Analysis of myosin heavy chains at the protein level in horse skeletal muscle

Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblot ting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocy tochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique al lowed the quantification of the three MHC isoforms known to be present in d ifferent horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-I IX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and M HC-IIA from the fastest to the slowest migrating band and a quantification by densitometry for each MHC isoform was also possible. The identity of the se three MHCs was confirmed by immunoblots with specific monoclonal antibod ies. Five fibre types were defined immunohistochemically according to their MHC content: I, I + IIA, IIA, the hybrid IIAX and IIX. When quantitative d ata obtained with the four different methodologies were combined and compar ed, they were consistent and, when considered together, showed significant correlation. Nevertheless, the percentage of MHC-IIA histochemically derive d was underestimated, while that of MHC-IIX was overestimated in comparison with the immunocytochemical determination of these MHC isoforms. The perce ntage of MHC-I obtained by ELISA technique was underestimated. In short, th ese integrated methods for the analysis of MHCs at the protein level demons trate that equine skeletal muscle does not express the MHC-IIB, so type II fibres have been misclassified in numerous previous studies based upon the very traditional mATPase histochemistry. They also offer new prospects for muscle fibre typing in equine experimental studies and veterinary medicine.