Excitation-contraction coupling and sarcoplasmic reticulum function in mechanically skinned fibres from fast skeletal muscles of aged mice

doi:10.1113/jphysiol.2002.022418

Excitation-contraction coupling and sarcoplasmic reticulum function in mechanically skinned fibres from fast skeletal muscles of aged mice

David R. Plant and Gordon S. Lynch

Department of Physiology, The University of Melbourne, Victoria 3010, Australia

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Ageing is generally associated with a decline in skeletal muscle mass and strength, and a slowing of muscle contraction, factors that impact upon the quality of life for the elderly. Alterations in Ca²⁺ handling are thought to contribute to these age-related changes in muscle contractility, yet the effects of ageing on sarcoplasmic reticulum (SR) Ca²⁺ handling and the Ca²⁺ transport system remain unresolved. We used mechanically skinned single fibres from the fast twitch extensor digitorum longus (EDL) muscles from young (4-month-old) and old (27- to 28-month-old) mice to test the hypothesis that the age-related changes in skeletal muscle contractility, especially the slower rate of contraction, are due to changes intrinsic to the muscle fibres. There were no age-related differences in the peak height of depolarization-induced contractile response (DICR) or the number of DICRs elicited before rundown (DICR < 50 % of initial). The time taken to reach peak DICR (TPDICR) was ~12 % slower in single muscle fibres from old compared with young mice (P < 0.05). The rate of relaxation following DICR was not different in young and old mice. Examination of SR function demonstrated that SR Ca²⁺ reloading in Ca²⁺-depleted skinned fibres was not different in young and old mice, nor was there any age-related difference in Ca²⁺ leak from the SR. However, low [caffeine] contracture in fibres from old mice was only half of that observed in fibres from young mice (P < 0.05), indicating a lower sensitivity of the SR Ca²⁺ release channel (CRC) to caffeine. We found no difference in maximum Ca²⁺-activated force (P_o) or specific force (sP_o; P_o corrected for cross-sectional area) in EDL muscle fibres from young and old mice. Impaired excitation-contraction (E-C) coupling and a decrease in SR CRC function are mechanisms which are likely to contribute to the overall slowing of muscle contraction with age.

(Resubmitted 15 April 2002; accepted after revision 30 May 2002)
Corresponding author G. S. Lynch: Department of Physiology, The University of Melbourne, Victoria 3010, Australia. Email: gsl{at}unimelb.edu.au

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Ageing is generally associated with a decline in skeletal muscle mass and strength (sarcopenia) and a slowing of muscle contraction, factors that impact upon the quality of life for the elderly (Larsson & Ramamurthy, 2000; Frontera et al. 2000; Lynch, 2002). Progressive muscle fibre denervation, the loss of fast motor units and subsequent motor unit remodelling have been implicated as mechanisms responsible for these deleterious effects of ageing on skeletal muscle (Einsiedel & Luff, 1992; Faulkner et al. 1995; Wineinger et al. 1995; Kadhiresan et al. 1996). However, the age-related reduction in the speed of contraction has been reported to occur before the onset of severe muscle wasting (Larsson & Edstrom, 1986; Narayanan et al. 1996), indicating that age-related changes intrinsic to skeletal muscle fibres cannot be excluded (Gonzalez et al. 2000).

The sequence of events linking an electrical signal at the neuromuscular junction to force development is defined as excitation-contraction (E-C) coupling. This sequence of events involves depolarization of the transverse-tubular (t)-system, which activates specialized voltage sensors (dihydropyridine receptors, DHPRs), which in turn, open ryanodine receptors/Ca²⁺ release channels (CRCs) in the adjacent sarcoplasmic reticulum (SR) membrane, allowing efflux of Ca²⁺ into the cytoplasm and binding of Ca²⁺ to troponin C and, subsequent crossbridge cycling and force development (Melzer et al. 1995). Age-related alterations in the proteins involved in E-C coupling are thought to contribute to the changes in muscle contractility (Margreth et al. 1999). These alterations include: a reduction in the amount of Ca²⁺ available for triggering contraction and a reduction in Ca²⁺ release due to DHPR-ryanodine receptor (RyR) uncoupling (Delbono et al. 1995); impairment of SR Ca²⁺ pump function (Narayanan et al. 1996); abnormalities in the regulation of RyRs (Damiani et al. 1996); and decreased turnover of Ca²⁺-ATPase and RyR protein (Ferrington et al. 1998). These studies have utilized different experimental approaches in their assessment of SR properties and contractility of aged skeletal muscle, including measurements of Ca²⁺ uptake and/or release from: isolated SR vesicle preparations (Damiani et al. 1996; Ferrington et al. 1997, 1998), single chemically skinned muscle fibres (Larsson & Salviati, 1989; Danieli-Betto et al. 1995) and small muscle fibre segments for use with the double Vaseline gap voltage clamp technique (Delbono et al. 1995; Wang et al. 2000). However, there are several effects of ageing on SR Ca²⁺ handling and the Ca²⁺ transport system that remain unresolved, particularly with respect to SR Ca²⁺ release and the functional coupling of RyRs (Margreth et al. 1999).

One muscle preparation that has not been used for the assessment of age-related effects on SR function and E-C coupling is the mechanically skinned muscle fibre (Lamb & Stephenson, 1990). Mechanical skinning leaves the muscle fibre segment with a sealed t-system, retaining normal voltage regulation of Ca²⁺ release with an endogenous level of SR Ca²⁺, allowing for manipulation of the intracellular environment (Posterino et al. 2000). The advantage of this technique is that it allows assessment of the DHPR-CRC interaction, SR Ca²⁺ release, as well as subsequent force production due to crossbridge cycling in a single fibre that has an approximately endogenous SR Ca²⁺ content, and retains normal voltage regulation of SR Ca²⁺ release (Lamb & Stephenson, 1994). The purpose of this study was to investigate the effects of age on E-C coupling and SR function in single fast twitch mechanically skinned muscles. Specifically, we tested the hypothesis that the age-related slowing of contraction and decrease in force output in muscles of elderly is due to changes in SR function and E-C coupling in mechanically skinned single muscle fibres.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

All experiments were approved by the Animal Experimentation Ethics Committee of The University of Melbourne and complied with the Code of Conduct for the Care and Use of Animals as stipulated by the National Health and Medical Research Council of Australia. Young (4-month-old; n = 10) and old (27- to 28-month-old; n = 8) male C57BL/10 ScSn mice were killed by rapid cervical dislocation. The extensor digitorum longus (EDL, fast twitch) muscles were quickly excised from one hindlimb, blotted on filter paper and then placed in a Petri dish filled with cooled liquid paraffin. The muscles were pinned at resting length to the base of a dish that was lined with Sylgard gel (Dow Corning, Midland, MI, USA) stored on ice and used intermittently for dissection.

