Stapedius muscle fibre composition in the rat

Abstract

The stapedius muscle (SM) is supposed to prevent cochlear damage by noise. Consequently functional demands are the ability of fast contraction with long endurance. This implies the presence of a large fraction of myosin type II fibres with an appreciable oxidative capacity. We determined the myosin composition of SM fibres using consecutive complete SM cross-sections (6 week old rats) which were processed by enzyme histochemistry (EHC) to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) or by immunohistochemistry (IHC) using myosin heavy chain (MyHC) antibodies. Method accuracy was determined in co-processed extensor digitorum longus (EDL). Four hundred SM and 200 EDL fibres were assigned to mATPase type I, IIA, IIB, IIX or ‘miscellaneous’ (‘Misc’) categories. Per mATPase category the fibres were attributed to groups with specific MyHC composition. In the EDL, mATPase type I and IIB fibres expressed only MyHC I and IIB respectively, whereas about 10% of the type IIA and 40% of the type IIX fibres expressed more than one MyHC. Thus IHC detects amounts of myosin isoforms which are not detected by EHC. The mATPase IIX category criterion leaves the possibility that this category contains fibres with myosin type IIA and/or IIB in larger amounts. The criteria of the mATPase categories type I, IIA or IIB preclude assignment to these categories of fibres which also contain other myosin isoforms in larger amounts. Such fibres were classified in one of the mATPase ‘Misc’ categories. Thus in the EDL the capability of the EHC criteria to select ‘pure’ fibres in terms of myosin differs per mATPase category. None of the SM fibres were assigned to the mATPase type I or IIB categories, about 25% to the type IIA, 60% to type IIX and 15% (including most fibres which expressed MyHC I) to a ‘Misc’ category. All SM fibres expressed two or more MyHC isoforms, MyHC IIB occurring in all fibres and substantial amounts of MyHC IIA and/or IIX in most. These findings confirm the hypothesis that such fibres have the capacity to contract fast and have the better fatigue resistance.

Introduction

Two small muscles are present in the middle ear of mammals, the stapedius (SM) and the tensor tympani. Contraction of these muscles will result in stiffening of the middle ear bone chain, hence altering the transmission of sound to the cochlea. These middle ear muscles contract upon acoustic and non-acoustic stimuli such as self-phonation, tactile stimuli, and during arousal (Borg et al., 1984, Djupesland, 1975). There are speculations about their function. A widely accepted theory is that the muscles prevent the auditory receptors in the inner ear from injury by intense noise (Fletcher and Riopelle, 1960, Hilding, 1961, Zakrisson, 1975). Acoustic stimulation above about 80 dB results in middle ear muscle contraction which correlates with the intensity of noise (Teig, 1973, van den Berge et al., 1990). The SM is supposed to be the most relevant in altering the transmission of sound (van den Berge et al., 1990). It should be able to contract fast and to sustain activity for longer periods if damage due to sudden and long-standing high-level noise exposure is to be prevented. Knowledge of the myosin composition of the SM fibres will contribute to the understanding of the functional properties of the SM.

SM fibres were characterised on the basis of myofibrillar ATPase (mATPase) lability after pre-incubation in acid or alkali media and on the basis of activity of enzymes involved in pathways of ATP production. Lyon and Malmgren (1982) reported in the cat 77% fast oxidative glycolytic fibres (supposed to be equivalent to type IIA fibres) and 23% smaller fibres, of which 13% type I-like fibres and 10% type II-like fibres, assumed to be comparable to slow oxidative type I and fast glycolytic type IIB (characterisation according to Peter et al., 1972). Vegetti et al. (1982) found in horse, sheep, cow, pig, dog, cat and rabbit type I and IIA fibres respectively, in ratios from 20/80% up to 50/50%, presumably belonging to slow and fast contracting fatigue-resistant motor units. van den Berge and Wirtz (1989) determined in the rat SM fast (87%) and slow fibres (5%), both showing a moderate to high level of succinic dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (GPox). Fast-twitch white fibres showing a low level of SDH activity like type IIB fibres were not found in the study by van den Berge and Wirtz (1989). Burgener and Mayr (1980) combined electron microscopy and mATPase methods; in the guinea pig two thirds of the fibres resembled myotubes and stained for mATPase-like IIC fibres, which suggests that they are still in a state of development. One third of the fibres appeared to be more mature, they stained like type I fibres.

It is assumed that the SM mainly contains muscle fibres that are fatigue-resistant and reasonably fast; this suggests, on the basis of findings in skeletal (Larsson and Moss, 1993, Rivero et al., 1998) and masticatory muscles (Bredman et al., 1991), that they contain (combinations of) myosin type IIA, IIX and IIB. The use of different histochemical methods, different animal species and partly superseded classification of contractile components impedes insight into the myosin composition (hybridism) of the SM fibres. Moreover, the presence of myosin type IIX fibres could not be found with the methods used thus far.

Recently developed enzyme histochemical (EHC) and immunohistochemical (IHC) methods allow the determination of the myosin composition of SM fibres more accurately. The EHC fibre characterisation was extended with a mATPase double pre-incubation procedure which enables the identification of muscle fibres expressing myosin type IIX (Sant’Ana Pereira et al., 1995a). Moreover, this method is said to detect fibres that contain myosin type IIX in combination with either type IIA or IIB.

EHC fibre characterisation as was done by Schiaffino et al. (1989) and Gorza (1990), supplemented with the EHC procedure to detect type IIX of Sant’Ana Pereira et al. (1995a), combined with IHC with monoclonal antibodies (mAbs) against myosin heavy chain (MyHC) I, IIA, IIX, IIB (Sant’Ana Pereira et al., 1995b, Schiaffino et al., 1989) and cardiac-α (Bredman et al., 1992), has not yet been applied on serial muscle cross-sections, either skeletal or stapedius muscle. Therefore, we investigated the SM of 6 week old rats (which already carries out the adult function) together with a skeletal muscle, the extensor digitorum longus (EDL).