Genetically confirmed patients with FSHD1 will be recruited from the Radboud university medical center. Healthy individuals without a history of neuromuscular disease will be included as healthy controls. Two disease control groups (one with muscular dystrophy and one with inflammatory myopathy) will be included to determine whether the results obtained in this study are specific for FSHD, or a general feature of muscular dystrophy or inflammation – both histological features of FSHD. For the muscular dystrophy disease control group, patients with genetically confirmed OPMD (12–17 trinucleotide repeats in PABPN1) will be included. OPMD was chosen because it is a relatively common muscular dystrophy, has an adult age of onset and has no associated systemic features which might influence muscle fiber function. Also, patients are not likely to be wheelchair bound, which is an exclusion criterion for this study. For the inflammatory myopathy control group, patient with sIBM fulfilling the modified 2010 Griggs criteria will be included . We have chosen sIBM because it is relatively common, has clearly delineated histological features and diagnostic criteria, and patients are not treated with corticosteroids, which is an exclusion criterion for this study. The full list of exclusion criteria is as follows:
Age <18 or >65 years
Chronic obstructive pulmonary disease
Chronic heart failure
Previous treatment with chemotherapy and/or radiation therapy
Use of corticosteroids during more than two weeks in the past 5 years
Use of statins in the past year
Contra-indications for MRI or muscle biopsy
In each group, 14 participants will be included for a total of 56 participants. Age and gender matching will be applied on the group level. The Medical Ethics Review Committee region Arnhem-Nijmegen approved the study protocol.
In vivo study procedures
Information on medical history and current medication use will be collected. Body weight and length will be measured. Parameters of muscle injury will be assessed in serum (creatine kinase (CK), lactic dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT)). In FSHD patients, disease severity will be assessed using the Ricci Clinical Severity Scale (CCS) and the CSS corrected for age: age-corrected CSS (ACCSS) [21–23].
Physical activity and muscle strength
Physical activity will be assessed in several ways. Participants will be equipped with an actometer, a device that is attached to the ankle to constantly measure physical activity during 12 days [24, 25]. Validated questionnaires will be administered to assess participants' daily living activities, disease impact and possible confounding factors such as pain: Health Assessment Questionnaire (HAQ) , International Physical Activity Questionnaire (IPAQ) , Sickness Impact Profile (SIP 68) , McGill’s Pain Questionnaire (MPQ) . Muscle strength will be graded with the Medical Research Council (MRC) scale . A trained examiner will obtain a Motor Function Measure . In addition, a six-minute walk test and timed up and go test will be performed [32, 33].
Quantitative muscle studies
Isometric force recordings of maximal voluntary and electrical evoked contractions of the quadriceps femoris will be performed as described previously . Participants will be seated in a custom-build chair with the knee angle set at 120°. To prevent compensatory movements, participants are strapped at the hips and upper body to maintain their position. Electrical pulses will be delivered through two self-adhesive surface electrodes placed proximally over the rectus femoris and distally over the vastus medialis portion of the quadriceps muscle. Two gel electrodes will be used to make a surface electromyogram (sEMG).
Maximum voluntary contraction
All measurements will be performed in the leg from which the muscle biopsy will be taken (i.e., the right leg, unless in the presence of asymmetrical weakness in which case the weakest leg will be biopsied). Participants will be asked to perform a Maximum Voluntary Contraction (MVC) during 3 seconds. The highest force from three contractions is used to represent the MVC. Next, the 100 Hz current that evokes 30% of MVC will be determined and used for all subsequent measurements.
After a 5-minute rest period, the force–frequency relation will be determined with 1 Hz, 100 Hz, 1 Hz, 10 Hz, 20 Hz, 30 Hz, and 50 Hz pulse trains of 1 s duration. There will be a 1-minute rest period after each contraction. The 1 Hz stimulation will be performed multiple times to verify that the 100 Hz pulse train causes no fatigue or potentiation. The contraction time, rate of force rise, and early and late relaxation times will be derived from the 100 Hz contraction (positively filtered with a 30 Hz filter frequency). These parameters provide a measure for the rate of cross-bridge cycling and the rates of Ca2 reuptake by the sarcoplasmic reticulum.