Muscle fibre preparation

Under a microscope a small bundle of fibres was dissected at one end and separated down to the mid-belly of the muscle using fine jeweller's forceps. This bundle of fibres was repeatedly separated until a single muscle fibre was isolated. Each fibre was mechanically skinned by peeling the sarcolemma away from the fibre, such that 70-90 % of the fibre bulk remained (Lamb & Stephenson, 1990). One end of the peeled fibre segment was mounted to a sensitive force transducer (AE801 SensoNor, Horten, Norway) with a braided surgical silk tie (10-0, Deknatel, New York, USA) and the other end of the fibre secured between the jaws of a pair of jeweller's forceps (No 5, Dumont & Fils, Switzerland). Average sarcomere length of the fibres was adjusted to a length slightly longer than optimal in order to reliably measure depolarization-induced contractile responses (Owen et al. 1997). The sarcomere length was estimated from the diffraction pattern produced when the beam of a He-Ne laser (Spectra Physics, OR, USA) was passed along the length of the fibre. Fibre diameter was estimated using a calibrated graticule in the eyepiece of a dissecting microscope.

Solutions for depolarization-induced responses of mechanically skinned fibres

The composition of the solutions and the procedures used for activation of the skinned muscle fibres has been described in detail previously (Lamb & Stephenson, 1990; Posterino & Lamb, 1996). Briefly, the t-system of the fibres was polarized by incubating the fibre in a potassium hexamethylenediamine-tetra-acetic acid solution (K-HDTA; composition (mM): Hepes 90, K⁺ 125, Na⁺ 36, HDTA 50, Mg (total) 8.5, NaN₃ 1, creatine phosphate 10, ATP 8) for 2 min. The t-system of the fibre was depolarized by rapidly substituting K-HDTA with Na-HDTA, an identical solution but with all of the K⁺ replaced by equimolar Na⁺. Maximum Ca²⁺-activated force (P_o) was determined in an approximately equimolar CaEGTA solution (composition (mM): Hepes 90, K⁺ 125, Na⁺ 36, Ca-EGTA 50, Mg (total) 8.12, NaN₃ 1, creatine phosphate 10, ATP 8; pCa < 4.7). All solutions contained 1 mM free [Mg²⁺] (unless otherwise stated) and had an osmolality of ~295 mosmol kg^-1 (Lamb & Stephenson, 1994). All experiments were performed at 22 ± 1 °C.

Experimental protocol

Protocol 1: contractility of mechanically skinned fibres. Freshly skinned fibres were incubated in the standard K-HDTA solution to polarize the sealed t-system. The t-system could be depolarized by replacing K-HDTA with Na-HDTA, eliciting a transient force response. The t-system was repolarized by 30 s incubation in K-HDTA. The depolarization and repolarization sequence was repeated until each fibre had rundown (i.e. when peak depolarization-induced contractile response (DICR) equals 50 % of the initial value), at which point fibres were bathed in a low [Mg²⁺] K-HDTA solution (0.1 mM free [Mg²⁺]), to assess the viability of SR CRC function (Posterino & Lamb, 1996). At the conclusion of the DICR measurement, P_o was determined in Ca-EGTA.

We examined the effects of age on E-C coupling and SR function in fast type II fibres from the EDL muscle. Our fibre analysis was based on that described by Stephenson et al. (1998) who showed that in rat skeletal muscle fibres the DICR reached a much smaller proportion of the maximum Ca²⁺-activated force (P_o) in slow twitch fibres (~10 %) than in fast twitch fibres, where they were typically above 80 %. The fibres were identified by SDS gel electrophoresis and by their sensitivity to Sr²⁺. In another set of experiments, Goodman et al. (2000) showed that DICR was significantly higher in fibres containing myosin heavy chain (MHC) IIb from EDL muscles of rats (62 ± 6 % of maximum Ca²⁺-activated force) than in soleus muscle fibres from rats containing MHC I (25 ± 5 % of maximum Ca²⁺-activated force). The number of responses developed by a fibre preparation until the response declined to 25 % of its highest level was significantly higher in fibres containing MHC IIb from EDL muscles of rats (22 ± 3) than in fibres containing MHC I from the soleus muscles from rats (10 ± 1). Our previous experiments using mechanically skinned single fibres isolated from rat muscles confirm these findings (Plant et al. 2002) and we have reported that under our experimental conditions maximum DICR from mechanically skinned type II fibres from rat EDL muscles is 72 ± 5 % of maximum Ca²⁺-activated force and 20 ± 1 depolarizations can be elicited before fibre rundown (DICR < 50 % of initial). In contrast, DICRs from type I fibres from the soleus muscle of rats have maximum DICR of 47 ± 5 % of maximum Ca²⁺-activated force and only 7 ± 1 can be depolarizations elicited before rundown. Note that in our experiments fibre type was determined from activating fibres in a solution of low [Sr²⁺], a well-established technique for fibre type identification that has been verified with the MHC isoform composition of single muscle fibres (Bortolotto et al. 2000).

To examine the effect of age on the rate of contraction we also measured the time taken to reach peak DICR (TPDICR). This parameter was determined from the average of the first three DICRs in each fibre. The TPDICR is comparable to a measurement of the time taken to reach maximum isometric force for an intact muscle or single muscle fibre, except that the magnitude of the DICR corresponds to ~85 % of maximum Ca²⁺-activated force.

Protocol 2: SR function. Another group of fibres was used to examine the ability of the Ca²⁺-depleted SR to load Ca²⁺. The SR of fibres was first completely depleted of Ca²⁺ by 2 min incubation in the release solution (low [Mg²⁺] K-HDTA, containing 30 mM caffeine and 0.5 mM EGTA). The SR of each fibre was then partially reloaded by incubation in a Ca²⁺-loading solution (K-HDTA with 50 µM added Ca²⁺) for a set period (10, 20 or 30 s). After each Ca²⁺ loading period the SR was again depleted of Ca²⁺ with the release solution. The area under the curve of force trace following SR Ca²⁺ release after each loading time was corrected for maximum (designated as the SR Ca²⁺ content after 60 s in Ca²⁺ loading solution in these experiments). The SR Ca²⁺ content vs. loading time was plotted from the average of the fibre responses and a curve fitted to the values to determine the rate of SR Ca²⁺ reloading.

