Unique expression of cytoskeletal proteins in human soft palate muscles

Abstract

The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for β-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, β-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles.

Keywords: cytoskeleton; desmin; dystrophin; muscle fiber; palatopharyngeus; sleep apnea; soft palate; uvula.

Figure 1

Des‐Neg and DysC‐Neg fibers in UV. Muscle sections from an adult (A–D, M–O) and infant (E–H) UV muscle and a limb muscle, musculus vastus lateralis (I–L). The sections are immunostained for desmin (Des, green color; A,E,I), the C‐terminal of dystrophin (DysC, red color; B,F,J) and laminin (Lam, white color; C,G,K). (D,H,L) and (M–O) show merged stainings. Sections (M) and (N) (Des, green color; Lam, red color) are also stained for DAPI (nucleus stained blue). (M) shows longitudinal sectioned muscle fibers double‐stained for desmin (green color) and laminin (red color). Des‐Neg/DysC‐Neg fibers are marked (*) and Des‐Pos/DysC‐Pos are marked (+). Insets from (E–H) highlight magnified Des‐Neg/DysC‐Neg fibers in UV from an infant (4 months old). Note the low or absent staining for desmin and dystrophin C‐terminal (_*), but distinct staining for laminin in the sections from both the adult and infant UV muscle, while in the limb muscle all sections are positively stained. The staining pattern for contractile slow MyHCI (I, green color) and fast MyHCII (II, red color) proteins is shown in (O), and the relation to Des‐Neg/DysC‐Neg fibers is shown in (N). Notice the typical expression of slow MyHCI in Des‐Neg/DysC‐Neg fibers (stars). Scale bar: 50 μm.

Figure 2

Des‐Neg and DysC‐Neg fibers in PP. Muscle cross‐sections from an adult (A,B) and infant (C,D) PP muscle multi‐stained for desmin (Des, green color), dystrophin C‐terminal (DysC, red color), laminin (Lam, white color) and DAPI (nuclei, blue color) (A,C) and just for dystrophin C‐terminal (DysC, red color; B,D). Des‐Neg/DysC‐Neg fibers are marked (*) and Des‐Pos/DysC‐Pos are marked (+). Note the high frequency of fibers unstained or weakly stained for both the dystrophin C‐terminal and desmin in adult and infant muscles. Scale bar: 50 μm.

Figure 3

Immunoreaction of antibodies against different domains of the dystrophin molecule in palate and limb muscles. Serial cross‐sections from palatopharyngeus (A–D) and biceps brachii (E–H) stained with antibodies directed against the N‐terminal (NCL‐DYS3, red color; A,E), the rod (NCL‐DYS1, green color; B,F) and the C‐terminal (GTX15227, red color; C,G and NCL‐DYS2, green color; D,H) domains of the dystrophin molecule. The schematic sketch shows domains of the dystrophin molecule corresponding to antibody affinity. Note that while all domain‐specific antibodies showed immunoreaction in the limb muscle, a subgroup of fibers was unstained or weakly stained for the two antibodies directed against the C‐terminus in the UV (asterisks). Muscle fibers stained for all four antibodies are marked (+). Scale bar: 50 μm.