In this important series of studies a collaboration between the research group in Amsterdam led by Anthony Sargeant and that in London under the direction of Professor Geoffrey Goldspink used new techniques based on microdissection of fragments of human muscle fibre obtained by needle biopsy.
One part is used to characterize the fibre type in respect of the heavy chain myosin isoform expressed. The other part of the fragment is analysed for high energy phosphate concentrations. Fibres are classified on the basis of expressing either type I, type IIA, or type IIX myosin heavy chain isoforms. It should be noted however that in the type II population many fibres co-express both IIA and the IIX isoforms and we therefore characterize these fibres on the basis of the degree of co-expression. We have used this technique to examine the time course of high energy phosphate concentration and fatigue in different fibre populations during exercise. The progressive reduction of power during maximal sprint efforts may be interpreted as the cumulative effect of metabolic depletion in successive fibre type populations from IIX to IIXa to IIAx to IIA to I. One important application of the micro-dissection technique is that PCr content may also be used as a very sensitive metabolic marker for fibre type recruitment during very short duration concentric, isometric and eccentric exercise
From the first biopsy single fibers were isolated and characterized as type I and II, and phosphocreatine-to-creatine (PCr/Cr) ratios and periodic acid-Schiff (PAS) stain intensities were measured. Cross sections were cut from the second biopsy, individual fibers were characterized as type I and II, and PAS stain intensities were measured. A decline in PCr/Cr ratio and in PAS stain intensity was used as indication of fiber recruitment. Within 1 min of exercise both type I and, although to a lesser extent, type II fibers were recruited. Furthermore, the PCr/Cr ratio revealed that the same proportion of fibers was recruited during the whole 45 min of exercise, indicating a rather constant recruitment. The PAS staining, however, proved inadequate to fully demonstrate fiber recruitment even after 45 min of exercise. We conclude that during cycling exercise a greater proportion of type II fibers is recruited than previously reported for isometric contractions, probably because of the dynamic character of the exercise. Furthermore, the PCr/Cr ratio method is more sensitive in determining fiber activation than the PAS stain intensity method.
We have shown that fatigue may be the consequence of a metabolic challenge to a relatively small population of fast fatigue-sensitive fibres, as indicated by [ATP] depletion to approximately 30% of resting values in those fibres expressing myosin heavy chain isoform IIX after just 10 s of maximal dynamic exercise. Since these same fibres will have a high maximal velocity of contraction, they also make a disproportionate contribution to power output in relation to their number, especially at faster movement rates. The microdissection technique can also be used to measure phosphocreatine concentration ([PCr]), which is an exquisitely sensitive indicator of muscle fibre activity; thus, in just seven brief maximal contractions [PCr] is depleted to levels < 50% of rest in all muscle fibre types. The technique has been applied to study exercise at different intensities, and to compare recruitment in lengthening, shortening and isometric contractions, thus yielding new information on patterns of recruitment, energy turnover and efficiency
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 samples