Additional muscle fibres were used to examine Ca²⁺ 'leak' from the SR. As for the fibres above, the SR of each fibre was first depleted of Ca²⁺ and the SR partially reloaded (30 s) and the SR depleted of Ca²⁺ to determine 'control' SR Ca²⁺ content. This sequence was repeated but with an additional 2 min equilibration (K-HDTA containing 10 mM free [Mg²⁺] and 0.5 mM EGTA (a solution in which Ca²⁺ uptake and leak is prevented)) and 30 s 'leak' period (standard K-HDTA with 1 mM total EGTA) prior to SR Ca²⁺ release. The area under the curve of the force response after the 'leak' period was compared with 'control' SR Ca²⁺ content to determine the relative amount of Ca²⁺ leaked from the SR.

The sensitivity of the SR to caffeine-induced Ca²⁺ release was determined by low [caffeine] contracture (K-HDTA solution containing 7 mM caffeine). A fresh group of fibres was dissected, and again the SR was completely depleted of Ca²⁺ (as described previously), and partially reloaded (30 s) before incubation in the 7 mM caffeine solution. P_o was determined after the caffeine contraction and the peak force response in 7 mM caffeine compared with P_o for each fibre as an indication of sensitivity to caffeine-induced Ca²⁺ release.

Statistical analysis

Values in the text are presented as means ± S.E.M. Comparisons of fibres from young and old mice were completed by either analysis of variance or Student's t test, where appropriate. Results were considered significant when P < 0.05.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Dissection and isolation of single fibres from EDL muscles proved more difficult in old than young mice due to the presence of greater amounts of connective tissue surrounding the fibres. Nonetheless, once single fibres were isolated, the sarcolemma was successfully peeled from the outside of the fibres and the t-system 'sealed' under oil so as to establish normal membrane polarity when bathed in K-HDTA. Rapid substitution of K-HDTA for Na-HDTA solution depolarized the t-system, causing rapid release of Ca²⁺ from the SR, initiating a depolarization-induced contractile response (DICR; Fig. 1).

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Figure 1. Depolarization-induced force responses of mechanically skinned fibres from young and old mice
Original recordings of depolarization-induced contractile responses from a mechanically skinned fibre from a young (A) and an old (B) mouse. Fibres were depolarized with Na-HDTA and the t-system repolarized in K-HDTA for 30 s between successive depolarizations. Many depolarizations could be elicited from fibres from both young and old mice before rundown. Incubation with a low [Mg²⁺] solution (0.1 mM free [Mg²⁺]) elicited a transient force response indicative of functional CRC, even at the point of fibre rundown. At the conclusion of all experiments, each fibre was bathed in a Ca-EGTA solution (pCa < 4.7) to determine maximum Ca²⁺ -activated force. Depol, depolarization with Na-HDTA solution; Low [Mg²⁺], 0.1 mM in K-HDTA; P_o, maximum Ca²⁺ activated force (pCa < 4.7).

DICR in mechanically skinned fibres from EDL muscles of young and old mice was very similar, with no differences in peak tension developed during DICR or in the number of depolarizations elicited before rundown (see Table 1), indicating that all fibres examined were type II MHC isoform composition (Stephenson et al. 1998; Goodman et al. 2000; Plant et al. 2002). Even at the point of rundown, incubation in the low [Mg²⁺] solution elicited a large force response indicating that the SR CRC was still functional (see Fig. 1). TPDICR was slower in fibres from old compared with young mice (971 ± 90 vs. 853 ± 38 ms, P < 0.05; See Fig. 2). No age-related differences were observed in the rate of relaxation of the DICR. In addition, no differences were observed in either cross-sectional area (CSA), P_o or sP_o of mechanically skinned fibres from the EDL muscles of young and old mice (see Table 1).

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Figure 2. Ageing slows time taken to reach peak of depolarization-induced force responses of mechanically skinned fibres
Original recordings of depolarization-induced contractile responses from the mechanically skinned fibres from a young (A) and an old (B) mouse shown in Fig. 1. On average, TPDICR was ~12 % slower in fibres from old compared with young mice (P < 0.05). Depol, depolarization in Na-HDTA solution. Depolarization sequence indicated in parentheses.

Another group of mechanically skinned fibres from young and old mice were used to examine SR Ca²⁺ loading properties. The area under the force-response trace following incubation in the release solution was used as an approximate indicator of SR Ca²⁺ content (Lamb & Stephenson, 1994). Initially the SR of the fibres was depleted completely of Ca²⁺ after 2 min equilibration in the release solution, and the SR partially reloaded with Ca²⁺ by incubation in the load solution for defined intervals (10, 20 or 30 s). The SR Ca²⁺ content at each 'load duration' was compared to maximum (60 s Ca²⁺ loading) to establish the load duration-SR Ca²⁺ content relationship. A curve was fitted to the relationship to determine the rate of SR Ca²⁺ reloading (see Fig. 3). No difference in the rate of SR Ca²⁺ reloading was observed in mechanically skinned fibres from young and old mice.

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Figure 3. Age does not affect SR Ca²⁺ reloading rate in mouse mechanically skinned fibres
Rate of SR Ca²⁺ reloading in mechanically skinned fibres from young ( small square , n = 11 fibres) and old ( filled square , n = 12 fibres) mice. Initially the SR of each fibre was depleted of Ca²⁺ by incubation in the release solution and then loaded for a designated period (10, 20 or 30 s) and the area under force trace of the subsequent exposure to the release solution used as an indicator of SR Ca²⁺ content. Values were represented according to maximum (60 s load) and a curve fitted to the average of the responses (continuous line, young; dotted line, old) to determine the Ca²⁺ reloading rate. The rate of SR Ca²⁺ reloading was not altered by age.

SR Ca²⁺ leak was also determined in another group of mechanically skinned fibres from young and old mice. The difference in the area under the force-response trace (approximate SR Ca²⁺ content) after the leak period and normal loading, was used to determine relative SR Ca²⁺ leak in mechanically skinned fibres from young and old mice (see Fig. 4). Under conditions where Ca²⁺ uptake was prevented, leak of Ca²⁺ from the SR was not different in fibres from young and old mice.

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Figure 4. SR Ca²⁺ permeability is not altered with age
SR Ca²⁺ leak was determined following 30 s 'leak' period (when all Ca²⁺ uptake was prevented) in mechanically skinned fibres from EDL muscles of young ( square , n = 11 fibres) and old mice ( filled square , n = 12 fibres). The SR of fibres was loaded to ~70 % maximum and SR Ca²⁺ content estimated from the area under the force trace in release solution, determined before and after the 'leak' period. There were no age related effects on SR Ca²⁺ permeability.

Low [caffeine] contracture was used to assess the sensitivity to caffeine-induced Ca²⁺ release in the mechanically skinned fibres from young and old mice. Ageing was associated with a depression in peak caffeine-induced Ca²⁺ release, with the response in old mice much lower than that observed in fibres from young mice (peak caffeine-induced force = 65 ± 3 % of P_o for young mice, n = 12 fibres vs. 34 ± 3 % for old mice, n = 10 fibres, P < 0.001; Fig. 5).

