Measurement of high-energy phosphates in tiny fragments of human muscle fibres

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This research method was  developed in the group headed by Professor Anthony J Sargeant in Amsterdam. Arnold de Haan had developed the basis of the technique in the 1980s as part of his own PhD work. This was subsequently refined to enable very small fragments of human muscle fibre obtained by needle biopsy to be analysed. The present research paper describes that refined techniques and its sensitivity. The work formed part of the PhD thesis the outstanding Greek PhD student, Christina Karatzaferi, who was supervised by Tony Sargeant and Arnold de Haan.

Improved high-performance liquid chromatographic assay for the determination of “high-energy” phosphates in mammalian skeletal muscle. Application to a single-fibre study in man

Christina Karatzaferi, Arnold de Haan, Carla Offringa, Anthony J Sargeant.

Journal of Chromotography

J Chromatogr B Biomed Sci Appl. 1999 Jul 9;730(2):183-91
Abstract
A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze-dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6+/-0.42, 2.11+/-0.06 (n=6) and 10.5+/-0.31 and 1.19+/-0.02 (n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing > or = 1 microg.
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Human Muscle Power

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The ability of generating muscle power power is important whether you are an Olympic athlete, a ballet dancer, or an elderly person wanting to climb the stairs to go to bed. In this comprehensive review of his research Anthony Sargeant points out the importance of different types of muscle fibres that make up the human skeletal muscles that produce power in legs and arms. Tony also points out that in research seeking to measure human muscle power it is essential to measure or control the speed at which the power is generated (this is because power is the product of work and velocity).

Structural and functional determinants of human muscle power
by Anthony J Sargeant

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.

Anthony J Sargeant

Method for measuring high energy phosphates in fragments of human muscle fibres

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This research publication describes a method developed in the Amsterdam Department of Anthony Sargeant. The data was collected by an outstanding Greek PhD student, Christina Karatzaferi, directed by Tony and his colleague Arnold de Haan.
Journal of Chromotography
J Chromatogr B Biomed Sci Appl. 1999 Jul 9;730(2):183-91

Abstract A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze-dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6+/-0.42, 2.11+/-0.06 (n=6) and 10.5+/-0.31 and 1.19+/-0.02 (n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing > or = 1 microg.

Muscle is less strong and less efficient in young when compared with older animals

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This research carried out in the Department of Anthony Sargeant in Amsterdam demonstrated how during rapid growth the muscle of young animals is weaker and less efficient compared with older animals.
Acta Physiologica Scandanavica
Acta Physiol Scand. 1993 Apr;147(4):347-55

We investigated the effect of age on (the reduction of) work output, efficiency and muscle fibre type composition. Rat medial gastrocnemius muscles of three age-groups performed a series of 15 repeated contractions within 6 s (blood flow was arrested). Stimulation and shortening velocities were chosen as optimal for each group, while all muscles shortened over the same relative fibre lengths.

The fibre type composition showed a higher proportion of the oxidative type IIBd fibres in the middle-aged group [5 months old; 39.8 +/- 6.8 vs. 23.6 +/- 4.2% of the fibre area in the young rats (1.3 months old)] in contrast to the type IIBm fibres (52.9 vs. 67.9%, respectively), while the old group (22 months old) was not different from the middle-aged group. Work output in the last contraction (relative to the first contraction) was not different between the age-groups (53.1 +/- 18.1; 48.0 +/- 6.5 and 61.1 +/- 6.2%, respectively). High-energy phosphate utilization was not different between the groups (150.6 +/- 11.2; 154.6 +/- 15.6 and 157.2 +/- 7.0 mumol g-1 dry wt, respectively). However, the efficiency was approximately 30% lower in the muscles of the youngest group, which corresponds with a lower specific power and specific tension. Since the change in fibre type composition is unlikely to be the cause of the low efficiency in the young animals, the causes remain unclear, but may be related to the rapid growth of the young rats in our study.

Age effects on muscle fatigue and recovery

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Research into muscle fatigue in older compared to young rats. There seemed to be differences which may be related to the population of faster muscle fibres which seemed to take longer to recover from fatigue in the older rat muscles. The data for this publication was collected by Arnold de Haan who had been PhD student of Professor Anthony Sargeant.
Quarterley Journal of Experimental Physiology
Q J Exp Physiol. 1989 Sep;74(5):715-26

Force-velocity, power-velocity and unloaded shortening data were obtained from in situ medial gastrocnemius muscle-tendon complexes (stimulated at 60 Hz) with intact circulation of mature male rats (approximately 125 days old). Measurements were carried out at the end of a long (15 s) contraction (fatigued muscles) or with a short (1 s) contraction either in the fresh state (fresh muscles) or in muscles which had recovered for 15 min after a long contraction. Compared to the fresh state fatigue reduced isometric force by 57%, maximal shortening velocity by approximately 40% and maximal power output by 81%.

