Cytoskeletal Proteins in Myotendinous Junctions of Human Extraocular Muscles

Abstract

Purpose: The purpose of this study was to investigate the cytoskeletal composition of myotendinous junctions (MTJs) in the human extraocular muscles (EOMs). Desmin and other major cytoskeletal proteins are enriched at the MTJs of ordinary myofibers, where they are proposed to be of particular importance for force transmission and required to maintain myofiber integrity.

Methods: EOM and limb muscle samples were analyzed with immunohistochemistry using antibodies against the intermediate filament proteins desmin, nestin, keratin 19, vimentin, and different myosin heavy chain (MyHC) isoforms. MTJs were identified by labeling with antibodies against laminin or tenascin.

Results: In contrast to MTJs in lumbrical muscle where desmin, nestin, and keratin 19 were always present, approximately one-third of the MTJs in the EOMs lacked either desmin and/or nestin, and all MTJs lacked keratin 19. Approximately 6% of the MTJs in the EOMs lacked all of these key cytoskeletal proteins.

Conclusions: The cytoskeletal protein composition of MTJs in human EOMs differed significantly from that of MTJs in limb muscles. These differences in cytoskeletal protein composition may indicate particular adaptation to meet the functional requirements of the EOMs.

Figure 1.

MTJs of lumbrical muscle doubled-labeled with antibodies (green) against desmin (A), nestin (D), keratin 19 (G), vimentin (J), and antibody against laminin (red; B, E, H, and K). The right column shows the merged images for each intermediate filament protein and laminin. Note the strong labeling at MTJs with the antibody against desmin (thick arrows in A and C), nestin (thick arrows in D and F), and keratin 19 (thick arrows in G and I). Notice the weak labeling with nestin at MTJs (thin arrows in D and F). Arrowheads (J–L) denote unlabeled MTJs with the antibody against vimentin on the muscle side of the MTJs. Bars = 50 µm.

Figure 2.

MTJs in the human EOMs labeled with the antibody against laminin. The arrowheads (A–C) denote MTJs facing the tendon. Arrow (C) denotes MTJ facing away from the tendon. Bars = 20 µm.

Figure 3.

MTJs in EOMs double-immunolabeled with antibodies against desmin (green in A, D, and G) and laminin (red in B, E, and H). Merged images for desmin and laminin are shown in C, F, and I. Immunolabeling with desmin is increased at MTJs in myofibers containing desmin (thick arrows in A–C) or present in the proximity of the MTJs in myofibers lacking desmin in the remaining of their length (thin arrows in D–F). Note the absence of desmin at MTJs in myofibers lacking desmin in the remaining of their length (arrowheads in G–I). Bars = 20 µm.

Figure 4.

Immunoreactivity with the antibody against nestin (green in A and D; red in G) at MTJs labeled with antibodies against laminin (red in B and E) or tenascin (green in H) in EOMs. Merged images for desmin and either laminin or tenascin are shown in C, F, and I. Strong immunolabeling with the antibody against nestin was present at MTJs in myofibers containing nestin (thick arrows in A–C) and in myofibers lacking nestin (thin arrows in D–F) in the remaining of their length. Note the absence of nestin in MTJs of myofibers completely lacking nestin in the remaining of their length (arrowheads in G–I). Bars = 20 µm.

Figure 5.

Five (I–V) different patterns of immunostaining with antibodies against desmin (green in A, K, O, and S; and gray in F) or nestin (gray in B, L, P, and T; and green in G) at MTJs in myofibers containing MyHCIIa (A–R) and MyHCI (S–V) in the EOMs. Merged images of desmin and laminin are shown in C, M, Q, and U. Merged image of nestin and laminin is shown in H. Bars = 20 µm.

Figure 6.

Schematic illustration of the major patterns of immunoreactivity regarding desmin and nestin at MTJ and in the remaining portion of the myofiber present in the muscle sections of the human EOMs. I: Desmin and nestin were present in both MTJs and the remaining portion of the myofibers. II: Desmin and nestin were present at MTJs but nestin was lacking in the remaining portion of the myofibers. This pattern is similar to that of myofibers and their MTJs in skeletal muscle in general. III: Desmin was present in both the MTJs and along the myofibers whereas nestin was totally absent. IV: Desmin and nestin were absent at MTJs and along the remaining of the myofibers. V: Only nestin was present at the MTJs whereas neither desmin nor nestin were present in the remaining portion of the myofiber present in the muscle section.

Figure 7.

Immunolabeling with antibodies against desmin (green in A, E, and I), laminin (red in B, F, and J) and keratin 19 (gray in D, H, and L) in EOMs. Merged images of desmin and laminin are shown in C, G, and K. Keratin 19 was absent from MTJs and the remaining parts of the myofibers containing desmin along their length (thick arrows in A–D) or only in the vicinity of the MTJs (thin arrows in E–H), or in myofibers completely lacking desmin (arrowheads in I–L). Bars = 20 µm.

Figure 8.

Immunolabeling with antibodies against vimentin (green in A, B, D, and E) and desmin (gray in C and F) at MTJs identified with the antibody against laminin (red in B, C, E, and F). Note that vimentin was absent from the MTJs and along the myofibers either containing (C) or lacking (F) desmin. Bar = 20 µm.