Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus later alis muscle biopsies of 15 young men (with an average age of 22 y) by corre lating immunohistochemistry with specific anti-MHC monoclonal antibodies, m yofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC beta -slow, MHC-IIA and MHC-IIX. The characterisati on of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories ac cording to the MHC isoform. they express at the protein level, types I, I+I IA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were cle arly underestimated by mATPase histochemistry. For a very high percentage o f fibres there was a precise correspondence between the MHC protein isoform s and mRNA transcripts. The integrated methods used demonstrate a high degr ee of precision of the immunohistochemical procedure used for the identific ation and quantification of human skeletal muscle fibre types. The monoclon al antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibre s. This protocol offers new prospects for muscle fibre classification in hu man experimental studies.