Enzyme histochemistry of skeletal muscle.

This is the first of three papers on the application of enzyme histochemical techniques to the study of developing and diseased muscle. This paper will deal with developing muscle in various laboratory animals, the second with developing human muscle, and the third with hereditary neurogenic atrophies in infancy and childhood. In 1678 Stefano Lorenzini had already observed that animal muscle could be subdivided into red and white muscle on the basis of differences in colour (Ciaccio, 1898). The extensive studies of Ranvier (1873, 1874, 1880) as well as of other physiologists (Kolliker, 1856; Griutzner, 1884; Knoll, 1891) during the last century helped to establish a firm correlation between the morphology of muscle and its function. It was shown that the red muscle fibres had a slow speed of contraction, but were capable of sustained activity, while the white fibres had a fast speed of contraction and were mainly adapted for short bursts of rapid activity. Following the introduction of a reliable histochemical method for the demonstration of succinate dehydrogenase in tissue sections (Seligman and Rutenburg, 1951), a number of authors demonstrated a variation in the content of this enzyme in the individual muscle fibres in various animals (Padykula, 1952; Wachstein and Meisel, 1955; Bufno and Germino, 1958; Nachmias and Padykula, 1958; Ogata, 1958; George and Scaria, 1958). In general, red muscle contained mainly fibres with a high content of succinate dehydrogenase and white muscle a high proportion of fibres poor in succinate dehydrogenase. In a comparative study of human and various animal muscles (Dubowitz and Pearse, 1 960a, 1960b, 1961), it was observed that in serial section the fibres rich in succinate dehydrogenase also had a high content of other oxidative enzymes such as cytochrome oxidase, nicotinamide-adenine dinucleotide (NADH) diaphorase, and NAD-linked lactate dehydrogenase, but were poor in phosphorylase. In contrast, the fibres with a weak reaction for succinate dehydrogenase also had a low content of the other oxidative enzymes but were rich in phosphorylase. It was suggested that these fibre types be referred to as type I and type II respectively. Type I fibres corresponded to 'red' muscle and probably depended on oxidative metabolism (Krebs cycle) for their energy, whereas the type II fibres, corresponding to 'white' muscle, probably obtained their energy mainly from the anaerobic glycolytic pathway. In most muscles, fibres were also observed with an intermediate activity between the strong and the weak fibres, for a particular enzyme reaction. Pearse (1961) demonstrated a high content of mitochondrial, non-coenzyme linked, oc-glycerophosphate dehydrogenase in type II fibres, and recently Blanchaer, van Wijhe, and Mozersky (1963) and van Wijhe, Blanchaer, and Jacyk (1963) have shown that, with the addition of phenazine metho-sulphate to the histochemical reagents, a high content of NAD-linked oc-glycerophosphate dehydrogenase as well as lactate dehydrogenase is found in type II fibres. These observations provide further evidence that type II fibres selectively utilize the substrates of the glycolytic cycle in their metabolism. Engel (1962) has also observed a high content of myosin adenosine triphosphatase (ATPase) in type II fibres. Attempts have recently been made, on the basis of the relative content of various enzymes, to subdivide further the gastrocnemius and soleus muscles of the rat into three fibre types (Stein and Padykula, 1962) or even eight fibre types (Romanul, 1964). While recognizing that fibres do occur with an intermediate activity for the different enzyme reactions, and that additional permutations are possible with the use of a larger number of enzyme methods, I think that the subdivision into two types, with a limited number of enzyme methods, is useful in the comparative study of normal and diseased human and animal muscle.