In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After I4C-labeling of the fiber proteins in v i m by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LCls and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LCls, LC2s and LClf. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained ; this slow myosin had the light chain composition LCls, LC2s and LClf.Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LClf, LC2f and LC3f but were found to have different myosin heavy chains.On the basis of the results presented, we suggest that thc histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable Fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.

To analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.

Using recently developed highly specific antisera to the full range of known adult mammalian skeletal muscle myosins, an immunohistochemical and histochemical examination was made of the middle ear muscle tensor tympani in the dog and cat. Approximately half the fibres were of the IIM type and there was a substantial population of apparently slow-tonic fibres, both these types being rare in mammals. In addition, some type I but no IIA nor IIB fibres were detected. Moreover, as no multiple end-plate innervation, thought to be typical of slow-tonic fibres, could be demonstrated in this muscle by acetylcholinesterase staining or by Ruffini gold impregnation, it is suggested that in tensor tympani the slow-tonic fibres are focally innervated. The very short length of the fibres, only 1-2 mm, is probably sufficient to permit adequate depolarization of a whole fibre by a single centrally situated end-plate. The functional implications of this combination of very rare fibre types in tensor tympani are unclear at present.