Metabolism changes in single human fibres during brief maximal exercise

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 +/- 1 and 18 +/- 4 mmol (kg dry mass)(-1), respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP].     

Skeletal muscle ATP turnover and single fibre ATP and PCr content during intense exercise at different muscle temperatures in humans

The effect of temperature on skeletal muscle ATP turnover, pulmonary oxygen uptake and single fibre ATP and PCr content was studied during intense cycling exercise. Six healthy male subjects performed 6-min intense (Δ50%LT-VO_2peak) cycling, at 60 rpm, under conditions of normal (N) and elevated muscle temperature (ET). Muscle biopsies obtained from the vastus lateralis at rest, 2 and 6 min were analysed for homogenate ATP, PCr, lactate and glycogen, allowing estimation of anaerobic ATP turnover. Freeze-dried single fibres from biopsies were characterised according to their myosin heavy chain composition (type I, IIA or IIAX) and analysed for ATP and PCr content. Pulmonary gas exchange was measured throughout. There was no difference in pulmonary oxygen uptake between the trials. The elevation of muscle temperature resulted in a lower (P < 0.05) PCr content, higher (P < 0.05) lactate content and greater (P < 0.05) anaerobic ATP turnover after 2 min of exercise. There was no effect of temperature on these measures at 6 min. In single fibres it was observed that in ET, there was a lower (P < 0.05) PCr content in type I fibres after 2 min with no differences between conditions after 6 min. The present study demonstrates that elevation of muscle temperature results in a greater anaerobic ATP turnover and type I fibre PCr degradation during the initial 2 min of intense exercise.     

Changes in PCr/Cr ratio in single characterized muscle fibre fragments after only a few maximal voluntary contractions in humans

AIM: This methodological study investigated the number of brief maximal voluntary isometric contractions (MVC) needed to show evidence of fibre activation, as indicated by changes in the phosphocreatine to creatine (PCr/Cr) ratio. METHODS: Subjects performed series of four, seven and/or 10 MVC (1 s on, 1 s off) of the m. quadriceps (60 degrees -flexion angle). Biopsy samples of the m. vastus lateralis were taken at rest and immediately post-exercise. Single muscle fibres were dissected from the freeze-dried samples and classified as types I, IIA or IIAX, using mATPase stainings. Fragments of characterized fibres were analysed for PCr and Cr content. Analyses of variance were performed to investigate changes in PCr/Cr per fibre group over time, followed by Bonferroni post-hoc test (P < 0.01). The fifth percentile of resting values of each fibre group was determined. RESULTS: Mean PCr/Cr ratio after four, seven and 10 MVCs were significantly lower for all fibre groups (P < 0.01). The mean decreases were 44, 64 and 76%, respectively. However, only after seven and 10 contractions PCr/Cr ratios of all, but three type I and two type IIAX fibres, individual fibres were below the fifth percentile. CONCLUSION: In very short duration exercise, involving seven brief maximal voluntary contractions, changes in the PCr/Cr ratio indicated activation of different characterized muscle fibre fragments. The results suggest that this approach may be useful for investigating the pattern of fibre type activation in exercise of very short duration.     

Fibre size and metabolic properties of myosin heavy chain-based fibre types in rat skeletal muscle

Cross-sectional area (CSA), succinate dehydrogenase (SDH), and -glycerophosphate dehydrogenase (GPD) activities were measured in single fibres of adult rat medial gastrocnemius muscle (deep region) using quantitative histochemical procedures. The same fibres were identified in serial sections stained by immunohistochemistry with monoclonal antibodies specific for selected myosin heavy chain (MyHC) isoforms. The values of CSA, SDH and GPD activities formed a continuum, but significant differences in mean values were observed among fibre types of presumed homogeneous MyHC content. Type IIA fibres were the smallest, type IIB fibres were the largest, and type I and IIX fibres were intermediate. Type IIA fibres had the highest SDH activity, followed in rank order by type IIX, type I and type IIB. The average GPD activity was ranked according to fibre type such that IIB>IIX>IIA>I. Hybrid fibres co-expressing two MyHC isoforms generally showed intermediate mean CSA, SDH a nd GPD values lying between their respective pure MyHC fibre types. Across all fibres, there was an inverse relationship between SDH activity and CSA and between GPD and SDH activities, and a positive correlation between GPD and CSA. Moreover, a significant interdependence between CSA, SDH activity, GPD activity and MyHC content existed on a fibre-to-fibre basis, suggesting that the MyHC isoform expressed in a fibre is associated with differences in size, oxidative and glycolytic capabilities of muscle fibres. In fact, most of the fibres could be discriminated into discrete groups with the same MyHC content when their CSA, SDH and GPD values were considered together.     

Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres

The maximum velocity of unloaded shortening (V₀) and the myosin heavy chain (MyHC) and light chain (MyLC) isoform composition were determined in single fibres from soleus and extensor digitorum longus (EDL) muscles of male and female rats 3–6 and 22–24 months old. In the soleus muscle, the β/slow (type I MyHC) isoform predominated in both young and old animals, irrespective of gender. In the EDL, fibres expressing type IIX MyHC or a combination of IIX and IIB (IIXB) MyHC isoforms were predominant in old rats, while type IIB MyHC fibres predominated in young individuals of both genders. The V₀ of soleus fibres expressing the type I MyHC isoform decreased (P < 0.001) by 40% with age in spite of an unchanged MyLC composition. In the EDL, the V₀ of fibres expressing IIX, IIXB and IIB MyHC isoforms did not change with age or differ between males and females. In conclusion, similar age-related changes in V₀ and MyHC composition were observed in single muscle cells from both male and female rats. The present results demonstrate that the relationship between V₀ and MyHC isoform composition at the single fibre level is similar in male and female rats, and that similar qualitative changes take place during ageing in both genders.     

Energy state and myosin heavy chain isoforms in single fibres of normal and transforming rabbit muscles

 Energy-rich phosphates, [ATP]/[ADP_free] ratios, and the myosin heavy chain (MHC) complement were determined in single fibres from normal rabbit muscles, and in fibres isolated from tibialis anterior muscle undergoing fast-to-slow conversion by chronic low-frequency stimulation (CLFS). In normal muscles, energy-rich phosphate contents and [ATP]/[ADP_free] ratios could thus be assigned to different MHC-based fibre types. Phosphocreatine (PCr) contents and [ATP]/[ADP_free] ratios differed markedly between fast- and slow-twitch fibres, as well as within the fast fibre subtypes. Both magnitudes were approximately twofold higher in the fastest (type IIB) fibres as compared to the slowest (type I) fibres. According to PCr contents and [ATP]/[ADP_free] ratios pure and hybrid fibres were aligned in an order similar to that determined by their contractile properties and myofibrillar ATPase activities. CLFS for up to 30 days induced pronounced decreases in PCr and [ATP]/[ADP_free] which attained levels twofold lower than in normal slow-twitch fibres. In both normal and stimulated muscles, PCr and [ATP]/[ADP_free] ratios were correlated, indicating their equilibrium in the different fibre types. The relationship detected between MHC isoform expression and the [ATP]/[ADP_free] ratio suggests that the drastic and persistent depression of the cellular energy state may act as an important signal initiating fast-to-slow transformation processes in muscle fibres. Received: 26 June 1998 / Accepted: 31 July 1998     

Metabolically assessed muscle fibre recruitment in brief isometric contractions at different intensities

This study investigated the recruitment of type I, IIA and IIAX fibres after seven isometric contractions at 40, 70 and 100% maximal voluntary knee extension torque (MVC, 1 s on/1 s off). Biopsies of the vastus lateralis muscle were collected from seven subjects at rest and immediately post-exercise. Fibre fragments were dissected from the freeze-dried samples and characterized as type I, IIA and IIAX using mATPase staining. Phosphocreatine (PCr) and creatine (Cr) content were measured in the remaining part of characterized fibres. A decline in the ratio of PCr to Cr (PCr/Cr) was used as an indication of activation. The mean peak torques were, respectively, 39 (2), 72 (2) and 87 (6)% MVC. Cumulative distributions of type I and IIA fibres were significantly shifted to lower PCr/Cr ratios at all intensities (Kolmogorov-Smirnov test, P<0.05). The cumulative distribution of type IIAX fibres showed a significant leftward shift only at 87% MVC (P<0.05). A hierarchical order of fibre activation with increasing intensity of exercise was found, with some indication of rate coding for type I and IIA fibres. Evidence for activation of type IIAX fibres was only found at 87% MVC.     

Structural and functional determinants of human muscle power

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.     

