Muscular Function Patients Facioscapulohumeral Dystrophy (FSHD)" began in May 2010 and a protocol amendment to permit additional analyses and enrolment of healthy controls to the present study was approved 9 months later. The present study allowed to compare changes in quadriceps oxygenation and hemodynamics during maximal voluntary quadriceps isometric contraction and to determine the relationships between these parameters and both systemic oxidative stress markers and muscle function. The inclusion criteria for the main study were published in Passerieux et al., 2015 [4]. Briefly, patients with FSHD between four and nine D4Z4 repeat units and with positive family history for FSHD, aged from 18 to 60 years and without HIV and/or hepatitis were included. Controls were sedentary individuals (i.e., less than 1h of physical activity per week). The exclusion criteria for all subjects were: cardiovascular contraindications to exercise, peripheral arterial vascular disease, cardiac or pulmonary disease, diabetes or human immunodeficiency virus infection and presence of current drug treatments, including vitamins and/or antioxidants.

Ethics committee approvals
The original full study protocol was approved by the institutional ethics committee of the Montpellier University Hospitals (Comité de Protection des Personnes de Montpellier -ref. 09 01 01; ID-RB: 2008-AOI1582-53) and the French National Agency Medicine and Health Products Safety (ANSM -B900956-40), and was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments and the European guidelines for good clinical practice. This study was registered on clinicaltrials.gov (NCT01596803). The subgroup of patients with FSHD and healthy controls study was approved by the institutional ethics committee of the Montpellier University Hospital (Comité de Protection des Personnes de Montpellier -ref. 09 01 01; ID-RB: 2008-AOI1582-53) as a separate amendment. The trial objectives, study design, risks, and benefits were explained and written informed consent was obtained from all participants. This amendment allowed to compare changes in quadriceps oxygenation and hemodynamics during maximal voluntary quadriceps isometric contraction and to determine the relationships between these parameters and both systemic oxidative stress markers and muscle structural parameters (localization of mitochondria, typology and muscle volume determined by magnetic resonance imaging in patients with FSHD and healthy controls).

Exercise protocol
All subjects (FSHD and CONT) came twice to the laboratory at the INSERM U1046, Centre Hospitalier Regional Universitaire (CHRU) of Montpellier, France. During the first visit, the written informed consent and baseline clinical and physical activity data were collected. Moreover, a magnetic resonance imaging (MRI) examination was performed. Then, subjects were familiarized with the testing procedure and performed the maximal voluntary quadriceps isometric contraction (MVC Q ) test associated with muscle oxygenation measurement by NIRS in a well-ventilated room at a temperature of 20-22°C. During the second visit, all subjects underwent a skeletal muscle biopsy and blood sampling ( Figure 1).

Figure 1
Study design and exercise protocol. Physical test consisted of 3 to 5 maximal voluntary quadriceps isometric contraction (MVC Q ) for about 6s to evaluate the maximal peak torque and the oxygenation parameters with the near-infrared spectroscopy (NIRS). The text between the boxes indicates the rest periods.

Force determination during maximal voluntary contraction
The exercise protocol (MVC Q ) was performed on an adapted exercise bench (Banc de Koch, Genin Medical, France) in seated position with knees and hips flexed at 90° and arms crossed over the chest, as routinely done in our laboratory [18]. All volunteers performed three maximum isometric knee extension trials to obtain at least two values with less than 10% variability and the best value was taken as the MVC Q , if they could not reach this variability, two others MVC Q were realized. Each participant was instructed to perform a maximum fast force rise and to maintain the maximum contraction force for about 6s during isometric contractions, with at least 1 min of rest between contractions ( Figure 1).

Determination of blood antioxidant status and stress markers
The antioxidant status and stress markers were evaluated as previously described [4]. Reduced (GSH) and oxidized glutathione (GSSG), glutathione peroxidase (GSH-Px), copper-zinc superoxide dismutase (CuZn-SOD) and marker of lipid peroxidation (lipid peroxides) were measured in venous blood samples. The detection of urinary 8-hydroxyguanosine (8-OH-dG, a marker of DNA oxidation (oxidized DNA)) levels was normalized to creatinine levels in the urine. Each parameter was routinely determined at Liege University Hospital, Belgium, as previously described [3]. The reference intervals of the mean values of antioxidant and oxidative stress markers were obtained from a large healthy population [15][16][17].

