| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0623, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 1 April 2004;
accepted after revision 2 July 2004; first published online 15 July 2004)
Corresponding author M. C. Hogan: Department of Medicine, 0623-A, University of California, San Diego, La Jolla, CA 92093-0623, USA. Email: mchogan{at}ucsd.edu
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Unlike whole-animal and whole-muscle models, in the isolated, intact, single skeletal muscle fibre preparation, problems associated with fibre type recruitment heterogeneity are eliminated. Furthermore, the extracellular environment is homogeneous and can be easily adjusted and determined, therefore eliminating complications associated with substrate availability, extracellular pH and/or metabolic waste product removal. van der Laarse et al. (1991) have previously demonstrated in these single fibres, using densitometric analyses, that succinate dehydrogenase activity correlates with resistance to fatigue (r= 0.83), suggesting that MVD may be a critical factor in maintaining the rephosphorylation of depleted high-energy phosphates and therefore the development of force (van der Laarse et al. 1989b). However, direct measurement of MVD has not been performed in these single fibres. In the present study, in order to test directly the hypothesis that MVD is associated with resistance to fatigue of skeletal muscle, we quantified MVD using electron microscopy after determining the rate of fatigue development of individual, isolated skeletal muscle fibres during conditions where O2 and substrate availability were nonlimiting.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Adult female Xenopus laevis were doubly pithed and decapitated. Lumbrical muscles II–IV were removed, and single living muscle fibres (n= 14) of varying fatigability were microdissected from the muscle according to appearance under dark-field illumination, which has previously been demonstrated to estimate fatigability (Lannergren & Smith, 1966; Westerblad & Allen, 2002). Dissections and experiments were performed in Ringer solution (mM: 112 NaCl, 1.87 KCl, 0.82 CaCl2, 2.38 NaHCO3, 0.07 NaH2PO4 and 0.1 EGTA) at 20°C and pH 7.0. Following dissection, platinum clips were attached to the tendons, and the fibres were mounted in a glass chamber perfused with Ringer solution of ambient O2 partial pressure (159 mmHg). Tetanic contractions were induced by direct stimulation (70 impulses s–1 of 1 ms duration at 9 V, with stimulus train duration of 200 ms) with platinum conducting electrodes on either side of the fibre, using a Grass S48 stimulator (Quincy, MA, USA). Force development was measured with a force transducer system (Aurora Scientific, Model 400 A, Aurora, Ontario, Canada). A Biopac Systems MP100WSW (Santa Barbara, CA, USA) A–D converter was used to transform the analogue force signal, and the digital data were collected and analysed with AcqKnowledgeIII v3.5 software (Biopac Systems). Fibres were stimulated at increasing frequencies (0.25, 0.33, 0.5 and 1 contractions s–1) in a sequential manner with each stimulation frequency lasting 2 min. For each fibre, force development was measured until the fatigue time point (time at which force production was <50% initial maximum force) was surpassed. Thirty seconds after cessation of contractions, a single tetanic stimulation was performed in order to test for recovery and to demonstrate fatigue.
Electron microscopy
Immediately after the end of the contractile protocol, fibres were immersed in 6.25% solution of glutaraldehyde in 0.1 M sodium cacodylate buffer for fixation, and processed for electron microscopy as previously described (Mathieu-Costello, 1987). Specimens were sectioned transversely (perpendicular to the fibre axis) into ultrathin sections (50–80 nm) with an LKB Ultratome III and contrast-stained with uranyl acetate and bismuth subnitrate (Riva, 1974). The volume density of mitochondria was estimated by standard point-counting at a final magnification of 24 000x of electron micrographs taken on 70 mm film using a Zeiss 10 electron microscope. Micrographs of a carbon grating replica were recorded for calibration on each film. A total of 20 fields per specimen were analysed.
Statistical analysis
Student's paired t test and regression of least squares were performed. In all analyses, a 0.05 level of significance was used. Results are reported as means ±S.E.M. Correlations were conducted using SigmaPlot 5.0 regression analysis.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The causes of fatigue during high-intensity contractions in skeletal muscle have been widely investigated, yet the precise mechanisms remain controversial. Studies in exercising humans and in isolated whole muscle have demonstrated that significant alterations in intracellular and extracellular metabolite concentrations (NADH, ADP, Pi, lactate, K+ and H+), substrate limitation, and O2 limitation can be factors associated with fatigue (Godt & Nosek, 1989;; Westerblad et al. 1991, 2002; Fitts, 1994). However, differences in muscle fibre type, blood flow heterogeneity and muscle fibre recruitment patterns have traditionally made determinations of metabolic events during fatigue at the cellular level difficult to determine. In the isolated, single skeletal muscle fibre model used in the present study, the extracellular environment was carefully maintained so that fatigue within a discrete fibre could be analysed. Moreover, in this model it is possible to measure discrete intracellular events simultaneously with force production, allowing a more accurate determination of those factors associated with the fatigue process. In the single-fibre model used in the present study it has been demonstrated that fatigue is associated with increases in intracellular Pi and changes in intracellular Ca2+ handling (Westerblad et al. 1991, 2002; Westerblad & Allen, 2002).
