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The superfast extraocular myosin (MYH13) is localized to the innervation zone in both the global and orbital layers of rabbit extraocular muscle

Margaret M. Briggs^* and Fred Schachat

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

View larger version (21K): [in a new window]   Fig. 1. Longitudinal variation in myosin heavy chain (MyHC) expression detected by high resolution SDS-PAGE. (A) Superior rectus muscle was cut into six parts as shown. The approximate location of the endplate band region (EPB) is marked. (B) Myofibrils prepared from each region identified in A were analyzed by SDS-PAGE and stained with Coomassie Blue G-250. The bands were identified by comparison with the migration positions in muscles of known myosin composition (Briggs and Schachat, 2000), and as described in Materials and methods. EO myosin is MYH13. (C) Relative amounts of each MyHC were determined by scanning with visible light and densitometry.

View larger version (34K): [in a new window]   Fig. 2. MYH13 mRNA is also localized to the central region of the muscle. Primers specific for MYH13 mRNA were used in semiquantitative RT-PCR of RNA prepared from different sections of the muscle. The resulting 320 bp product is abundant in the central region (a), but greatly diminished in the distal (b) and far distal (c) regions. The muscle used for RNA preparation did not include as much of the proximal region as that used for the protein samples.

View larger version (16K): [in a new window]   Fig. 3. Production of two antibodies specific for MYH13. Comparison of MYH13 with other MyHC sequences identified two highly divergent regions (EO-1, amino acids 1827-1938, and EO-2, amino acids 1210-1382). (A) Antibodies directed against those regions recognized only MYH13 on western blots containing extraocular muscle (EOM) and diaphragm muscle (Dia). (B) The migration positions of the myosin isoforms are shown on a duplicate gel stained for protein with Coomassie Blue G-250.

View larger version (132K): [in a new window]   Fig. 4. Distribution of MYH13 in global and orbital layers of rabbit extraocular muscle (EOM). (A) The specificity of EO-1 was established by staining sections from control muscles that express the other skeletal and cardiac MyHC isoforms as described in text: adductor magnus (Add. Mag.), semitendinosus (ST), Diaphragm and fetal muscle (gest. day 27). (B) In the central region of EOM containing the endplate band, EO-1 stains most fibers in the orbital region near the top of the section, as well as many fibers in the global layer in the lower portion of the section. Scale bar, 200 µm.

View larger version (147K): [in a new window]   Fig. 5. Localization of the endplate band (innervation zone). (A) High levels of MYH13 in this region were detected by staining with EO-1. (B) In a semiserial section the neuromuscular junctions were detected by labeling acetylcholine receptors with fluorescently labeled alpha-bungarotoxin. N, nerve.

View larger version (57K): [in a new window]   Fig. 6. Expression of MYH13 persists in distal portions of the global layer. Sections taken from the endplate and more distal regions were stained with EO-1. (A) A portion of the section shown in Fig. 5, within the endplate band, (B) approximately 3 mm distal, and (C) approximately 6 mm distal to (A), where the muscle is considerably thinner because many fibers do not extend the full length of the muscle. Scale bar, 100 µm. Orbital (upper) and global (lower) layers are marked by arrows.

View larger version (58K): [in a new window]   Fig. 7. Molecular confirmation that MYH13 is present in both orbital and global layers. (A) Schematic illustration of dissection of a 120 µm frozen section into two samples: the inner global layer (Glob.; light gray) and the outer (or intermediate) global layer plus the orbital surface layer (Orb.; dark gray). (B) The myosin isoforms present in EOM and in the two dissected samples (Orb. and Glob.) were analyzed by SDS-PAGE, which showed that MYH13 is present in the global as well as orbital layer.

View larger version (21K): [in a new window]   Fig. 8. SDS-PAGE analysis of single extraocular muscle (EOM) fibers. Single fibers were dissected from the global region in the central region of the muscle. The five representative fibers show that MYH13 (EO) is coexpressed with different levels of IIb and, at lower levels, with IIx and IIa. Sar, sartorius.