Force data analysis
Force was indicated visually on the computer screen and a computer beep was used to denote the timing of muscle contraction and relaxation. MVC Q signal was recorded through a strain gauge linked to a computer interface (Biopac MP150WSW, Acknowledge, France). For each participant, MVC Q and Δtime were determined from the force-time curve as the absolute maximum value (N) and the time to reach the MVC Q (s), respectively. The rate of force development (ROFD) was defined as the ability to generate the fastest force development [19]. ROFD (N.s -1 ) corresponds to the maximum value of the MVC Q /Δtime ratio, and ROFD t200 corresponds to the rate of force development 200ms after the beginning of the contraction [20]. Both ROFD and MVC Q were normalized to the participant's body weight (kg). The difference between the end and the beginning of each contraction was considered as the contraction duration.

Determination of muscle oxygenation by near-infrared spectroscopy and data analysis
The NIRS signal reflects the balance between O 2 supply by the circulatory system and O 2 consumption by the muscle and thus allows measuring the quantitative variations in muscle oxygenation, but also the kinetics of deoxygenation and reoxygenation (for a review, see [6,7]). For this study, a spectrometer NIRO 200 was used (Hamamatsu Photonics, Japan). This apparatus measures concentration changes in oxyhemoglobin (O 2 Hb), deoxyhemoglobin (HHb), and total hemoglobin (tHb) that are expressed in µmol.l -1 .cm. The tissue oxygenation index (TOI) = O 2 Hb/tHb, expressed as a percentage, gives an absolute measure of O 2 Hb saturation. The optical probe consisted of one emitter with three laser-emitting diodes (775, 810, 850nm) and one detector with two silicon photodiodes (4 mm between photodiodes) separated by a distance of 4 cm. The probe unit supported by a rubber shell was firmly attached to the skin of the distal region of the dominant leg, 15 cm above the knee joint, parallel to the major axis of the quadriceps muscle. Double-sided adhesive tape was used to prevent the probe from sliding on the skin during the exercise. A soft black cloth covered the rubber shell, and all wires were taped down to minimize their movement during exercise.
Prior to each testing session, the NIRO-200 initialization procedure was carried out to set each laser power automatically in order to establish the optimum measurement conditions, and all used procedures were previously described by [21,22]. NIRS signals were sampled at 2 Hz by the NIRO-200 apparatus and collected simultaneously with torque data using the Biopac Acqknowledge Data Acquisition software. They were saved using the NIRO-200 Online software (Version 1.20.03) and stored on the computer for later analysis.

NIRS data analysis
The ΔTOI deoxygenation slope (ΔTOI DSlope ), which represents muscle O 2 consumption per unit of time, was determined as the negative slope of the least squared regression line of ΔTOI during the contraction phase. The minimum ΔTOI amplitude (ΔTOI min ) was the difference between the minimum TOI value reached at the end of the contraction phase and the mean 30s baseline TOI value. To provide an estimation of efficiency, ΔTOI min values were normalized to the force peak (i.e., ΔTOI min /MVC Q ) in both groups. The ΔTOI reoxygenation slope (ΔTOI RSlope ), which represents the muscle oxidative capacity and/or O 2 supply, was the average positive slope of the least squared regression line of ΔTOI during the 6s after reaching ΔTOI min [21,22]. The maximum ΔTOI (ΔTOI max ) was the difference between the maximum TOI value reached during the 15s relaxation phase and the corresponding mean 30s baseline TOI value. The mean ΔtHb amplitude (ΔtHb mean ) was the difference between the mean tHb value during the contraction phase and the corresponding mean 30s tHb. The t 50 was the half time of oxygenated hemoglobin (O 2 Hb) recovery to baseline values [23]. The physiological significance of these NIRS parameters was previously described by [21][22][23]. We also included the maximum ΔtHb amplitude during the relaxation period (ΔtHb max ) and the ΔtHb analysis at specific time points: at the start of contraction (ΔtHb 0 ), at the end of contraction (ΔtHb end ), at 6s and 15s after the end of contraction (ΔtHb 6 and ΔtHb 15 ).

