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PURPOSE: To investigate the myosin heavy chain (MyHC) composition of human extraocular (EOM) and levator palpebrae (LP) muscle fibers. METHODS: Adult human EOMs and LP were studied with SDS-PAGE, immunoblots, and immunocytochemistry, with antibodies against six MyHC isoforms. Myofibrillar adenosine triphosphatase (mATPase) and reduced nicotinamide adenine dinucleotide (NADH)-TR activity and fiber area were also determined. RESULTS: Most of the fibers in both layers stained strongly with anti-MyHCIIa. Approximately 14% of the fibers in the global layer and 16% in the orbital layer were labeled with anti-MyHCI. The remaining 24% of the fibers in the global layer and 3% in the orbital layer were not stained with either of these two antibodies, but were reactive to anti-MyHCeom (MyHCeom(pos)/MyHCIIa(neg) fibers). The fibers stained with anti-MyHCI had acid-stable mATPase activity, and the remainder of the fibers had alkaline-stable mATPase activity. Almost all the slow fibers stained with both anti-MyHCI and anti-MyHCslow tonic in both layers. Anti-MyHCalpha-cardiac stained approximately 26% of these slow fibers in the orbital layer and 7% in the global layer. Some slow fibers in both layers lacked staining with anti-MyHCslow tonic or with anti-MyHCalpha-cardiac. MyHCemb and/or MyHCeom were also present in some of the fibers of all the groups. The LP did not stain with anti-MyHCslow tonic. CONCLUSIONS: The present study revealed that the human EOMs have a very complex fiber type and MyHC composition and differ significantly from the EOMs of other species. The features of the LP were distinct from those of the four recti, the obliquus superior, and the limb muscles.

PURPOSE: To investigate the composition of the fibers in human extraocular muscles (EOMs) with respect to the sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA)-1 and -2 and to investigate possible correlations between SERCA and myosin heavy chain (MyHC) composition. METHODS: EOM samples were processed for immunocytochemistry with monoclonal antibodies specific against SERCA1 (fast isoform), SERCA2 (slow isoform), or different MyHCs. A total of 1571 fibers were analyzed. Microsomal EOM fractions were analyzed with SDS-PAGE and immunoblots. RESULTS: The fast fibers, containing MyHCIIa, accounted for 79% of the fibers in the orbital layer (OL) and 74% in the global layer (GL). More than 99% of these fibers contained SERCA1, and 86% of them coexpressed SERCA1 and -2. Almost all slow fibers stained with SERCA2; 54% of those in the GL and all in the OL coexpressed SERCA1 and -2. Fifteen percent of the fibers in the GL and less than 1% in the OL were MyHCeom(pos)/MyHCIIa(neg) fibers. All these contained SERCA1 and in the OL also stained strongly with anti-SERCA2. Biochemically SERCA2 was more abundant than SERCA1. CONCLUSIONS: The human EOMs had a very complex pattern of expression of the major protein regulating fiber relaxation rate. The coexistence of SERCA1 and -2, together with complex mixtures of MyHCs in most of the fibers provide the human EOMs with a unique molecular portfolio that allows a highly specific fine-tuning regimen of contraction and relaxation.

PURPOSE: To examine the distribution of myosin-binding protein C (MyBP-C) in human extraocular muscles (EOMs) and to correlate the myosin heavy chain (MyHC) and the MyBP-C composition of the fibers. METHODS: Samples from 17 EOMs, 3 levator palpebrae (LP), and 6 limb muscles were analyzed with SDS-PAGE and immunoblot or processed for immunocytochemistry with monoclonal antibodies (mAbs) against MyBP-C-fast, MyBP-C-slow, MyHCIIa, MyHCI, MyHCsto, MyHCalpha-cardiac, and MyHCemb. RESULTS: In the limb muscle samples, fast fibers were labeled with anti-MyBP-C-fast and anti-MyBP-C-slow, whereas the slow fibers were immunostained with anti-MyBP-C-slow only, in accordance with previous studies. In 11 EOM samples MyBP-C-fast was not detected, and weak staining with anti-MyBP-C-fast was seen only in a few fibers in the proximal part of 2 muscles. The mAb against MyBP-C-slow labeled all fibers, but fibers containing MyHCI were generally more strongly stained. In the levator palpebrae, immunostaining with anti-MyBP-C-fast was present in some fibers labeled with anti-MyHCIIa and/or anti-MyHCeom. MyBP-C-fast and -intermediate were not detected biochemically in the EOMs. CONCLUSIONS: The lack of MyBP-C-fast and intermediate is an additional feature of the human EOM allotype. The true EOMs have a unique myofibrillar protein isoform composition reflecting their special structural and functional properties. The levator palpebrae muscle phenotype is intermediate between that of the EOMs and the limb muscles.

PURPOSE: To determine the laminin isoform composition of the basement membranes (BMs) in the human extraocular muscles (EOMs) and relate it to the fact that EOMs are spared in laminin alpha2-chain-deficient congenital muscular dystrophy. METHODS: Samples from adult human EOMs and limb muscle were processed for immunocytochemistry, with monoclonal antibodies against laminin chains (Ln) alpha1 to -5, beta1 and -2, and gamma1. Neuromuscular junctions (NMJs) were identified with acetylcholinesterase reaction. The capillary density was measured in sections stained with anti-Lnalpha5. RESULTS: The extrasynaptic BM of the EOM muscle fibers contained Lnalpha2, -beta1, -beta2, and -gamma1, and, in contrast to limb muscle, it also contained Lnalpha4 and -alpha5, to some extent. The distinct laminin composition of the EOMs was confirmed by the presence of Lutheran protein, an alpha5-chain-specific receptor not found in limb muscle. At the NMJs, there was increased expression of Lnalpha4 and expression of Lnalpha2, -alpha5, -beta1, -beta2, and -gamma1 was also maintained. The capillary density was very high (1050 +/- 190 capillaries/mm(2)) in the EOMs and significantly (P < 0.05) higher in the orbital (1170 +/- 180 capillaries/mm(2)) than in the global (930 +/- 110 capillaries/mm(2)) layer. CONCLUSIONS: The human EOMs showed important differences in laminin isoform composition and capillary density when compared with human limb muscle and muscles of other species. The presence of additional laminin isoforms other than laminin-2 in the BM of the extrasynaptic sarcolemma could partly explain the sparing of the EOMs in Lnalpha2-deficient congenital muscular dystrophy.