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Figure 5. Age decreases the sensitivity to caffeine-induced Ca²⁺ release
Original trace of a mechanically skinned fibre from a young (A) and an old (B) mouse indicating low [caffeine] contracture and maximum Ca²⁺-activated force (P_o). C, the average response to low [caffeine] from old mice ( filled square , n = 10 fibres) was only 52 % of that in fibres from young mice ( square , n = 9 fibres). ***P < 0.001.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

Depolarization-induced contractile responses (DICR) were elicited from mechanically skinned EDL muscle fibres from both young and old mice, indicating functional E-C coupling. All fibres examined had DICR properties of type II fibres, as indicated by the peak DICR and number of depolarizations elicited before rundown (Stephenson et al. 1998; Goodman et al. 2000; Plant et al. 2002). Despite the similarities in DICR, the time taken to reach peak DICR in fibres from old mice was slower than in fibres from young mice. There was also a marked age-related reduction in the sensitivity of the SR to caffeine-induced Ca²⁺ release in these fibres. We found that in fast twitch skeletal muscle fibres the ability of the SR to load Ca²⁺ and the leak rate of Ca²⁺ from the SR were not affected by age. The results indicate that despite the conservation of SR function of single muscle fibres with age, the slowing of DICR in single fibres is likely to contribute to the overall slowing of muscle contraction with age.

This study demonstrated that mechanically skinned fibres from the EDL muscles of mice are suitable for examining E-C coupling and SR function, producing similar data to that described previously for similar preparations from the skeletal muscles of rats (Lamb & Stephenson, 1994; Posterino & Lamb, 1996; Plant et al. 2002). In functional fibres lacking a sarcolemmal membrane, we found that most of the properties of the SR were preserved in the skeletal muscles of aged mice, with few differences in muscle contractility between young and old animals.

The reduced caffeine-induced Ca²⁺ release in fast twitch fibres from old mice might be mediated by decreased SR Ca²⁺ release, reduced myofibrillar sensitivity to Ca²⁺ in the presence of 7 mM caffeine, reduced SR Ca²⁺ concentration in fibres from old mice, or increased Ca²⁺-buffering capacity of the myofibrillar component. We also demonstrated that SR Ca²⁺-pump activity is maintained with age and is therefore not likely to contribute to the decrease in caffeine-induced Ca²⁺ release in fast twitch fibres from old mice. Our finding of a decreased sensitivity of the SR to caffeine-induced Ca²⁺ release in mechanically skinned fibres differs from that of Damiani et al. (1996), who reported an increased sensitivity to caffeine in RyR1 of SR membrane vesicle preparations from fast twitch skeletal muscles of aged rats. The differences in the muscle preparations and the animal species used could account for the disparity between the two studies. Our finding of no difference in the leak of Ca²⁺ from the SR with age is in good agreement with previous biochemical studies that have reported no effect of age on the conformational stability of the SR membrane lipid bilayer (Ferrington et al. 1997).

Uncoupling of the DHPR and RyRs has been reported in the skeletal muscles of aged rats based on high-affinity ligand binding studies (Renganathan et al. 1997). It was suggested that DHPR-RyR uncoupling at the t-tubule-SR triadic junction would result in an absolute reduction in SR Ca²⁺ release in response to sarcolemmal depolarization and consequently a reduced contractile strength in aged skeletal muscle (Renganathan et al. 1997). We found that functional E-C coupling in mechanically skinned fibres was affected by age as evidenced by the slower depolarization-induced force responses for EDL muscle fibres from old compared with young mice. The advantage of the mechanically skinned fibre preparation is that the E-C coupling CRC-RyR system is preserved in its normal configuration and is functionally intact. Our finding of a slower DICR indicates some level of DHPR-RyR uncoupling in fast twitch muscle fibres from aged mice. However, this slower rate of force development did not affect the magnitude of the depolarization-induced force responses of EDL muscle fibres from old compared with young mice. The TPDICR reported in our experiments were slower than the times reported for spontaneous force responses that sometimes occur before a mechanically skinned fibre is depolarized (Posterino et al. 2000). We also measured similar spontaneous force responses in some of the mechanically skinned fibres used in our experiments and, on average, the time to reach peak force of these of these spontaneous contractions was 220 ± 26 ms (n = 5), identical to that reported previously (Posterino et al. 2000). It should also be noted that depolarization-induced force production in mechanically skinned fibres is a coordinated contractile response with peak force corresponding to 85 % of maximum isometric force. Force production following depolarization is dependent on release of Ca²⁺ from the SR and binding of Ca²⁺ to troponin to allow crossbridge cycling. If there was an age-related slowing of SR Ca²⁺ release, then there would be a slower rise in [Ca²⁺]_i and a slower formation of active crossbridges, factors that would both increase the time taken to reach peak contraction. Our TPDICR data are consistent with, and confirm that SR CRC function is compromised with increasing age, and an underlying mechanism that contributes to the slowing of force development with age (Larsson & Edstrom, 1986; Narayanan et al. 1996).

The aim of this study was to determine whether the discrete events of E-C coupling are altered as a consequence of age. The proteins regulating depolarization-induced force and SR Ca²⁺ release and reuptake are not the myosin heavy chains (MHC). Changes in MHC within single fibres with increasing age have been well documented in rodents (Li & Larsson, 1996; Degens et al. 1998) and humans (Larsson et al. 1997). Our purpose was not to repeat the findings of previous investigations where MHC isoform composition has been correlated with contractile events that are most influenced by them, i.e. maximum velocity of shortening (V_max). As for previous investigations of SR function in well-described fast twitch mammalian skeletal muscle fibres (Lamb & Stephenson, 1994) we did not determine MHC composition in these fibres and instead focussed our examination on whether the events controlling depolarization-induced contraction are affected by age. Fibre type transitions have been reported in both fast and slow muscles of the rat with increasing age, and in addition to our findings of altered E-C coupling especially the slowed DICRs, changes in MHC are also likely to contribute to the age-related decrease in the speed of muscle contraction (Sugiura et al. 1992). Given the possibility of type IIb to type IIx MHC isoform transitions in the EDL muscles of ageing mice, a bias in the selection of fibres is possible. The correlation of MHC composition and TPDICR is clearly worthy of further investigation.

