Muscle Fibre can co-express different isoforms of Myosin Heavy Chain

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Anthony Sargeant directed this work on skeletal muscle as Head of the Amsterdam research group. The meticulous work was carried out by Jose Sant’Ana Pereira who was one of Tony Sargeant’s PhD students.

The mATPase histochemical profile of rat type IIX fibres: correlation with myosin heavy chain immunolabelling

Jose A A Sant’Ana PereiraArnold de HaanWessels AAnton F MoormanAnthony J Sargeant.

Journal of Histochemistry
Histochem J. 1995 Sep;27(9):715-22
In the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively differentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPase) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characterized on Western blots of muscle homogenates. Enzyme histochemical reactions with the traditional pH buffers were compared to those of the new method and, in conjunction with the immunoreactions, used to confirm the relationship between MyHC expression and the distinct profiles for mATPase. Immunohistochemical reactions demonstrated that the new method only differentiates those fibres expressing myosin heavy chain IIX. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chain IIX in combination with either the IIA or IIB forms. Quantitative histochemistry and immunohistochemistry (by image analysis), used to examine the relationship between staining intensities for mATPase and amounts of myosin heavy chain IIX expression, revealed that the new method discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r2 = 0.93)
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Human Muscle Fibre Types

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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.
Characterization of human skeletal muscle fibres according to the myosin heavy chains they express

Steven EnnionJose A A Sant’ana PereiraAnthony J SargeantArchie YoungGeoffrey Goldspink.

Journal of Muscle Research and Cell Motility
J Muscle Res Cell Motil. 1995 Feb;16(1):35-43
Using a method of single muscle fibre analysis, we investigated the presence of RNA transcripts for various isoforms of the myosin heavy chain (MyoHC) gene in histochemically, immunohistochemically and electrophoretically characterized individual muscle fibres (n = 65) from adult human vastus lateralis muscle. A cDNA clone isolated in this study was shown to contain the 3′ end of a previously uncharacterized human MyoHC gene which is expressed specifically in human fast IIA muscle fibres and we conclude that this clone contains part of the human fast IIA MyoHC gene. In all the fibres histochemically, immunohistochemically and electrophoretically characterized as containing the previously classified IIB MyoHC (n = 23), it was shown that the human equivalent to the rat type IIX MyoHC gene is expressed. This observation was taken to suggest that the previously classified IIB muscles fibres in human muscle express a MyoHC isoform equivalent to the rat IIX, not the IIB, and would therefore be more accurately classified as IIX fibres.

Sustaining Human Muscle Power and Resisting Fatigue

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This review paper is based on an invited key-note lecture given by Professor Anthony J Sargeant to the Polish Physiological Society. As a review it deals with one aspect of the research interests and developments initiated by Professor Sargeant over many years.
Journal of Physiology and Pharmacology 2006 Nov;57 Suppl 10:5-16

During human locomotion the ability to generate and sustain mechanical power output is dependent on the organised variability in contractile and metabolic properties of the muscle fibres that comprise the active muscles. In studies of human exercise we have used a micro-dissection technique to obtain fragments of single muscle fibres from needle biopsies before and after exercise. Each fibre fragment is divided into two parts.

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

How are different human muscle fibre types recruited in exercise?

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This study using a PCr/Cr ratio analysis of single human muscle fibre fragments obtained from needle biopsy during exercise was part of along term interest of Professor Anthony J Sargeant. It is based on techniques developed in his research team over many years.
Journal of Applied Physiology
J Appl Physiol. 2007 Nov;103(5)

In the literature, an inconsistency exists in the submaximal exercise intensity at which type II fibers are activated. In the present study, the recruitment of type I and II fibers was investigated from the very beginning and throughout a 45-min cycle exercise at 75% of the maximal oxygen uptake, which corresponded to 38% of the maximal dynamic muscle force. Biopsies of the vastus lateralis muscle were taken from six subjects at rest and during the exercise, two at each time point.

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.

Professor Anthony J Sargeant reviewed one aspect of his research for The Physiological Society

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An important review of of human muscle power which is an important for the elderly wishing to climb the stairs to bed as it is for the elite athlete or ballet dancer.
Experimental Physiology
Exp Physiol. 2007 Mar;92(2):323-31

Measurements of human power need to be interpreted in relation to the movement frequency, since that will determine the velocity of contraction of the active muscle and hence the power available according to the power-velocity relationship. Techniques are described which enable movement frequency to be kept constant during human exercise under different conditions. Combined with microdissection and analysis of muscle fibre fragments from needle biopsies obtained pre- and postexercise we have been able ‘to take the muscle apart’, having measured the power output, including the effect of fatigue, under conditions of constant movement frequency.

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

Characterization of single human skeletal muscle fibres – Research supervised by Professor Anthony J Sargeant

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Professor Anthony J Sargeant was the Head of Department and supervisor of Jose Sant’ana Pereira (very sadly now deceased) who carried out this meticulous work under his supervision in the early 1990s.
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 samples

http://www.pubfacts.com/author/Anthony+J+Sargeant

Research by Anthony J Sargeant illuminating energetics of human muscle fibre types

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Conceived, initiated, and written by Professor Anthony J Sargeant but using techniques and laboratory work carried out by his post-doctoral student, Jose Sant’ana Pereira, this research contributes to our understanding of the energy turnover in different muscle fibre types and the nature of fatigue in human muscle.
Journal of Physiology
J Physiol. 1996 Oct 15;496 ( Pt 2):583-588

Abstract

1. The relationship between myosin heavy chain (MyHC) isoforms and high energy phosphate content was studied in human muscle fibres at rest and following maximal dynamic exercise lasting 25 s.

2.Single fibre fragments were characterized as type I, type IIA, type IIX or type IIAX. These latter fibres were subdivided into five groups on the basis of the proportion of MyHC IIX isoform present.

3. Resting ATP concentration in type I fibres was 10% lower than in type II fibres (P < 0.05), but no differences were found amongst type IIA, IIX and IIAX fibre groups. Phosphocreatine (PCr) content was lower in type I than in type II fibres (P < 0.01) and, amongst type II fibres, increased progressively with the amounts of MyHC IIX expressed.

4. After 25 s of maximal dynamic exercise ATP concentration was reduced in all fibres. The decrease was approximately 25% in type I fibres and between 47 and 66% in the type II subgroups.

5. Post-exercise PCr content was low in all fibre types. Fibre groups with the lowest post-exercise PCr also had the lowest ATP and the highest inosine monophosphate contents. delta PCr (rest to post-exercise) was smallest in type I fibres and showed a progressive increase in the type II fibre groups as the proportion of the faster IIX myosin heavy chain isoform increased.

http://www.pubfacts.com/author/Anthony+J+Sargeant