Formation of inosine monophosphate (IMP) in human skeletal muscle during incremental dynamic exercise

The influence of exercise intensity on the accumulation of inosine monophosphate (IMP) in human skeletal muscle has been investigated. Ten men cycled at workloads corresponding to 40%, 75% and 100% of their maximal oxygen uptake (VO2 max). Muscle IMP was below the detection limit (less than 0.01 mmol kg-1 dry wt) at rest and after exercise at 40% of VO2 max, but increased to 0.26 +/- 0.06 (mean +/- SEM) and 3.50 +/- 0.51 mmol kg-1 dry wt after exercise at 75% and 100% of VO2 max respectively. Accumulation of IMP corresponded to a similar decrease in the total adenine nucleotide content. The muscle content of IMP was positively related to lactate and negatively related to phosphocreatine (PCr). IMP was formed in both fibre types, but the IMP content at fatigue was about twice as high in type II fibres as in type I fibres. It was concluded that the IMP content of human skeletal muscle is very low at rest and after low-intensity exercise, but increases after moderate and high-intensity exercise. In contrast to rat muscle, where deamination of AMP predominantly occurs in the fast-twitch muscle fibres, IMP is formed during exercise in both fibre types in human muscle. Accumulation of IMP appears to reflect an imbalance between the rate of utilization and the rate of regeneration of ATP.     

Increased IMP content in glycogen-depleted muscle fibres during submaximal exercise in man

To study the relationship between glycogen depletion and IMP accumulation in different fibre types, single fibres were dissected from biopsies taken at rest and after one hour of exercise at 70% of maximal oxygen uptake. These fibres were analysed histochemically for glycogen and fibre types and pooled into classes of type I or type II fibres with low, medium or high glycogen content, in a total of six classes. These pools were analysed for ATP, ADP, AMP and IMP contents by high performance liquid chromatography. The contents of ATP, ADP and AMP at rest, and immediately after exercise, were not significantly different between the six fibre classes. The IMP content in glycogen-depleted fibres obtained after exercise was, however, higher than in pools of glycogen-filled fibres obtained both at rest and after exercise. In conclusion, the elevated IMP content in glycogen-depleted but not in glycogen-filled type I and type II muscle fibres during prolonged submaximal exercise indicates a decreased ATP regeneration rate in glycogen-depleted fibres, which may be a factor limiting exercise duration during prolonged submaximal exercise.     

Changes in phosphocreatine concentration of skeletal muscle during high-intensity intermittent exercise in children and adults

Purpose

The aim of the present study was to test the hypotheses that a greater oxidative capacity in children results in a lower phosphocreatine (PCr) depletion, a faster PCr resynthesis and a lower muscle acidification during high-intensity intermittent exercise compared to adults.

Methods

Sixteen children (9.4 ± 0.5 years) and 16 adults (26.1 ± 0.3 years) completed a protocol consisting of a dynamic plantar flexion (10 bouts of 30-s exercise at 25 % of one repetition maximum separated by 20-s recovery), followed by 10 min of passive recovery. Changes of PCr, ATP, inorganic phosphate, and phosphomonoesters were measured by means of ³¹Phosphorous-magnetic resonance spectroscopy during and post-exercise.

Results

Average PCr (percentage of [PCr] at initial rest (%[PCr]_i)) at the end of the exercise (adults 17 ± 12 %[PCr]_i, children 38 ± 17 %[PCr]_i, P < 0.01) and recovery periods (adults 37 ± 14 %[PCr]_i, children 57 ± 17 %[PCr]_i, P < 0.01) was significantly lower in adults compared to children, induced by a stronger PCr decrease during the first exercise interval (adults ?73 ± 10 %[PCr]_i, children ?55 ± 15 %[PCr]_i, P < 0.01). End-exercise pH was significantly higher in children compared to adults (children 6.90 + 0.20, ?0.14; adults 6.67 + 0.23, ?0.15, P < 0.05).

Conclusions

From our results we suggest relatively higher rates of oxidative ATP formation in children’s muscle for covering the ATP demand of high-intensity intermittent exercise compared to adults, enabling children to begin each exercise interval with significantly higher PCr concentrations and leading to an overall lower muscle acidification.     

ATP content in single fibres from human skeletal muscle after electrical stimulation and during recovery

The ATP content was measured in type I and type II fibres from human vastus lateralis muscle at rest, after electrical stimulation and during recovery. At rest the mean values were 25.2 ± 4.02 and 25.9 ± 3.62 mmol kg^-1 dry muscle (mean ± SD) for type I and type II fibres respectively. Normal distribution curves were found for both types I and II fibres. After intermittent electrical stimulation for 83 s (1.6 s Stimulation, 1.6 s pause) with occluded blood flow, the force generation decreased to 22± of the initial value and the muscle tissue showed a mean decrease in ATP to 14.8 and in phosphocreatine to 5.44 mmol kg^-1 dry muscle; lactate increased to 128.9 mmol kg^-1 dry muscle. The ATP content in isolated fibres was equally decreased in both fibre types to 16 mmol kg^-1 dry muscle. In 11± of the fibres the ATP content was lower than 10 mmol kg^-1 dry muscle. After 15 min rest with intact blood circulation ATP was completely resynthesized in type I fibres and to 91± in type II fibres.     