In addition to examining depolarization-induced contractile responses, we also examined the properties of the intact functional SR of mechanically skinned fibres from EDL muscles of young and old mice. Under our experimental conditions, no differences were observed in SR Ca²⁺ reloading to the Ca²⁺ depleted SR of fast twitch EDL muscle fibres. A lower rate of Ca²⁺ uptake in SR vesicle preparations has been reported for slow twitch soleus muscles from old compared with young F344 rats (Narayanan et al. 1996), which may be responsible for the slower rate of relaxation of aged slow twitch muscles. Age also had no effect on the conformational stability of the SR membrane lipid bilayer (Ferrington et al. 1997), which was confirmed by our report of no age-related difference in the leak of Ca²⁺ from the SR of mechanically skinned muscle fibres. Our finding of a decreased sensitivity of the SR to caffeine-induced Ca²⁺ release in mechanically skinned fibres differs from that of Damiani et al. (1996), who reported an increased sensitivity to caffeine in RyR1 of SR membrane vesicle preparations from fast twitch skeletal muscles of aged rats. The differences in the muscle preparations and the animal species used could account for the disparity between the two studies.

In addition to the slowing of contraction, a decrease in the intrinsic force producing capacity of intact skeletal muscles has been observed with increasing age (Brooks & Faulkner, 1988; Phillips et al. 1993; Lynch et al. 2001). The results from our experiments on mechanically skinned fibres indicate that there were no differences in absolute maximum Ca²⁺-activated force production or force per cross-sectional area (sP_o) in fast twitch EDL muscle fibres from old and young mice. The skinned fibre preparation allows direct investigation of the contractile apparatus without the confounding effects of neural influences, changes in fibre architecture, or alterations in intercellular connective tissue (Lamb & Stephenson, 1994; Stephenson et al. 1998). Our finding of no difference in the sP_o of single fast twitch EDL muscle fibres is consistent with the majority of previous studies using chemically permeabilized or mechanically skinned muscle fibre preparations (Eddinger et al. 1986; Lynch et al. 1993a; Brooks & Faulkner, 1994; Plant & Lynch, 2001) although it should be noted that some investigators have reported an age-related decrease in sP_o in different species (Larsson et al. 1997; Thompson & Brown, 1999; Frontera et al. 2000). Differences in the experimental conditions and the methods used to prepare single muscle fibres are likely factors that can attribute for the discrepancy in the sP_o data between the different studies. Where changes in sP_o have been reported, these differences are often fibre type specific (Larsson et al. 1997; Frontera et al. 2000). A decrease in SR Ca²⁺ release following sarcolemmal depolarization, mediated by DHPR-RyR uncoupling at the t-tubule-SR triadic junction (Renganathan et al. 1997) or a decrease in SR CRC activity, could also contribute to a reduction in maximum force production. The advantage of the mechanically skinned muscle fibre preparation is that the E-C coupling CRC-RyR system is preserved in its normal configuration and is functionally intact. Our finding of no effect of age on force production (peak DICR, P_o, sP_o) in EDL muscle fibres from old compared with young mice indicates that despite such uncoupling, normal force production is maintained. DHPR-RyR uncoupling appears to more likely be implicated in the mechanism underlying the slowed contractility in fibres from aged mice.

There are conflicting reports as to whether there are changes in the number of Ca²⁺ channels or Ca²⁺ pumps, or in their intrinsic molecular properties as a consequence of age. In a series of studies examining E-C coupling in ageing muscle, Delbono and colleagues have asserted that there is an age-related reduction in peak intracellular [Ca²⁺] due to a larger number of RyRs uncoupled to DHPRs in skeletal muscle (Delbono et al. 1995; Renganathan et al. 1997). However, in their review of sarcoplasmic reticulum function in aged skeletal muscle, Margreth et al. (1999) state that experimental evidence excludes the occurrence of age-related changes in the molecular properties of Ca²⁺ channels or Ca²⁺ pumps, and that the problem of Ca²⁺ homeostasis in aged skeletal muscle is one of altered regulation. Our finding that the activity of the CRC was decreased in mechanically skinned fibres of aged mice is consistent with the reported decrease in L-type Ca²⁺ channel charge movement in (fast twitch) flexor digitorum brevis muscle fibres from aged mice (Wang et al. 2000).

The present study focussed on an examination of the age-related changes to E-C coupling and SR function in fast twitch single fibres from the EDL muscle of the mouse, but did not examine these effects in slow twitch muscle fibres. Preparation of mechanically skinned fibres for studying E-C coupling and SR function is extremely labour intensive since the muscle fibres must be prepared fresh daily and can be used only for a limited period (Lynch et al. 1993a,b). Therefore, for this series of experiments it was possible to examine the age-related changes only in fast twitch single fibres, and an examination of the age-related changes to slow twitch fibres is the subject of future studies.

The findings of the present study have demonstrated that although the force producing capacity and functional E-C coupling of single fast twitch skeletal muscle fibres is essentially preserved with age, the slower time-to-peak of the depolarization-induced force responses and the decrease in SR CRC activity indicates some impairments in fibre contractility and SR function with increasing age. The results support the notion that the problem of Ca²⁺ homeostasis in aged skeletal muscle is due to a combination of altered regulation (Margreth et al. 1999) and minor alterations in E-C coupling (Delbono et al. 2000). These alterations are likely mechanisms contributing to the age-related slowing of muscle contraction.

REFERENCES

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Abstract
Introduction
Methods
Results
Discussion
References

BORTOLOTTO, S. K., CELLINI, M., STEPHENSON, D. G. & STEPHENSON, G. M. (2000). MHC isoform composition and Ca²⁺- or Sr²⁺ -activation properties of rat skeletal muscle fibers. American Journal of Physiology - Cell Physiology 279, C1564-1577 [Medline]

BROOKS, S. V. & FAULKNER, J. A. (1988). Contractile properties of skeletal muscles from young, adult and aged mice. Journal of Physiology 404, 71-82 [Abstract]

BROOKS, S. V. & FAULKNER, J. A. (1994). Isometric, shortening, and lengthening contractions of muscle fiber segments from adult and old mice. American Journal of Physiology 267, C507-513 [Medline]

DAMIANI, E., LARSSON, L. & MARGRETH, A. (1996). Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle. Cell Calcium 19, 15-27 [Medline]

DANIELI-BETTO, D., BETTO, R., MEGIGHIAN, A., MIDRIO, M., SALVIATI, G. & LARSSON, L. (1995). Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiologica Scandinavica 154, 59-64 [Medline]

DEGENS, H., YU, F., LI, X. & LARSSON, L. (1998). Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres. Acta Physiologica Scandinavica 163, 33-40 [Medline]

DELBONO, O., O'ROURKE, K. S. & ETTINGER, W. H. (1995). Excitation-calcium release uncoupling in aged single human skeletal muscle fibers. Journal of Membrane Biology 148, 211-222 [Medline]