After another 5-minute rest period, a series of 30 Hz 1 s on 1 s off pulses will be applied for a period of 2 minutes to determine muscle fatigability. Fatigue is determined as the relative decline of force between the first three contractions compared with that of the last three contractions.
A MR exam of the upper and lower leg will be performed in all participants using a 3 Tesla MR system (Tim TRIO, Siemens, Erlangen, Germany) with the spine array coil and two phased-array coils placed around the legs. A fish-oil marker will be placed on the site where the muscle biopsy will be taken from, to ensure that MRI findings correspond to the approximate site of muscle biopsy. Patients will be placed in the scanner feet first supine. The table will be positioned to have first the upper and subsequently the lower leg in the isocenter of the magnetic field for imaging of the thigh and calf muscles respectively.
Scout images will be acquired in three orthogonal directions for accurate positioning of the MRI slices. Thereafter 8 transversal slices (FOV 175×175 mm2, thickness 4 mm, gap 6 mm, baseresolution 256) will be acquired with a T2 multi spin echo sequence (TR: 3000 ms, 16 equally spaced echo times 7.7 – 123.2 ms). Subsequently followed by 23 transversal slices (thickness 4 mm, gap 0.4 mm) that will be obtained with a T1 turbo spin echo sequence (FOV 250×244.5 mm2, TR/TE 600 ms/13 ms, baseresolution 448), and with a Turbo Inversion Recovery sequence (TIRM) (FOV 175x175 mm2, TR/TE/TI 4100 ms/41 ms/220 ms, baseresolution 256). The same imaging protocol will be used for both the upper and lower leg.
Hyperintensities on the TIRM images will be detected visually as an indicator for edema and/or inflammation . Muscle fraction at the slice corresponding to the approximate site of muscle biopsy will be quantified using the method described by Kan .
In vitro study procedures
One needle muscle biopsy of the quadriceps femoris (vastus lateralis), and one of the tibialis anterior will be obtained from each participant. The tibialis anterior is frequently affected in FSHD, but early involvement of the quadriceps femoris is uncommon [3, 37]. In the presence of symmetrical muscle strength, the right side will be biopsied. In case of asymmetrical weakness based on neurological examination, the weakest muscle will be biopsied.
An experienced neurologist or neurology resident, taking routine antiseptic precautions, will perform the muscle biopsies. Biopsy material intended for histology will be frozen in isopentane and stored at −80°C. Material for protein analysis will be immediately frozen in liquid nitrogen and stored at −80°C. Specimens intended for contractile studies are stored at −20°C in a glycerinating solution containing half glycerol and half relaxing solution. For composition of this solution, see below.
Muscle fiber preparation
For contractile studies, biopsy material will be placed in a relaxing solution containing 1% (v/v) Triton X-100 at 4°C . Triton is used to permeabilize the plasma membranes, resulting in “skinned” muscle fibers. The skinning process enables studies of sarcomeric function in isolation by eliminating the confounding effects of upstream processes of excitation-contraction coupling, such as sarcoplasmatic reticulum Ca2+ handling. Protease inhibitors will be added to the solution to prevent protein degradation. Skinned single muscle fibers will be isolated and fiber ends will be attached to aluminum t-clips. The clips are then mounted to a length motor on one end, and a force transducer on the other.
Muscle fiber mechanics
Maximum force generation and cross-bridge cycling kinetics
Skinned muscle fibers will be activated by sequentially bathing them in a relaxing solution, pre-activation solution and activation solution. The composition of these solutions has been described previously . Force generating capacity will be measured at a sarcomere length of 2.5 μm. Maximal force generating capacity will be measured at pCa 4.5 and corrected for cross-sectional area. To determine whether changes in maximal force are due to changes in the kinetics of cross-bridge attachment and detachment, a rapid stretch-release protocol will be imposed on an activated fiber. The rate constant of tension redevelopment (Ktr) will be determined by fitting the rise of tension to the following equation: F = Fss(1 – e-Ktr·t) . After force redevelopment reaches the steady-state maximal force again, active stiffness will be determined. Length changes of sequentially 0.3, 0.6, 0.9, -0.3, -0.6 and −0.9% will be applied, during which peak force is measured. Corrected peak force will be linearly plotted against the imposed length changes, with the slope representing the fraction of strongly attached cross bridges .