These reductions were similar to data previously obtained with younger rats (40 days old). However, the velocity data of the muscles which had recovered for 15 min after a long contraction showed a greater reduction in the mature rats. This difference between the two age groups together with a difference in the changes in the initial parts of the isometric force time curves suggest an age-dependent response of the fast-fatigable fibre population of these mixed muscles. In a separate series of experiments the underlying mechanism of the recovery from fatigue was studied in a group of young rats. Fatigue was induced with five long (15 s) contractions (each at 5 min intervals). The recovery of isometric force and power output was monitored with short contractions which indicated a plateau of recovery but the absolute values were still reduced after 60 min (85 and 71% of prefatigue values, respectively). Phosphocreatine concentration recovered rapidly, whereas the ATP concentration was still markedly reduced after 1 h of recovery. The time courses of recovery of inosine-5′-monophosphate (IMP) and lactate concentrations resembled those of force and power output. Thus it is possible that age-dependent differences in IMP and/or lactate production may play a role in fatigue and recovery from fatigue.

The effect of age on muscle performance

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Research into the effects of ageing on muscle function. This work was carried out by Arnold de Haan (later Professor) as part of his PhD supervised by Professor Anthony Sargeant.

Changes in isometric force, power output and relaxation rate have been measured during repetitive tetanic contractions in 2 groups of rats of different ages. During the first 5 contractions there were no differences between a young and mature group. In contrast to isometric force production, which decreased about 3% per contraction, power output initially increased to 108% of the power output in the first contraction.

A greater reduction in power output and relaxation rate after the 5th contraction indicated a greater reduction of the cross-bridge cycling rate in the younger rats. ATP, phosphocreatine and lactate concentrations after the last contraction were not different between the age-groups. In contrast IMP production, which has been suggested may play a regulatory role during fatigue was twice as high in the young rats. Judged by isometric force production there is no age-related difference in fatiguability. However, profound differences were observed in power output, which indicates that quantification of fatigue as a loss of isometric force may be seriously misleading when considering the functional status of the muscle for normal dynamic contractions.

Fundamental Research into Muscle – important insights for understanding muscle function in health and disease

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Meticulous research carried out by Arnold de Haan (later Professor) as part of his PhD completed under the supervision of Professor Anthony Sargeant. It might be noticed that although the experimental work in the laboratory was conducted entirely by Arnold de Haan there are eight authors on this paper. Research by committee is not always productive but it was very much the tradition in the Faculty which Tony joined in 1985. After a few years he managed to achieve a more realistic approach with only those who made a ‘significant’ contribution being listed as authors.
European Journal of applied Physiology
Eur J Appl Physiol Occup Physiol. 1988;57(1):64-9

The effect of muscle dimensions on economy (force-time integral divided by the amount of energy utilized) was investigated in male rats (body mass range 95-490 g), anaesthetized with pentobarbital. The medial gastrocnemius muscle in situ performed 6 maximal isometric contractions of 350 ms duration (1.s-1) at twitch optimum length at 35 degrees C.

The areas under the 6 time-force curves were added to obtain force-time integral of the experiment. Differences of concentrations of ATP, phosphocreatine and lactate between experimental and contralateral (resting) muscles were used to calculate high-energy phosphate consumption due to stimulation. Muscle mass and cross-sectional area increased (approximately +400% and +300%, respectively) over the rat body mass range studied. Muscle length and length of the most distal fibre bundle increased by approximately 17 mm and 4 mm, respectively. Force-time integral (N.s) increased proportional to cross-sectional area whereas high-energy phosphate consumption (mumoles) increased proportional to muscle mass. The relative fraction of the total energy consumption utilized for force-independent processes was independent of rat body mass. The economy of the actomyosin system was unaffected during growth, whereas economy of the whole muscle decreased during growth by approximately 30% (p less than 0.001). The effect of muscle dimensions on economy is discussed with respect to human endurance capacity measured by voluntary isometric contractions