EDDINGER, T. J., CASSENS, R. G. & MOSS, R. L. (1986). Mechanical and histochemical characterization of skeletal muscles from senescent rats. American Journal of Physiology 251, C421-430 [Medline]

EINSIEDEL, L. J. & LUFF, A. R. (1992). Alterations in the contractile properties of motor units within the aging rat medial gastrocnemius. Journal of the Neurological Sciences 112, 170-177 [Medline]

FAULKNER, J. A., BROOKS, S. V. & ZERBA, E. (1995). Muscle atrophy and weakness with aging: contraction-induced injury as an underlying mechanism. Journal of Gerontology 50A, 124-129

FERRINGTON, D. A., JONES, T. E., QIN, Z., MILLER-SCHLYER, M., SQUIER, T. C. & BIGELOW, D. J. (1997). Decreased conformational stability of the sarcoplasmic reticulum in aged skeletal muscle. Biochimica et Biophysica Acta 1330, 233-247 [Medline]

FERRINGTON, D. A., KRAINEV, A. G. & BIGELOW, D. J. (1998). Altered turnover of calcium regulatory proteins of the sarcoplasmic reticulum in aged skeletal muscle. Journal of Biological Chemistry 273, 5885-5891 [Abstract/Full Text]

FRONTERA, W. R., SUH, D., KRIVICKAS, L. S., HUGHES, V. A., GOLSTEIN, R. & ROUBENOFF, R. (2000). Skeletal muscle fiber quality in older men and women. American Journal of Physiology - Cell Physiology 279, C611-618 [Medline]

GONZALEZ, E., MESSI, M. L. & DELBONO, O. (2000). The specific force of single intact extensor digitorum longus and soleus mouse muscle fibers declines with aging. Journal of Membrane Biology 178, 175-183 [Medline]

GOODMAN, C. A., PATTERSON, M. F., CELLINI, M. & STEPHENSON, G. M. M. (2000). MHC isoform expression and E-C coupling in mechanically skinned single muscle fibres of the rat. The Physiologist 43, 26

KADHIRESAN, V. A., HASSETT, C. A. & FAULKNER, J. A. (1996). Properties of single motor units in medial gastrocnemius muscles of adult and old rats. Journal of Physiology 493, 543-552 [Abstract]

LAMB, G. D. & STEPHENSON, D. G. (1990). Calcium release in skinned muscle fibers of the toad by transverse tubule depolarisation or by direct stimulation. Journal of Physiology 423, 495-517 [Abstract]

LAMB, G. D. & STEPHENSON, D. G. (1994). Effects of intracellular pH and [Mg²⁺] on excitation-contraction coupling in skeletal muscle fibres of the rat. Journal of Physiology 478, 331-339 [Abstract]

LARSSON, L. & EDSTROM, L. (1986). Effects of age on enzyme-histochemical fibre spectra and contractile properties of fast- and slow-twitch skeletal muscles in the rat. Journal of the Neurological Sciences 76, 69-89 [Medline]

LARSSON, L. & SALVIATI, G. (1989). Effects of age on calcium transport activity of sarcoplasmic reticulum in fast- and slow-twitch rat muscle fibres. Journal of Physiology 419, 253-264 [Abstract]

LARSSON, L., LI, X. P. & FRONTERA, W. R. (1997). Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells. American Journal of Physiology 273, C638-649

LARSSON, L. & RAMAMURTHY, B. (2000). Aging-related changes in skeletal muscle - Mechanisms and interventions. Drugs and Aging 17, 303-316 [Medline]

LI, X. & LARSSON, L. (1996). Maximum shortening velocity and myosin isoforms in single muscle fibers from young and old rats. American Journal of Physiology 270, C352-360 [Medline]

LYNCH, G. S. (2002). Novel therapies for sarcopenia: ameliorating age-related changes in skeletal muscle. Expert Opinion on Therapeutic Patents 12, 11-27

LYNCH, G. S., RODGERS, B. J. & WILLIAMS, D. A. (1993a). The effects of age and low-intensity endurance exercise on the contractile properties of single skinned fast- and slow-twitch skeletal muscle fibers. Growth, Development & Aging 57, 147-161

LYNCH, G. S., STEPHENSON, D. G. & WILLIAMS, D. A. (1993b). Muscle samples obtained by needle biopsy are suitable for studying single fibre contractile properties. Acta Physiologica Scandinavica 148, 27-35 [Medline]

LYNCH, G. S., HINKLE, R. T., CHAMBERLAIN, J. S., BROOKS, S. V. & FAULKNER, J. A. (2001). Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old. Journal of Physiology 535, 591-600 [Abstract/Full Text]

MARGRETH, A., DAMIANI, E. & BORTOLOSO, E. (1999). Sarcoplasmic reticulum in aged skeletal muscle. Acta Physiologica Scandinavica 167, 331-338 [Medline]

MELZER, W., HERRMANN-FRANK, A. & LUTTGAU, H. C. (1995). The role of Ca²⁺ ions in excitation-contraction coupling of skeletal muscle fibres. Biochimica et Biophysica Acta 1241, 59-116 [Medline]

NARAYANAN, N., JONES, D. L., XU, A. & YU, J. C. (1996). Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat. American Journal of Physiology 271, C1032-1040 [Medline]

OWEN, V. J., LAMB, G. D., STEPHENSON, D. G. & FRYER, M. W. (1997). Relationship between depolarization-induced force responses and Ca²⁺ content in skeletal muscle fibres of rat and toad. Journal of Physiology 498, 571-586 [Abstract]

PHILLIPS, S. K., WISEMAN, R. W., WOLEDGE, R. C. & KUSHMERICK, M. J. (1993). Neither changes in phosphorus metabolite levels nor myosin isoforms can explain the weakness in aged mouse muscle. Journal of Physiology 463, 157-167 [Abstract]

PLANT, D. R. & LYNCH, G. S. (2001). Rigor force responses of permeabilized fibres from fast and slow skeletal muscles of aged rats. Clinical and Experimental Pharmacology and Physiology 28, 779-781 [Medline]

PLANT, D. R., LYNCH, G. S. & WILLIAMS, D. A. (2002). H₂O₂ increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles. Journal of Physiology 539, 883-891 [Abstract/Full Text]

POSTERINO, G. S. & LAMB, G. D. (1996). Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibers of rat and toad. Journal of Physiology 496, 809-825 [Abstract]

POSTERINO, G. S., LAMB, G. D. & STEPHENSON, D. G. (2000). Twitch and tetanic force responses and longitudinal propagation of action potentials in skinned skeletal muscle fibres of the rat. Journal of Physiology 527, 131-137 [Abstract/Full Text]