Cardiovascular reflexes during sustained handgrip exercise: role of muscle fibre composition,potassium and lactate

Summary Six healthy men performed sustained static handgrip exercise for 2 min at 40% maximal voluntary contraction followed by a 6-min recovery period. Heart rate (f _c), arterial blood pressures, and forearm blood flow were measured during rest, exercise, and recovery. Potassium ([K⁺]) and lactate concentrations in blood from a deep forearm vein were analysed at rest and during recovery. Mean arterial pressure (MAP) andf _c declined immediately after exercise and had returned to control levels about 2 min into recovery. The time course of the changes in MAP observed during recovery closely paralleled the changes in [K⁺] (r=0.800,P<0.01), whereas the lactate concentration remained elevated throughout the recovery period. The close relationship between MAP and [K⁺] was also confirmed by experiments in which a 3-min arterial occlusion period was applied during recovery to the exercised arm by an upper arm cuff. The arterial occlusion affected MAP whilef _c recovered at almost the same rate as in the control experiment. Muscle biopsies were taken from the brachioradialis muscle and analysed for fibre composition and capillary supply. The MAP at the end of static contraction and the [K⁺] appearing in the effluent blood immediately after contraction were positively correlated to the relative content of fast twitch (% FT) fibres (r=0.886 for MAP vs %FT fibres,P<0.05 andr=0.878 for [K⁺] vs %FT fibres,P<0.05). Capillary to fibre ratio showed an inverse correlation to % FT fibres (r=–0.979,P<0.01). These results indicated that activation of FT rather than slow twitch fibres during static contraction induced a more marked arterial pressure reflex. It was concluded that the arterial pressure reflex would seem to be mediated through stimulation of unmyelinized free nerve endings in the contracted muscle. The [K⁺] would appear to be a more likely candidate than lactate as a mediator for this pressure reflex.     

Bio-energetic changes in human gastrocnemius muscle 1-2 days after strenuous exercise.

[31P]magnetic resonance spectroscopy was used to study the metabolic sequelae of intense muscular activity in gastrocnemius of seven subjects 1-2 days after a 67-mile bicycle ride. The muscle was examined at rest, during a test exercise and during recovery from test exercise. Post-ride and pre-ride results were compared. At rest, the ratio of phosphocreatine to ATP (PCr/ATP) was increased post-ride; during test exercise PCr/(PCr+Pi) was lower post-ride; and the recoveries of PCr, Pi and PCr/(PCr+Pi) after test exercise were delayed, with decreased 'overshoot' of PCr/(PCr+Pi) (which is due to recovery of Pi to below its resting value). Mild mitochondrial damage (perhaps due to exposure to high cytosolic [Pi] during the bicycle ride) may explain some of these results. In contrast to reports of largely eccentric exercise there was no increase in resting Pi/ATP. We have thus demonstrated perturbations of muscle bio-energetics 1-2 days after strenuous exercise, in the absence of convincing enzymological evidence of muscle damage.     

Phosphocreatine recovery overshoot after high intensity exercise in human skeletal muscle is associated with extensive muscle acidification and a significant decrease in phosphorylation potential

The phosphocreatine (PCr) recovery overshoot in skeletal muscle is a transient increase of PCr concentration above the resting level after termination of exercise. In the present study [PCr], [ATP], [P_i] and pH were measured in calf muscle during rest, during plantar flexion exercise until exhaustion and recovery, using the ³¹P NMR spectroscopy. A significantly greater acidification of muscle cells and significantly lower phosphorylation potential (ΔG _ATP) at the end of exercise was encountered in the group of subjects that evidenced the [PCr] overshoot as well as [ADP] and [P_i] undershoots than in the group that did not. We postulate that the role of the PCr overshoot-related transiently elevated [ATP]/[ADP_free] ratio is to activate different processes (including protein synthesis) that participate in repairing numerous damages of the muscle cells caused by intensive exercise-induced stressing factors, such as extensive muscle acidification, a significant decrease in ΔG _ATP, an elevated level of reactive oxygen species or mechanical disturbances.     

