Muscle biopsy and autopsy tissues were obtained and prepared using standard histological protocols . The rectus femoris, psoas, quadriceps, diaphragm and cardiac muscles were obtained from patient 16-2, and the diaphragm and abdominal wall muscles were obtained from patient 16-4. Briefly, fresh muscle was frozen in isopentane and stored at -80°C until sectioning. Eight-micrometer cryostat sections were stained with hematoxylin and eosin, Gomori trichrome, ATPase (at pH 4.3 and pH 9.2) or NADH. Photomicrographs were obtained by using a Nikon Eclipse 50i microscope (Melville, NY, USA) equipped with a SPOT Insight 4 Meg FW Color Mosaic camera and SPOT 188.8.131.52 software from Diagnostic Instruments (Sterling Heights, MI, USA). For electron microscopy, samples were fixed and processed according to standard histological techniques, and ultrastructural examination was performed on lead-stained, 95-nm sections at the time of autopsy and repeated during the preparation of this article.
Mutation analysis of the NEB gene (GenBank:NG_009382-1) was performed by dHPLC and sequencing as previously described . The dHPLC analyses were carried out using the automated Transgenomic WAVE Nucleic Acid Fragment Analysis System (Transgenomic, Omaha, NE, USA) with associated Navigator software. Primer data are available upon request (from VLL). All 159 of the 183 nebulin exons that could be analyzed by dHPLC were amplified using 148 primer pairs. PCR reactions were performed in 96-well plates suitable for dHPLC equipment. Each 35-μL reaction mixture contained 60 to 90 ng of genomic DNA (3 μL), 10× PCR buffer supplied with the AmpliTaq Gold PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) containing 15 mmol MgCl2, 5 nmol each of deoxyribonucleotide triphosphate, 20 pmol forward primer, 20 pmol reverse primer and 0.8 U AmpliTaq Gold polymerase enzyme (Applied Biosystems, Carlsbad, CA, USA). Reactions were carried out in a PTC-225 DNA Engine Tetrad Thermocycler (MJ Research, Waltham, MA, USA) starting with denaturation for 10 minutes at 95°C, followed by annealing at 55°C to 60°C depending on the amplicon and extension at 72°C. The lengths of the denaturation, annealing and extension steps varied depending on the amplicon. A final extension was performed at 72°C for 10 minutes. Amplification of the PCR products was confirmed by agarose gel electrophoresis before dHPLC analysis.
Before dHPLC analysis, PCR samples were denatured for 3 minutes at 95°C and then slowly reannealed by lowering the temperature from 95°C to 37°C over a period of 1 hour. Two to five microliters of the PCR amplicon on the 96-well plate were injected into a heated reverse-phase DNASep Column (Transgenomic, Omaha, NE, USA). The column temperature of the dHPLC was set for partially denaturing conditions. The melting profiles of the amplicons were calculated using the Navigator software, but the exact temperature was determined empirically. Conditions used for dHPLC analysis of each amplicon are available on request (from VLL).
Following dHPLC analysis, samples showing abnormal peaks were sequenced. The PCR products were purified using Exonuclease I and shrimp alkaline phosphatase (USB Corp., Cleveland, OH, USA), and the purified products were sequenced using BigDye version 3.1 sequencing chemistry and an ABI 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Sequences were analyzed using Sequencher 4.1 software (Gene Codes Corp, Ann, Arbor, MI, USA).
Western blot analysis
For determination of nebulin content, muscle samples (a biopsy of the quadriceps muscle from patient 16-2 and an autopsy specimen from the abdominal wall of patient 16-4) were first homogenized in buffers containing protease inhibitors (phenylmethylsulfonyl fluoride, 0.5 mmol; leupeptin, 0.04 mmol; E64, 0.01 mmol) to prevent protein degradation during the homogenization process. The homogenized muscle samples were run on 2.6% to 7% SDS-PAGE gels, and transferred onto polyvinylidene fluoride membrane using a semidry transfer unit (Bio-Rad Laboratories, Hercules, CA, USA). Blots were stained with Ponceau S to visualize total transferred protein. The blots were then probed with primary antibodies against nebulin's N terminus (rabbit polyclonal antibody, x35-x36a 1843x, provided by Dr Carol C Gregorio) and its C terminus (rabbit polyclonal antibody 6963, provided by Dr Siegfried Labeit) [21, 32] or against MHC. To control for loading differences, nebulin labeling was normalized to MHC as determined from the Ponceau S-stained membrane. Secondary antibodies conjugated with fluorescent dyes with infrared excitation spectra were used for detection. One-color IR western blots were scanned (Odyssey Infrared Imaging System, LI-COR Biosciences, Lincoln, NE, USA) and the images analyzed with One-D scan EX.