REGANATHAN, M., MESSI, M. L. & DELBONO, O. (1997). Dihydropyridine receptor-ryanodine receptor uncoupling in aged skeletal muscle. Journal of Membrane Biology 157, 247-253 [Medline]

STEPHENSON, D. G., LAMB, G. D. & STEPHENSON, G. M. (1998). Events of the excitation-contraction-relaxation (E-C-R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue. Acta Physiologica Scandinavica 162, 229-245 [Medline]

SUGIURA, T., MATOBA, H., MIYATA, H., KAWAI, Y. & MURAKAMI, N. (1992). Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiologica Scandinavica 144, 419-423 [Medline]

THOMPSON, L. V. & BROWN, M. (1999). Age-related changes in contractile properties of single skeletal fibers from the soleus muscle. Journal of Applied Physiology 86, 881-886 [Abstract/Full Text]

WANG, Z.-M., MESSI, M. L. & DELBONO, O. (2000). L-Type Ca²⁺ channel charge movement and intracellular Ca²⁺ in skeletal muscle fibers from aging mice. Biophysical Journal 78, 1947-1954 [Abstract/Full Text]

WINEINGER, M. A., SHARMAN, R. B., STEVENSON, T. R., CARLSEN, R. C. & MCDONALD, R. B. (1995). Peripheral nerve and muscle function in the aging Fischer 344/Brown Norway rat. Growth Development & Aging 59, 107-119

Acknowledgements

This work was supported by the Australian Research Council and The Rebecca L. Cooper Medical Research Foundation.

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BORTOLOTTO, S. K., CELLINI, M., STEPHENSON, D. G. & STEPHENSON, G. M. (2000). MHC isoform composition and Ca²⁺- or Sr²⁺ -activation properties of rat skeletal muscle fibers. American Journal of Physiology - Cell Physiology 279, C1564-1577	[Medline]
BROOKS, S. V. & FAULKNER, J. A. (1988). Contractile properties of skeletal muscles from young, adult and aged mice. Journal of Physiology 404, 71-82	[Abstract]
BROOKS, S. V. & FAULKNER, J. A. (1994). Isometric, shortening, and lengthening contractions of muscle fiber segments from adult and old mice. American Journal of Physiology 267, C507-513	[Medline]
DAMIANI, E., LARSSON, L. & MARGRETH, A. (1996). Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle. Cell Calcium 19, 15-27	[Medline]
DANIELI-BETTO, D., BETTO, R., MEGIGHIAN, A., MIDRIO, M., SALVIATI, G. & LARSSON, L. (1995). Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiologica Scandinavica 154, 59-64	[Medline]
DEGENS, H., YU, F., LI, X. & LARSSON, L. (1998). Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres. Acta Physiologica Scandinavica 163, 33-40	[Medline]
DELBONO, O., O'ROURKE, K. S. & ETTINGER, W. H. (1995). Excitation-calcium release uncoupling in aged single human skeletal muscle fibers. Journal of Membrane Biology 148, 211-222	[Medline]
EDDINGER, T. J., CASSENS, R. G. & MOSS, R. L. (1986). Mechanical and histochemical characterization of skeletal muscles from senescent rats. American Journal of Physiology 251, C421-430	[Medline]
EINSIEDEL, L. J. & LUFF, A. R. (1992). Alterations in the contractile properties of motor units within the aging rat medial gastrocnemius. Journal of the Neurological Sciences 112, 170-177	[Medline]
FAULKNER, J. A., BROOKS, S. V. & ZERBA, E. (1995). Muscle atrophy and weakness with aging: contraction-induced injury as an underlying mechanism. Journal of Gerontology 50A, 124-129
FERRINGTON, D. A., JONES, T. E., QIN, Z., MILLER-SCHLYER, M., SQUIER, T. C. & BIGELOW, D. J. (1997). Decreased conformational stability of the sarcoplasmic reticulum in aged skeletal muscle. Biochimica et Biophysica Acta 1330, 233-247	[Medline]
FERRINGTON, D. A., KRAINEV, A. G. & BIGELOW, D. J. (1998). Altered turnover of calcium regulatory proteins of the sarcoplasmic reticulum in aged skeletal muscle. Journal of Biological Chemistry 273, 5885-5891	[Abstract/Full Text]
FRONTERA, W. R., SUH, D., KRIVICKAS, L. S., HUGHES, V. A., GOLSTEIN, R. & ROUBENOFF, R. (2000). Skeletal muscle fiber quality in older men and women. American Journal of Physiology - Cell Physiology 279, C611-618	[Medline]
GONZALEZ, E., MESSI, M. L. & DELBONO, O. (2000). The specific force of single intact extensor digitorum longus and soleus mouse muscle fibers declines with aging. Journal of Membrane Biology 178, 175-183	[Medline]
GOODMAN, C. A., PATTERSON, M. F., CELLINI, M. & STEPHENSON, G. M. M. (2000). MHC isoform expression and E-C coupling in mechanically skinned single muscle fibres of the rat. The Physiologist 43, 26
KADHIRESAN, V. A., HASSETT, C. A. & FAULKNER, J. A. (1996). Properties of single motor units in medial gastrocnemius muscles of adult and old rats. Journal of Physiology 493, 543-552	[Abstract]
LAMB, G. D. & STEPHENSON, D. G. (1990). Calcium release in skinned muscle fibers of the toad by transverse tubule depolarisation or by direct stimulation. Journal of Physiology 423, 495-517	[Abstract]
LAMB, G. D. & STEPHENSON, D. G. (1994). Effects of intracellular pH and [Mg²⁺] on excitation-contraction coupling in skeletal muscle fibres of the rat. Journal of Physiology 478, 331-339	[Abstract]
LARSSON, L. & EDSTROM, L. (1986). Effects of age on enzyme-histochemical fibre spectra and contractile properties of fast- and slow-twitch skeletal muscles in the rat. Journal of the Neurological Sciences 76, 69-89	[Medline]
LARSSON, L. & SALVIATI, G. (1989). Effects of age on calcium transport activity of sarcoplasmic reticulum in fast- and slow-twitch rat muscle fibres. Journal of Physiology 419, 253-264	[Abstract]
LARSSON, L., LI, X. P. & FRONTERA, W. R. (1997). Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells. American Journal of Physiology 273, C638-649
LARSSON, L. & RAMAMURTHY, B. (2000). Aging-related changes in skeletal muscle - Mechanisms and interventions. Drugs and Aging 17, 303-316	[Medline]
LI, X. & LARSSON, L. (1996). Maximum shortening velocity and myosin isoforms in single muscle fibers from young and old rats. American Journal of Physiology 270, C352-360	[Medline]
LYNCH, G. S. (2002). Novel therapies for sarcopenia: ameliorating age-related changes in skeletal muscle. Expert Opinion on Therapeutic Patents 12, 11-27
LYNCH, G. S., RODGERS, B. J. & WILLIAMS, D. A. (1993a). The effects of age and low-intensity endurance exercise on the contractile properties of single skinned fast- and slow-twitch skeletal muscle fibers. Growth, Development & Aging 57, 147-161
LYNCH, G. S., STEPHENSON, D. G. & WILLIAMS, D. A. (1993b). Muscle samples obtained by needle biopsy are suitable for studying single fibre contractile properties. Acta Physiologica Scandinavica 148, 27-35	[Medline]
LYNCH, G. S., HINKLE, R. T., CHAMBERLAIN, J. S., BROOKS, S. V. & FAULKNER, J. A. (2001). Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old. Journal of Physiology 535, 591-600	[Abstract/Full Text]
MARGRETH, A., DAMIANI, E. & BORTOLOSO, E. (1999). Sarcoplasmic reticulum in aged skeletal muscle. Acta Physiologica Scandinavica 167, 331-338	[Medline]
MELZER, W., HERRMANN-FRANK, A. & LUTTGAU, H. C. (1995). The role of Ca²⁺ ions in excitation-contraction coupling of skeletal muscle fibres. Biochimica et Biophysica Acta 1241, 59-116	[Medline]
NARAYANAN, N., JONES, D. L., XU, A. & YU, J. C. (1996). Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat. American Journal of Physiology 271, C1032-1040	[Medline]
OWEN, V. J., LAMB, G. D., STEPHENSON, D. G. & FRYER, M. W. (1997). Relationship between depolarization-induced force responses and Ca²⁺ content in skeletal muscle fibres of rat and toad. Journal of Physiology 498, 571-586	[Abstract]
PHILLIPS, S. K., WISEMAN, R. W., WOLEDGE, R. C. & KUSHMERICK, M. J. (1993). Neither changes in phosphorus metabolite levels nor myosin isoforms can explain the weakness in aged mouse muscle. Journal of Physiology 463, 157-167	[Abstract]
PLANT, D. R. & LYNCH, G. S. (2001). Rigor force responses of permeabilized fibres from fast and slow skeletal muscles of aged rats. Clinical and Experimental Pharmacology and Physiology 28, 779-781	[Medline]
PLANT, D. R., LYNCH, G. S. & WILLIAMS, D. A. (2002). H₂O₂ increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles. Journal of Physiology 539, 883-891	[Abstract/Full Text]
POSTERINO, G. S. & LAMB, G. D. (1996). Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibers of rat and toad. Journal of Physiology 496, 809-825	[Abstract]
POSTERINO, G. S., LAMB, G. D. & STEPHENSON, D. G. (2000). Twitch and tetanic force responses and longitudinal propagation of action potentials in skinned skeletal muscle fibres of the rat. Journal of Physiology 527, 131-137	[Abstract/Full Text]
REGANATHAN, M., MESSI, M. L. & DELBONO, O. (1997). Dihydropyridine receptor-ryanodine receptor uncoupling in aged skeletal muscle. Journal of Membrane Biology 157, 247-253	[Medline]
STEPHENSON, D. G., LAMB, G. D. & STEPHENSON, G. M. (1998). Events of the excitation-contraction-relaxation (E-C-R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue. Acta Physiologica Scandinavica 162, 229-245	[Medline]
SUGIURA, T., MATOBA, H., MIYATA, H., KAWAI, Y. & MURAKAMI, N. (1992). Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiologica Scandinavica 144, 419-423	[Medline]
THOMPSON, L. V. & BROWN, M. (1999). Age-related changes in contractile properties of single skeletal fibers from the soleus muscle. Journal of Applied Physiology 86, 881-886	[Abstract/Full Text]
WANG, Z.-M., MESSI, M. L. & DELBONO, O. (2000). L-Type Ca²⁺ channel charge movement and intracellular Ca²⁺ in skeletal muscle fibers from aging mice. Biophysical Journal 78, 1947-1954	[Abstract/Full Text]
WINEINGER, M. A., SHARMAN, R. B., STEVENSON, T. R., CARLSEN, R. C. & MCDONALD, R. B. (1995). Peripheral nerve and muscle function in the aging Fischer 344/Brown Norway rat. Growth Development & Aging 59, 107-119