Calcium sensitivity of force generation
During daily-life muscle activation, individual fibers typically function at a submaximal level. Therefore, the calcium sensitivity of force generation provides additional information on contractile properties. The calcium sensitivity of force generation will be measured by bathing skinned muscle fibers in activation solutions with different pCas ranging from 4.5-7.0 and measuring steady-state force. The obtained force-pCa data will be fitted to the Hill equation, providing the pCa50 (the pCa giving 50% of maximal active tension) and the Hill coefficient, nH (an index of myofilament cooperativity) .
Passive force generation
Passive force generation will be measured by placing skinned muscle fibers in a chamber filled with relaxing solution and imposing length changes as previously described . The fibers will be set to their slack length (i.e. the length at which passive force is zero) and from there they will be stretched with a constant velocity to a sarcomere length of 3.2 μm, held for 90 s and then released back to slack length. Tension development during stretch will be determined.
Muscle protein analysis
To study whether changes in sarcomere contractile function are a consequence of proteomic alterations, extensive protein analysis using SDS-PAGE and Western blotting will be performed as described previously [43, 44]. We will pay special attention to myosin heavy chains, actin, titin, nebulin and regulatory proteins such as troponin and tropomyosin. In addition we will determine oxidation/nitrosylation (i.e. posttranslational modifications in response to oxidative stress) of sarcomeric proteins by Oxyblot and Western blotting, as described previously .
To study whether decreased maximal force generation is a result of decreased contractile protein content, we will determine myosin content per half sarcomere and will relate the results to the force generated by those fibers as described previously . In brief, myosin heavy chain (MHC) content and MHC isoform composition will be determined using SDS-PAGE. We will calculate fiber myosin concentration by dividing total MHC content by fiber volume (at a sarcomere length of 2.5 μm). This fiber myosin concentration will be multiplied by half-sarcomere volume to calculate MHC content per half sarcomere. Finally, MHC content per half sarcomere will be related to muscle fiber mechanics.
For analysis of titin and nebulin exon composition, we will use a titin/nebulin exon microarray that consists of 50-mer oligonucleotides representing all exons in the titin gene, spotted in triplicate on the array. The array contains human titin and nebulin genes as well as all known titin-binding proteins .
Histology will be used for light-microscopic evaluation of frozen sections. We will study size, shape, type and cytoarchitecture of muscle fibers, presence of internalized nuclei, destruction of muscle fibers and regeneration, as well as supporting connective tissue.
The primary outcome measurement of this study is the maximum force generating capacity (mN/mm2) of permeabilized single muscle fibers of FSHD patients and healthy control subjects. Secondary outcome measurements are:
The maximum force generating capacity of OPMD and sIBM fibers in relation to FSHD fibers.
Contractile properties of FSHD fibers such as cross-bridge cycling kinetics, calcium sensitivity and passive force generation in relation to fibers of healthy controls.
Maximum force generating capacity and contractile properties of muscle affected early (tibialis anterior) compared to muscle affected late (quadriceps femoris) in the disease course.
Analysis of sarcomeric proteins and titin and nebulin exon composition of FSHD muscle.
In vivo clinical parameters such as physical activity, muscle strength, disease severity, residual D4Z4 fragment length, muscle/fat fraction and quantitative muscle studies.
Statistical analysis and power calculation
Differences between the above-mentioned outcome measurements will be analyzed using analysis of covariance (ANCOVA). A p value <0.05 will be considered significant.
Due to the exploratory nature of this study, there are virtually no previous studies to refer to for power calculations. Based on our pilot study, we aim to test the hypothesis that there is a difference in maximum force generating capacity of 7 mN/mm2 (~10% of FSHD maximum force generation), with standard deviations of 6 mN/mm2, α = 0.05 and β = 0.20. This hypothesis requires a sample size of 13 subjects per group. Because the muscle tissue can be damaged during the biopsy procedure, we anticipate that ~10% of the collected biopsies will not be useful for experiments. This brings the total sample size to 14 subjects per group.