This paper describes the combination of a number of complementary techniques applied to the characterisation of human muscle fibre types using microdissection of fragments of fibres obtained by needle biopsy. The work was a collaboration between the Amsterdam Research Groups directed by Professors Anthony Sargeant and Anton Moorman and that of Professor Geoffrey Goldspink in London. The work was submitted by Jose Sant’ana Pereira as part of his PhD completed under the direction of Anthony Sargeant in the Vrije University of Amsterdam. Sadly Jose died some years later while working in Madison, Wisconsin.
New method for the accurate characterization of single human skeletal muscle fibres demonstrates a relation between mATPase and MyHC expression in pure and hybrid fibre type
Journal of Muscle Research and Cell Motility
J Muscle Res Cell Motil. 1995 Feb;16(1):21-34
In the present study we have developed a method which, by combining histochemical, immunohistochemical, electrophoretic and immunoblotting analyses on a single fibre, enables a sensitive characterization of human skeletal muscle fibres dissected from freeze-dried biopsy samples.
For histochemical (and immunohistochemical) analysis fibre fragments (500 microns) of individual fibres were mounted in an embedding medium to allow cryostat sections of normalized thickness to be reproducibly obtained. The specificity of the myofibrillar Ca2+ ATPase (mATPase) staining profiles in gelatin-embedded single fibre sections was tested by immunohistochemical reactions with anti-myosin heavy chain (MyHC) monoclonal antibodies specific to human MyHC I, IIA, IIB and IIA + IIB and by gel electrophoresis. The combined methodologies demonstrated the specificity of the mATPase staining patterns which correlated to the expression of distinct MyHC isoforms. In addition the results provide evidence that many fibres co-expressed different MyHC isoforms in variable relative amounts, forming a continuum. Staining intensities for mATPase, converted into optical density values by image analysis revealed that a relationship between mATPase and MyHC expression holds for hybrid fibres even when displaying one MyHC type with overwhelming dominance. 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 alone, with the current protocols, does not allow an accurate characterization of the specific MyHC-based fibre type(s). Although some hybrid fibres may have displayed a non-uniform expression of myosins along their lengths, most fibres from the IIA/B group (type) remained very stable with respect to the relative amounts of the MyHCs expressed. Finally, a second slow MyHC isoform was recognized on immunoblots of a mixed muscle sample.