Determination of muscle volume by MRI
MRI was performed using a 1.5 Tesla MRI apparatus (Magnetom Area Avento, Siemens Medical Erlangen Germany) with "lower limbs" surface antennas without injection of contrast dye and the participants in supine dorsal position, lower limbs at rest. T1-weighted 3 dimensional (3D) flash sequences were obtained in three orthogonal planes centered on the thighs. All images were treated with the Myrian ® 64 1.14.2 software (Intrasense, Montpellier, France). MRI images were analyzed by drawing a two-dimensional (2D) "mask" for each ten transversal leg images. Then, the software recognized all transversal masks and determined a 3D mask for the muscle group volume in both legs. To build the 3D mask, first we verified in six patients how many 2D masks (one 2D mask/each 5, 10, 15, 20 or 30 images, respectively) were required to sustain the 3D mask quality. The same procedure was used for assessing the fat infiltration and muscular tissues.
The muscle morphological analysis was carried out by using the thresholding technique and by contour recognition allowing the quantification of morphologically healthy muscle volume (i.e. without evidence of fatty degeneration). To detect and quantify the volume of muscle not affected by the dystrophic processes, we determined an intensity threshold (55-130 cm 3 ) for the identification and quantification of the nonaffected muscle volume. Volumes were expressed in cm 3 . More than 300 images per participant were treated.

Determination of muscle regions
To standardize and minimize the probability of erroneous interpretations of the MRI images, all measurements were determined from a standardized bone position between the little trochanter and the proximal extremity of the patella. The determination of the regions of interest (ROI) was performed in each muscle group, outside the fascia and vascular structures. The first ROI, named QD, included all quadriceps muscles (rectus femoris, vastus lateralis, vastus intermedius and vastus medialis). The second ROI, named POST, included the medial muscles (pectineus, gracilis and the three adductors: long, short and wide), hamstrings (semimembranous, semi-tendon and biceps femoris) and sartorius. The lipid ratio (RL) was determined by the formula:

Skeletal muscle biopsy
A muscle biopsy of the lateral part of the vastus lateralis was performed under local anesthesia (xylocaine) by using the percutaneous Bergstrom technique currently employed in our laboratory. Samples were then dissected free of visible connective tissue and fat. Each muscle biopsy was divided in two parts: one part to evaluate the tissue organization by hematoxylineosin staining of transversal sections and to investigate the mitochondria morphology (30mg) by transmission electron microscopy (TEM) and one part to assess the fiber typology.

Muscle fiber typology
To determine the muscle fiber typology, muscle tissues were frozen in precooled isopentane and then liquid nitrogen. Tissues were cut in fine slices of 10 microns thickness, mounted and stored at -20 o C. For immunofluorescence analysis, all incubations were carried out at room temperature on a shaker and in the dark, in the case of fluorescent immunoreagents. The following primary antibodies were used: anti-myosin heavy chain slow (MHCs) (1:1000 in PBS; Sigma ref M8421) and anti-myosin heavy chain fast (MHCf) (1:800 in PBS; Sigma ref M4276). After washes, samples were incubated with the secondary goat anti-mouse Alexa 488 antibody (1:1000 in PBS; Invitrogen ref A11017). Images were acquired with an Axio Imager M1 fluorescence microscope (Zeiss) and treated with Image J 1.47K and Adobe Photoshop CS3.

Mitochondrial morphology
The mitochondrial morphology was determined by TEM. Fresh muscle samples (5 mg) were immediately fixed in 2% paraformaldehyde, 2.5% glutaraldehyde and 0.1M cacodylate buffer, at 4°C overnight, and then rinsed with 0.1M cacodylate buffer. Samples were then prepared as previously described [24]. TEM analyses were carried out at the Centre Régional d'Imagerie Cellulaire (CRIC) of Montpellier (France).

Statistical Analysis
In this exploratory ancillary study, a total of 17 patients with FSHD completed the study (evaluation of muscle oxygenation by NIRS, muscle function parameters and oxidative stress markers). These patients were individually matched with healthy controls on age and BMI. No sample size was calculated. Data were summarized as mean ± standard deviation (SD). Groups were compared by the unpaired Student ttest or by the non-parametric Kruskal-Wallis (KW) test in case of skewed distributions. The association between any two variables was assessed by the Spearman correlation coefficient. Results were considered significant at the 5% critical level (p<0.05). All statistical calculations were performed with SAS version 9.4 (SAS Institute, Cary, North Carolina). In order to verify the effect size in the comparisons of averages between CONT and FSHD groups, Cohen's d was observed, and the values of d were considered small if 0.20 ≤ d <0.50; 0.50 ≤ d <0.80 and large if d ≥ 0.80 [25]. Analysis of covariance (ANCOVA using SigmaPlot 13 software) was performed, with "NIRS parameters" as the dependent variable and MVC Q and group (patient with FSHD/control) as independent covariates

Subjects
Among 53 adult patients with FSHD enrolled within the main study (Passerieux et (Table  1).

