NEW METHOD FOR THE ACCURATE CHARACTERIZATION OF SINGLE HUMAN SKELETAL-MUSCLE FIBERS DEMONSTRATES A RELATION BETWEEN MATPASE AND MYHC EXPRESSION IN PURE AND HYBRID FIBER TYPES

In the present study we have developed a method which, by combining hi stochemical, immunohistochemical, electrophoretic and immunoblotting a nalyses on a single fibre, enables a sensitive characterization of hum an skeletal muscle fibres dissected from freeze-dried biopsy samples. For histochemical (and immunohistochemical) analysis fibre fragments ( 500 mu m) of individual fibres were mounted in an embedding medium to allow cryostat sections of normalized thickness to be reproducibly obt ained. The specificity of the myofibrillar Ca2+ ATPase (mATPase) stain ing profiles in gelatin-embedded single fibre sections was tested by i mmunohistochemical reactions with anti-myosin heavy chain (MyHC) monoc lonal antibodies specific to human MyHC I, IIA, IIB and IIA + IIB and by gel electrophoresis. The combined methodologies demonstrated the sp ecificity of the mATPase staining patterns which correlated to the exp ression of distinct MyHC isoforms. In addition the results provide evi dence that many fibres co-expressed different MyHC isoforms in variabl e relative amounts, forming a continuum. Staining intensities for mATP ase, converted into optical density values by image analysis revealed that a relationship between mATPase and MyHC expression holds for hybr id fibres even when displaying one MyHC type with overwhelming dominan ce. The results also revealed that three MyHC isoforms I, IIA and IIB can be co-expressed on a single muscle fibre. In such a case mATPase a lone, with the current protocols, does not allow an accurate character ization of the specific MyHC-based fibre type(s). Although some hybrid fibres may have displayed a non-uniform expression of myosins along t heir lengths, most fibres from the IIA/B group (type) remained very st able with respect to the relative amounts of the MyHCs expressed. Fina lly, a second slow MyHC isoform was recognized on immunoblots of a mix ed muscle sample.