				B. J. Gurd, S. J. Peters, G. J. F. Heigenhauser, P. J. LeBlanc, T. J. Doherty, D. H. Paterson, and J. M. Kowalchuk O2 uptake kinetics, pyruvate dehydrogenase activity, and muscle deoxygenation in young and older adults during the transition to moderate-intensity exercise Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R577 - R584. [Abstract] [Full Text] [PDF]

				J. D. Schertzer, C. v. d. Poel, T. Shavlakadze, M. D. Grounds, and G. S. Lynch Muscle-specific overexpression of IGF-I improves E-C coupling in skeletal muscle fibers from dystrophic mdx mice Am J Physiol Cell Physiol, January 1, 2008; 294(1): C161 - C168. [Abstract] [Full Text] [PDF]

				S. Baudry, M. Klass, and J. Duchateau Postactivation potentiation influences differently the nonlinear summation of contractions in young and elderly adults J Appl Physiol, April 1, 2005; 98(4): 1243 - 1250. [Abstract] [Full Text] [PDF]

				J. D. Schertzer, D. R. Plant, J. G. Ryall, F. Beitzel, N. Stupka, and G. S. Lynch {beta}2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E526 - E533. [Abstract] [Full Text] [PDF]

				P. Gregorevic, D. R. Plant, N. Stupka, and G. S. Lynch Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration J. Physiol., July 15, 2004; 558(2): 549 - 560. [Abstract] [Full Text] [PDF]

				B. K. Barry and R. G. Carson The Consequences of Resistance Training for Movement Control in Older Adults J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2004; 59(7): M730 - M754. [Abstract] [Full Text] [PDF]

				B. L. Allman and C. L. Rice An age-related shift in the force-frequency relationship affects quadriceps fatigability in old adults J Appl Physiol, March 1, 2004; 96(3): 1026 - 1032. [Abstract] [Full Text] [PDF]

				J. G. Ryall, D. R. Plant, P. Gregorevic, M. N. Sillence, and G. S. Lynch {beta}2-Agonist administration reverses muscle wasting and improves muscle function in aged rats J. Physiol., February 15, 2004; 555(1): 175 - 188. [Abstract] [Full Text] [PDF]

				D. R. Plant and G. S. Lynch Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice Am J Physiol Cell Physiol, September 1, 2003; 285(3): C522 - C528. [Abstract] [Full Text] [PDF]