Quadriceps mechanical performance
Results obtained during quadriceps isometric contraction at the maximum voluntary intensity (see representative curves in Figure 3) indicated that MVC Q (160 ±111 vs 308 ± 103 N), ROFD (80.5 ± 75.4 vs. 362 ± 264 N.s -1 ) and ROFD t200 (402 ± 396 vs. 1642 ± 941 N.s -1 ) were significantly lower in the FSHD than in the CONT group (Table 1). These results did not change after normalization to body weight ( Table 1). The Δtime also was significantly higher in patients with FSHD than in CONT subjects. All mechanical parameters presented a large effect size (Cohen's d).
The t 50 value was significantly lower in the FSHD than in the CONT group (9.91 ± 5.97 vs. 15.8± 3.96 s; p<0.01, d=1.15). All muscle oxygenation parameters also presented a large effect size (Cohen's d).
The ΔtHb decreased rapidly at the onset of contraction in both groups ( Figure 4B). ΔtHb was nearly constant during the contraction phase (ΔtHb mean ) in both groups, suggesting blood flow occlusion due to muscle contraction. Then, ΔtHb quickly increased after the end of the contraction (reperfusion), and ΔtHb 15 and ΔtHb max were significantly higher in the FSHD than in the CONT group (ΔtHb 15    Data are the mean ± SD of 17 patients with FSHD and 14 controls (CONT). * p<0.05, **p<0.01; tHb rest : total hemoglobin volume at rest; ΔtHb 0, ΔtHb end, ΔtHb 6, and ΔtHb max, : total hemoglobin difference before the contraction start, at the end of contraction, at 6s and 15s after the end of the contraction and maximum amplitude, respectively.

Muscle structure determined by MRI
The total volumes of the QD and POST muscle regions (TV QD and the TV POST ) were not significantly different between groups. However, the absolute volume and the percentage of healthy QD (MV QD and %MV QD ) and POST muscles (MV POST and %MV POST ) were significantly lower in the FSHD than in the CONT group (Table 3). Conversely, the volume of fat infiltration in QD and POST muscle regions (FV QD and FV POST ) was significantly higher in the FSHD than in the CONT group (FV QD : 292 ± 193 vs. 18.5 ± 7.5 cm 3 ; FV POST : 746 ± 425 vs. 155 ± 59.9 cm 3 ) as observed also in the MRI images ( Figure 5). Excepted MV QD that presented a medium size effect, all others significantly different parameters presented a large effect size. Data are the mean ± SD of 17 patients with FSHD and 7 controls (CONT). *p<0.05, **p<0.01 and ***p<0.001 (vs CONT). MV: volume of healthy muscle; FV: volume of fat infiltrations and connective tissue; TV: MV + FV; % MV: percentage of healthy muscle volume. QD: quadriceps muscles (rectus femoris, vastus lateralis, vastus intermedius and vastus medialis); POST: medial muscles (pectineus, gracilis and the three adductors: long, short and wide), hamstrings (semi-membranous, semi-tendon and biceps femoris) and sartorius.

Vastus lateralis fiber typology
The vastus lateralis fiber typology parameters (number, diameter, surface and percentage of MHCs and MHCf fibers) were not significantly different between FSHD and CONT groups (Table 4).

Mitochondrial morphology by TEM
TEM analysis showed morphological changes in muscle mitochondria, particularly aggregation of subsarcolemmal mitochondria near blood capillary ( Figure 6).

Correlations between MVC Q and muscle oxygenation parameters in FSHD AND CONT
Analysis of the correlations between mechanical and muscle oxygenation parameters in all study healthy controls (n=14) and FSHD (n=17) showed significant correlations between MVC Q and both ΔTOI Dslope and ΔTOI min in FSHD group (r = -0.78, p<0.005) ( Table 5,  Fig7a). Similarly, these correlations were also observed in CONT group (r = -0.609, p<0.05). For ΔTOI Dslope and ΔTOI min , analysis of variance with an interaction term showed that there was no significant interaction between the groups (patients with FSHD and healthy controls) and MVC Q (P=0.816 and P = 0,097 respectively). However, for ΔTOI min this interaction tended to be significant suggesting that the relation between ΔTOI min and MVC Q differed between groups. In the model without interaction ΔTOImin adjusted on MVC Q remained significantly different (P < 0,001) between control and patients with FSHD and the coefficient of covariate MVC Q was significantly different from zero (P <0,001) suggesting that the covariate MVC Q significantly affects the ΔTOImin.  Values are mean ± SD of histological parameters analysed in fibers of vastus lateralis biopsies stained with anti-Myosin Heavy Chain Slow (MHCslow) and Myosin Heavy Chain Fast (MHCfast) antibodies. Data are the means ± SD of 17 patients with FSHD and 7 controls (CONT).