Small strips dissected from muscle biopsies were skinned overnight at about 4°C in relaxing solution (20 mmol N,N-Bis-(2-hydroxyethyl)-2-aminoethane sulfonic acid (BES), 10 mmol ethylene glycol tetraacetic acid (EGTA), 6.56 mmol MgCl2, 5.88 mmol Na+-ATP, 1 mmol dithiothreitol, 46.35 mmol K+-propionate, 15 mmol creatine phosphate, pH 7.0, at 20°C) containing 1% (vol/vol) Triton X-100. Control samples for muscle mechanics studies were isolated from the quadriceps muscles of three living individuals between 30 and 40 years of age, and all results were comparable to previously published results for control and experimental specimens representing a variety of ages, muscle groups and postmortem or postbiopsy intervals [17, 22]. To ensure that measurements were taken from representative and comparable fiber types, fibers and fiber bundles were typed and analysis was restricted to type 2 fibers (the predominant type found in the patient specimens). The skinning procedure renders the membranous structures in the muscle fibers permeable, which enables activation of the myofilaments with exogenous Ca2+. Preparations were washed thoroughly with relaxing solution and stored in 50% glycerol relaxing solution at -20°C for up to approximately 8 weeks. Small muscle bundles (diameter approximately 0.07 mm) were dissected from the skinned strips and mounted between a displacement generator and a force transducer element (AE 801; SensoNor, Horten, Norway) using aluminum T clips. Sarcomere length (SL) was set using a He-Ne laser diffraction system. Mechanical experiments performed on contracting muscle were carried out at a SL of about 2.5 μm for control muscle and at just over slack length for NM muscle, a length selected on the basis of our prior studies. By constructing force-SL relationships, we previously showed that at a SL of 2.5 μm, human muscle fibers from controls produced maximal force, whereas nebulin-deficient muscle fibers from NM patients produced maximal force just over slack length because of their shorter thin filaments . Thus, by performing our mechanical studies on NM muscle set just over slack length, we aimed to minimize force differences due to shorter thin-filament lengths. Fiber width and diameter were measured at three points along the fiber, and the cross-sectional area was determined by assuming an elliptical cross-section. Three different bathing solutions were used during the experimental protocols: a relaxing solution, a preactivating solution with low EGTA concentration and an activating solution. The composition of these solutions was described previously .
Simultaneous force-ATPase measurement
We used the system described by Stienen et al. to measure simultaneous force-ATPase activity . To measure ATPase activity, a nearby UV light was projected through the quartz window of the bath (30 μL volume and temperature controlled at 20°C) and detected at 340 nm. The maximum activation buffer (at -log[Ca2+] (pCa) 4.5) contained 10 mmol phosphoenol pyruvate with 4 mg mL-1 pyruvate kinase (500 U mg-1), 0.24 mg mL-1 lactate dehydrogenase (870 U mg-1) and 20 μmol diadenosine 5'-pentaphosphate. For efficient mixing, the solution in the bath was continuously stirred by means of motor-driven vibration of a membrane positioned at the base of the bath. ATPase activity of the skinned fiber bundles was measured as follows. ATP regeneration from adenosine diphosphate (ADP) was coupled to the breakdown of phosphoenol pyruvate to pyruvate and ATP was catalyzed by pyruvate kinase, which is linked to the synthesis of lactate catalyzed by lactate dehydrogenase. The breakdown of NADH, which is proportional to the amount of ATP consumed, was measured online by UV absorbance at 340 nm. The ratio of light intensity at 340 nm (sensitive to NADH concentration) and the light intensity at 410 nm (reference signal) was obtained by means of an analog divider. After each recording, the UV absorbance signal of NADH was calibrated by multiple rapid injections of 0.25 nmol of ADP (0.025 μL of 10 mmol ADP) into the bathing solution with a stepper motor-controlled injector. The slope of the ATP concentration versus time trace during steady-state tension development of a calcium-induced contraction (Figure 4B) was determined from a linear fit, and the value was divided by the fiber volume (in cubic millimeters) to determine the fiber's ATPase rate. ATPase rates were corrected for the basal ATPase measured in relaxing solution. The ATPase rate was divided by tension (force ÷ cross sectional area (CSA) to determine the tension cost.
To measure ktr, we used the large slack/release approach  to disengage force-generating cross-bridges from the thin filaments, which were isometrically activated. Fast activation of the fiber was achieved by transferring the skinned muscle fibers from the preactivation solution containing a low concentration of EGTA (pCa 9.0) to a pCa 4.5 activating solution. Once the steady state was reached, a slack equivalent to 10% of the muscle length was rapidly induced at one end of the muscle using the motor. This was followed immediately by an unloaded shortening lasting 30 milliseconds. The remaining bound cross-bridges were mechanically detached by rapidly (1 millisecond) restretching the muscle fiber to its original length, after which tension redeveloped. The rate constant of monoexponential k
was determined by fitting the rise in tension to the following equation: F = F ss(1 - e-ktr·t), where F is force at time t and ktr is the rate constant of tension redevelopment.