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
Jaas. Pereira et al., 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, Journal of muscle research and cell motility, 16(1), 1995, pp. 21-34
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.