Central to the fatigue process is the ability of the myocyte to balance ATP utilization with ATP generation (van der Laarse et al. 1989b). For example, it has been demonstrated that the rate of ATP consumption, as determined by myosin ATPase rates, correlates (r= 0.74) with fatigability of individual fibres in the single-cell model (van der Laarse et al. 1991). Similarly, research has been focused on the relationship between fatigability and the potential to regenerate hydrolysed ATP (Kugelberg & Lindegren, 1979; Burke, 1981; Nemeth et al. 1981; Hamm et al. 1988; Enad et al. 1989). This is accomplished through substrate level phosphorylation (anaerobic glycolysis and phosphocreatine hydrolysis) and oxidative phosphorylation. Mitochondria contain the pathway for oxidative phosphorylation and are intimately associated with fatigue-inducing factors, such as Ca2+ and Pi (Bose et al. 2003; Lannergren & Bruton, 2003), therefore playing a pivotal role in maintaining force production. However, in most studies using human or whole-muscle models, the relationship of MVD to fatigue resistance has been difficult to assess directly, and it remains uncertain whether the loss of force production during fatigue is due to metabolic inhibition, inadequate mitochondrial concentration, substrate limitation to activated mitochondria or inadequate availability of O2 to mitochondria. We have demonstrated in these Xenopus isolated skeletal muscle fibres that an earlier onset of fatigue resulting from inadequate O2 availability is associated with similar changes in Ca2+ handling, suggesting that fatigue during reductions in O2 supply operates through similar mechanisms to fatigue when the O2 supply is nonlimiting (Stary & Hogan, 2000). Furthermore, it has been demonstrated previously in similar single skeletal muscle fibres that the amount of succinate dehydrogenase activity, an estimate of MVD, is associated with the maximal rate of O2 consumption (van der Laarse et al. 1989a) and correlates (r= 0.83) with resistance to fatigue (van der Laarse et al. 1991), suggesting that MVD is a determinant of resistance to fatigue. However, technical limitations of the densitometric techniques employed in measuring succinate dehydrogenase activity, such as variability in tissue section thickness and incubation time, make an accurate estimate of MVD difficult (Gollnick & Hodgson, 1986).
In the present study, in order to evaluate the relationship between resistance to fatigue and MVD directly, we assessed MVD using electron microscopy after determining the rates of fatigue in corresponding isolated single skeletal muscle fibres during conditions of high extracellular O2 availability, and where metabolic waste product removal was not a limiting factor. A broad distribution of fatigue rates was evident (Fig. 1). During this type of stimulation protocol, leading to relatively rapid fatigue rates, it has been determined that substrate availability is not a limiting factor in these single fibres (Nagasser et al. 1992).
Similar to the fatigue rates, a broad distribution of MVD was observed (Fig. 2), from 2.7 to 9.2%. This is somewhat lower than that previously reported in other hindlimb muscles (flexor tarsi and ilio fibularis) of Xenopus laevis (Smith & Ovalle, 1973). One reason for this may be that the direct measurement of mitochondria using electron microscopy is more specific than the indirect densitometric methods employed to estimate oxidative enzyme concentrations in previous studies (Gollnick & Hodgson, 1986). However, this potential difference should not affect the comparative analysis with fatigue rates, since only the relative difference in MDV was used in determination of the correlation coefficient. When compared with the corresponding fatigue rates, a positive correlation (r= 0.93) with MVD was evident (Fig. 3). Since O2 availability and substrate limitation were not factors in the development of fatigue in this study, these results suggest that MVD per se is a critical determinant of the capacity for work in skeletal muscle fibres, probably due to the ability of mitochondria to maintain the balance between ATP supply and demand via oxidative phosphorylation (van der Laarse et al. 1989b), and to mitochondrial buffering of cytosolic Ca2+ and other ions.
In summary, the results of the present study, in which MVD in individual isolated fibres was assessed with electron microscopy and compared to the corresponding rates of fatigue, directly demonstrate that myocyte-specific MVD correlates with resistance to fatigue. These results also suggest that in these single skeletal muscle fibres during similar conditions of high O2 and substrate availability, and nonlimiting waste product removal, a standard fatigue protocol can be used as an estimate of oxidative capacity.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Burke RE (1981). Motor units: anatomy, physiology, and functional organization. In Handbook of Physiology, section 1, The Nervous System, vol. II, Motor Control, part 1, ed. Brooks, V. B., pp. 345–422. American Physiological Society, Bethesda, MD, USA.
Enad
JG, Fournier
M
&
Sieck
GC (1989). Oxidative capacity and capillary density of diaphragm motor units. J Appl Physiol
67, 620–627.
Fitts
RH (1994). Cellular mechanisms of fatigue. Physiol Rev
74, 49–94.