Correlations between muscle parameters, oxidative stress and antioxidant markers in FSHD and CONT
No correlation between muscle parameters and antioxidant status or oxidative stress markers could be found in healthy controls (n=7). In patients with FSHD, the blood level of lipid peroxides was negatively correlated with the MVC Q (r = -0.69, p<0.01) and with MVC Q /BW (r = -0.64, p <0.01). By contrast, blood concentrations of the antioxidant marker GSH-Px increased with MVC Q /BW values (r = 0.46, p <0.05) (Table 5). Similarly, a negative correlation was observed between TOI Dslope and GSSG values (r = -0.64, p<0.01). The TOI Dslope was also positively correlated with the GSH/GSSG ratio and the blood levels of lipid peroxides (oxidative stress markers) (r = 0.57, p<0.05 and r = 0.43, p<0.05, respectively). A significant positive correlation was also observed between ΔTOI min /MVC Q and the concentration of the antioxidant GSH (r = 0.45, p<0.05) ( Table 5). MVC Q and MVC Q /BW were significantly related with MV QD (r = 0.92, p<0.005 and r = 0.83, p<0.001, respectively) and % MV QD (r = 0.82, p<0.001 and r = 0.73, p<0.05, respectively) ( Table 5 and Figure 7b). Moreover, ROFD and ROFD/BW were correlated with MV QD (r = 0.69, p<0.05 and r = 0.63, p<0.05, respectively). Finally, the reduction in muscle volume in patients with FSHD resulted in a reduced O 2 consumption during muscle contraction. Hence, a negative and significant correlation between the MV QD and the deoxygenation rate (r = -0.68, p<0.01) was observed in this group (Figure 7c).