Effects of removal of weight-bearing function on contractility and myosin isoform composition in single human skeletal muscle cells

The purpose of this study was to investigate the effects of a 6-week period without weight bearing, achieved by bed rest, on the contractile behaviour, myosin isoform expression and myofibrillar protein content of single human muscle fibres. Percutaneous biopsied specimens of the quadriceps muscle were taken from three healthy male volunteers before and at the end of the experimental period. Maximum force normalised to cross-sectional area (specific tension), maximum velocity of unloaded shortening (V  ₀), and myosin heavy chain (MyHC) and light chain (MyLC) isoform composition were measured in single membrane-permeabilised muscle cells obtained from these specimens. At the end of the experimental period, specific tension was reduced (P < 0.001) by 40% and there was a parallel decline in myofibrillar protein content per muscle cell volume. V  ₀ did not change significantly in response to bed rest when data from all muscle cells were pooled. In two of the subjects, however, V  ₀ decreased (P < 0.01–0.001) in muscle cells expressing the β/slow (type I) MyHC isoform, but there was no change in fibres expressing type IIA or a combination of type IIA and IIB MyHCs. The slowing in type I MyHC fibres was associated with a change in the isoform composition of the regulatory MyLC. Received: 5 October 1995/Received after revision and accepted: 3 January 1996     

Fibre type composition of soleus and extensor digitorum longus muscles in normal female inbred Lewis rats

We have analysed the fibre type composition of soleus and extensor digitorum longus (EDL) muscles of normal female 4-6-month-old inbred Lewis rats. This rat strain is used in our ongoing study of the effects of thyroid hormone on myosin heavy chain (MyHC) isoform expression. On the basis of the mATPase reaction, soleus muscles contained 96.1 +/- 2.9% of type 1 fibres supplemented by 2A fibres. EDL muscles contained type 1 (5.5 +/- 1.0%), type 2A (18.8 +/- 1.7%) and type 2B (75.7 +/- 2.2%) fibres. Immunohistochemical analysis and SDS gel electrophoresis confirmed that most fibres in the soleus muscle expressed the type 1 (slow) MyHC isoform and that only a small proportion of fibres expressed the fast 2a MyHC isoform. Immunohistochemical analysis and SDS gel electrophoresis demonstrated that almost half of the 2B fibres of EDL muscles expressed the 2x/d MyHC isoform. In both muscle types, many fibres expressed more than one MyHC isoform. The content of slow fibres in the soleus muscle of female inbred Lewis rats was slightly higher than that reported for Wistar rats, but was considerably higher than that of Sprague-Dawley rats, whereas substantial differences were not found in the proportion of slow and fast fibre types in EDL muscles in these strains.     

A gated 31P NMR method for the estimation of phosphocreatine recovery time and contractile ATP cost in human muscle

Muscle phosphocreatine (PCr) recovery time constant (an index of muscle aerobic capacity) and contractile ATP cost were estimated from a gated (31)P NMR protocol which does not require intense, repetitive exercise. Subjects performed 2-s duration, maximum voluntary isometric ankle dorsiflexion contractions at 30-s intervals for 8 min (total 15 contractions), while single-shot (31)P spectra (51.7 MHz, TR 3 s) were acquired from the anterior compartment muscle. Spectra from the sixth through 15th contractions were retrospectively sorted, yielding 10 spectra (each 10 averages) gated to times before and after contraction. There was no significant decrease in muscle pH, allowing the calculation of contractile ATP cost directly from the percentage change in PCr during contraction cycles [8.86 +/- 0.82% (SE, n = 11) of PCr at rest], corresponding to an ATP cost of 1.69 +/- 0.16 mM/s (range 0.99-2.49 mM/s), assuming an 8.2 mM ATP concentration. The time constant for PCr recovery (tau 41.8 +/- 4.2 s, range 22.0-60.8 s) was calculated from tau = -Deltat/ln[D/(D + Q)], where Q is the percentage change in PCr due to contraction, D is the additional steady-state percentage drop in PCr from rest and Deltat is the interval between contractions. In the same subjects, the monoexponential PCr recovery time constant after more intense, repetitive isometric ankle dorsiflexion exercise (30 s at 0.5 Hz, 50% duty cycle) was similar to (36.2 +/- 3.5 s, range 16.5-58.8 s) and well correlated with (r = 0.82) the gated result. In contrast to the gated protocol, muscle pH decreased from 7.01 +/- 0.01 to 6.78 +/- 0.04 during recovery after the repetitive protocol. Hence the gated protocol allows the estimation of muscle ATP cost and PCr recovery without intense exercise or muscle acidification.