The histochemical myofibrillar ATPase (mATPase) method is used routinely for identification of equine skeletal muscle fiber types, but important problems have been observed with the subdivision of fast fiber population when using this method. To verify the use of this qualitative method, a number of equine muscle biopsies were analyzed with a combination of histochemical, immunohistochemical, electrophoretic, and morphometric techniques. The influence of training on these interrelations was also evaluated.
Five young (2-3 years old) thoroughbred horses were intensively trained for 8 months on a high-speed treadmill. Biopsies were taken from the gluteus medius muscle at the beginning, after 4 months, and at the end of the training program. Serial sections of the samples were stained by mATPase histochemistry and immunohistochemistry by using a number of monoclonal antibodies specific to selected myosin heavy chain (MyHC) isoforms. The histochemical and immunohistochemical categorization of a large number of fibers (N = 2,078) was compared fiber by fiber. The MyHC content of homogenates of the same biopsies were quantified by densitometry of a sensitive gel electrophoretic technique and compared with histochemical and immunohistochemical fiber types.
A large proportion of fibers examined (approximately 20%) were misclassified by traditional mATPase histochemistry. Many fibers histochemically identified as type IIB displayed both type IIa and type IIb MyHC isoforms, and nearly all type IIAB fibers in mATPase contained only the type IIa MyHC isoform by immunohistochemistry. Correlation analyses suggested a weak relation between the histochemically assessed relative cross-sectional area occupied by the three major fiber types (I, IIA, and IIB) and the electrophoretically assessed MyHC content, whereas a stronger relation was found between immunohistochemically defined fiber types and electrophoretic data. The four fiber type populations delineated according to MyHC content (I, IIA, IIAB, and IIB) had sizes and oxidative capacities significantly different from each other. No adaptation of any parameter measured to training was found. Training had no significant effect on the number of fibers misclassified by mATPase histochemistry.
These data demonstrate a significant limitation in mATPase histochemistry for assessing fibers containing fast MyHC isoforms. The use of monoclonal antibodies against specific MyHC isoforms seems to be a more sensitive and less subjective method.