Godt
RE
&
Nosek
TM (1989). Changes in intracellular mileu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J Physiol
412, 155–180.
Gollnick PD & Hodgson DR (1986). The identification of fiber types in skeletal muscle: a continual dilemma. Exer Sports Sci Rev 14, 81–104.
Hamm TM, Nemeth PM, Solanki L, Gordon DA, Reinking RR & Stuart DG (1988). Association between biochemical and physiological properties of single motor units. Muscle Nerve 11, 245–254.[CrossRef][Medline]
Holloszy
JO
&
Coyle
EF (1984). Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J Appl Physiol
56, 831–838.
Hood
D (2001). Contractile activity-induced mitochondrial biogenesis in skeletal muscle. J Appl Physiol
90, 1137–1157.
Hoppeler H & Fluck M (2003). Plasticity of skeletal muscle mitochondria: structure and function. Med Sci Sports Exer 35, 95–104.[Medline]
Kugelberg
E
&
Lindegren
B (1979). Transmission and contraction fatigue of rat motor units in relation to succinate dehydrogenase activity of motor unit fibers. J Physiol
288, 285–300.
Lannergren J & Bruton JD (2003). Mitochondrial Ca2+ in mouse soleus single muscle fibres in response to repeated tetanic contractions. Adv Exp Med Biol 538, 557–562.[Medline]
Lannergren J & Smith RS (1966). Types of muscle fibers in toad skeletal muscle. Acta Phys Scand 68, 263–274.
Mathieu-Costello O (1987). Capillary tortuosity and degree of contraction or extension of skeletal muscles. Microvas Res 33, 98–117.
Nagasser
AS, van der Laarse
WJ
&
Elzinga
G (1992). Metabolic changes with fatigue in different types of single muscle fibres of Xenopus laevis. J Physiol
448, 511–523.
Nemeth
PM, Pette
D
&
Vrbova
G (1981). Comparison of enzyme activities among single muscle fibers within defined motor units. J Physiol
311, 489–495.
Riva A (1974). A simple and rapid staining method for enhancing the contrast of tissue previously treated with uranyl acetate. Pflugers Arch 380, 153–158.
Smith RS & Ovalle WK (1973). Varieties of fast and slow extrafusal fibers in amphibian hind limb muscles. J Anat 116, 1–24.[Medline]
Stary CM & Hogan MC (2000). Phosphorylating pathways and fatigue development in contracting Xenopus single skeletal muscle fibers. Am J Physiol 278, R587–R591.
van der Laarse WJ, Diegenbach PC & Elzinga G (1989a). Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibers of Xenopus leavis. J Mus Res Cell Motil 10, 221–228.[CrossRef][Medline]
van der Laarse WJ, Elzinga G & Woledge RC (1989b). Energetics at the single cell level. News Physiol Sci 4, 91–93.
van der Laarse WJ, Lannergren J & Diegenbach PC (1991). Resistance to fatigue of single skeletal muscles from Xenopus related to succinate dehydrogenase and myofibrillar ATPase activities. Exp Physiol 76, 589–596.[Abstract]
Westerblad H & Allen DG (2002). Recent advances in the understanding of skeletal muscle fatigue. Curr Op Rheumatol 14, 648–652.
Westerblad
H, Allen
DG
&
Lannergren
J (2002). Muscle fatigue: lactic acid or inorganic phosphate the major cause?News Physiol Sci
17, 17–21.
Westerblad H & Lannergren J (1986). Force and membrane potential during and after fatiguing, intermittent tetanic stimulation of single Xenopus muscle fibers. Acta Physiol Scan 128, 369–378.[Medline]
Westerblad H, Lee JA & Allen DG (1991). Cellular mechanisms of fatigue in skeletal muscle. Am J Physiol 261, C195–C209.
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  B. Walsh, C. M. Stary, R. A. Howlett, K. M. Kelley, and M. C. Hogan Glycolytic activation at the onset of contractions in isolated Xenopus laevis single myofibres Exp Physiol, September 1, 2008; 93(9): 1076 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. E. R. Philipp, M. Schmidt, C. Gsottbauer, A. M. Sanger, and D. Abele Size- and age-dependent changes in adductor muscle swimming physiology of the scallop Aequipecten opercularis J. Exp. Biol., August 1, 2008; 211(15): 2492 - 2501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Stary, B. J. Walsh, A. E. Knapp, D. Brafman, and M. C. Hogan Elevation in heat shock protein 72 mRNA following contractions in isolated single skeletal muscle fibers Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R642 - R648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Kinsey, K. M. Hardy, and B. R. Locke The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle J. Exp. Biol., October 15, 2007; 210(20): 3505 - 3512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Kindig, B. Walsh, R. A. Howlett, C. M. Stary, and M. C. Hogan Relationship between intracellular PO2 recovery kinetics and fatigability in isolated single frog myocytes J Appl Physiol, June 1, 2005; 98(6): 2316 - 2319. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