Discussion
The results of this study suggest that, compared to healthy controls, patients with FSHD evidence a number of salient findings: 1) oxidative stress markers and antioxidant values are altered; 2) torque production is significantly decreased and is associated with reduced local O 2 consumption per unit of time; 3) muscle oxygenation and muscle performance parameters (ΔTOI Dslope , MVC Q ) are significantly correlated with oxidative stress markers; 4) skeletal muscle volume is significantly reduced and fat tissue increased for the same volume, although muscle typology is comparable between groups; 5) the reduction in muscle volume is related to reduced force production. The present data provide novel insights into the muscle oxidative metabolism, muscle volume and the correlations of these parameters with oxidative stress in patients with FSHD and contribute to explaining the reduction in force production observed in these patients.
The levels of oxidative stress markers and antioxidant molecules (GSH, GSSG, GSH-Px, CuZn-SOD, lipid peroxides, and oxidized DNA) were significantly altered in the FSHD group compared with the CONT group. These findings confirm the higher oxidative stress levels previously observed in patients with FSHD [3,4]. The higher oxidative stress levels are correlated with lower force development and ROFD in the FSHD group compared with the CONT group. A significant betweengroup difference in the oxidative metabolic demands (i.e., O 2 consumption relative to O 2 supply) in the quadriceps was observed during MVC Q , in accordance with a previous study [14].
The difference in O 2 demand between groups could be explained by the negative correlation between MVC Q and ΔTOI Dslope (Fig. 7a), observed also in control group. This suggests that the different muscle O 2 demands represent a different activation of the quadriceps muscle volume [21,25] and/or that in FSHD, less muscle mass is involved during contraction (due to the presence of muscle fatty infiltration). However, ΔTOI Dslop was not significantly different between groups after normalization by MVC Q and there was no significant interaction between groups (patients with FSHD and healthy controls) and the covariate MVC Q . But we cannot exclude lack of statistical power to detect between group differences. The finding that ΔTOI min was significantly different between groups only after normalization to force production (Table 3) confirms that dystrophic quadriceps muscles in FSHD produce less force for a lower O 2 consumption. Therefore, the reduced ΔTOI DSlope, in conjunction with arterial occlusion during MVC Q [28], as indicated by the similar values of ΔtHb 0 and ΔtHb end , shows that the volume of O 2 consumption by dystrophic quadriceps is reduced in the FSHD group compared with controls. This suggests a reduction in the ability of mitochondria to provide energy during MVC Q , which is consistent with previous results from our laboratory [3]. Indeed, in FSHD muscle biopsies, we observed mitochondrial dysfunction (decreased cytochrome c oxidase and ATP synthesis activities) that was correlated with lower quadriceps force production [3].
A novel finding of the present study is that the significant difference in deoxygenation kinetic parameters during MVC Q between the FSHD and CONT groups is linked to the oxidative stress status in patients with FSHD. Mitochondrial dysfunction and oxidative stress might play a crucial role in FSHD pathology [4,[29][30][31][32]. Interestingly, the ΔTOI Dslope was negatively correlated with the blood level of lipid peroxides and GSSG and positively correlated with the GSH/GSSG ratio in the FSHD group ( Table 5). The link between lower O 2 demand and higher lipid peroxide levels could be the result of morphological alterations in muscle mitochondria induced by oxidative stress that might lead to lower ATP synthesis in patients with FSHD [3]. Indeed, it was previously shown that NIRS data are correlated with the mitochondrial capacity in healthy subjects [5]. Moreover, the imbalance between lower blood GSH and higher blood GSSG values, observed in the FSHD group, could be explained by modifications in the intracellular redox environment and is consistent with the observation that GSH reduction reflects an increase in oxidative stress and mitochondrial dysfunction [33].
The significant reduction in force production and ROFD in patients with FSHD compared with controls is consistent with the muscle loss and weakness that characterize this disease [3,[34][35][36]. This is associated with alterations in the energy metabolite levels [37] and lower quadriceps force development during MVC Q in patients with FSHD compared with healthy subjects [3]. An additional novel aspect of the current study was to investigate muscle volume and fat infiltration in the thigh by MRI and not only the cross-section surface area [37,38]. To our knowledge, this is the first study assessing muscle volume in patients with FSHD. We did not find any difference in the total volume of quadriceps and posterior muscles (TV QD and TV POST ) between the FSHD and CONT groups. However, we report a significant reduction in the percentage of healthy muscle volume (MV QD and MV POST ) in patients with FSHD compared with CONT. This could be explained by the significant increase in fat infiltration and fibrous tissue (FV QD and FV POST ) in FSHD muscle (Table 3) and is consistent with previous observations obtained on cross-sectional surface area [35]. Moreover, muscle volume and fat infiltration as well as the deoxygenation kinetics were linked to force production (Fig. 7c) and were correlated with oxidative stress markers and the antioxidant status in patients with FSHD (Table 5). Therefore, our results suggest that the relationship between increased fat infiltration and lower muscle strength is coherent with the loss of muscle mass [37]. Additional studies are needed to confirm that muscle atrophy occurs in response to oxidative stress and the effects of antioxidant supplementation to counteract dystrophy progression.
In addition to these structural reorganizations inside FSHD muscles, we observed morphological changes characterized by abnormal aggregation of subsarcolemmal mitochondria near blood capillaries. This reorganization occurred without changes in muscle fiber typology. Indeed, we found similar muscle fiber typology in vastus lateralis biopsies from both groups, in accordance with the typology observed in healthy muscles [39]. Furthermore, Statland et al. (2015) observed a decrease in the capillary density in the vastus lateralis of patients with FSHD that could result in lower intracellular O 2 tension. Thus, mitochondria aggregation near blood capillaries could be the result of a migratory movement inside skeletal muscle regions with a better O 2 pressure gradient. The exact mechanism of this internal mitochondria reorganization is an important question for future studies.

Conclusions
The findings of this study provide clear evidence that patients with FSHD present a lower O 2 demand during maximal isometric contraction related to higher oxidative stress and reduced muscle volume, leading to reduced muscle functional performance compared with healthy sedentary controls. While the present work provides clear evidence that NIRS can be a useful tool to assess the kinetics of deoxygenation and reoxygenation in FSHD during maximal isometric exercise, other studies are necessary to determine the mechanisms by which muscle function is altered in FSHD. Nevertheless, this study supports the idea that oxidative stress could play an important role in FSHD pathophysiological mechanisms [29][30][31][32].