Muscles that are stretched during contraction (eccentric

contractions) are susceptible to damage (Hough, 1902),

particularly when the eccentric exercise is prolonged and

unaccustomed. For example, when walking down a

mountain, the quadriceps group of muscles is used to

deaccelerate the body and undergo eccentric contraction.

Characteristically the muscles involved are weak but pain-

free immediately after the exercise, but in the subsequent

days the muscle becomes stiff, tender and may become

even weaker. This latter collection of symptoms is called

delayed onset muscle soreness (DOMS). Full recovery may

require several weeks and there is a pronounced training

effect; thus if the same exercise is repeated the symptoms

are substantially reduced (Balnave & Thompson, 1993).

The mechanisms involved in this damage are complex and

multifactorial (for recent reviews see Morgan & Allen,

1999; Proske & Morgan, 2001; Warren et al. 2001).

Early ultrastructural studies established that there are

characteristic changes in the sarcomere patterns including

localized regions of overstretched sarcomeres and irregular

and distorted Z lines (Fridén et al. 1981). Morgan

(1990) proposed a mechanism to explain the sarcomere

inhomogeneities on the basis of the mechanical properties

of eccentrically contracting muscle. He pointed out that

when the force on a muscle exceeded about 1.6 Po (Po is the

maximum isometric force), the stretching velocity increased

uncontrollably (Katz, 1939). Furthermore, sarcomeres

on the descending limb of the force–length curve are

intrinsically unstable (Huxley, 1980); this arises when a

sarcomere on the descending limb becomes longer than its

neighbour; it is then weaker than the neighbour and

therefore inclined to stretch further. As a consequence

during stretch on the descending limb of the force–length

curve, individual half-sarcomeres can stretch suddenly

and dramatically (the popping sarcomere hypothesis) and

much of the increase in muscle length can occur in a small

number of abnormally stretched sarcomeres (Brown &

Hill, 1991; Talbot & Morgan, 1996). Most of these

overstretched sarcomeres reinterdigitate when the muscle

relaxes (Talbot & Morgan, 1996), but during repeated

eccentric contractions it would be expected that the

regions of overstretched sarcomeres can extend laterally

and longitudinally through the muscle leading to the

characteristic structural changes (Allen, 2001).

Development of T-tubular vacuoles in eccentrically damagedmouse muscle fibresElla W. Yeung *, Christopher D. Balnave †, Heather J. Ballard‡, J.-P. Bourreau ‡ and David G. Allen †

† Institute for Biomedical Research and Department of Physiology, University of Sydney F13, NSW 2006, Australia, *Department of RehabilitationSciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong and ‡ Department of Physiology, University of Hong Kong, Li Shu FanBuilding, 5 Sassoon Road, Hong Kong

Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol

of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres

showed a reduced force, a shift in the peak of the force–length relation and a steepening of the

force–frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular

space enabled the T-tubular system to be visualized. In unstimulated fibres and fibres subjected to

10 isometric tetani, the T-tubules were clearly delineated. Sulforhodamine B diffused out of the

T-tubules with a half-time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the

T-tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of

eccentrically damaged fibres extremely slowly with a half-time of 6.3 ± 2.4 min and diffused out of

the T-tubules with a half-time of 39 ± 4 s. Vacuole production was eliminated by application of

1 m ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles

appeared over a period of 1 h and were more numerous and more widely distributed than in the

absence of ouabain. We propose that T-tubules are liable to rupture during eccentric contraction

probably because of the relative movement associated with the inhomogeneity of sarcomere

lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by

the sodium pump, the volume load of Na+ and water exceeds the capacity of the T-tubules and

causes vacuole production. The damage to the T-tubules may underlie a number of the functional

changes that occur in eccentrically damaged muscle fibres.

(Received 14 November 2001; accepted after revision 17 January 2002)

Corresponding author D. G. Allen: Institute for Biomedical Research and Department of Physiology, University of Sydney F13,NSW 2006, Australia. Email: davida@physiol.usyd.edu.au

Journal of Physiology (2002), 540.2, pp. 581–592 DOI: 10.1113/jphysiol.2001.013839

© The Physiological Society 2002 www.jphysiol.org

It is less clear how these early structural changes lead to the

reductions of force and to the inflammation and even cell

death that are apparent after several days (McCully &

Faulkner, 1985). Sarcomeres are connected in series in a

muscle so that damage to some sarcomeres would not

necessarily affect the maximum force. Instead one might

predict that they would act as increased compliance and

cause the force–length curve to shift to longer lengths as

has been observed (Katz, 1939; Talbot & Morgan, 1998).

However even when muscles are stretched to the peak of

the new force–length relation, force is often reduced.

Warren et al. (1993) suggested that damage to excitation–

contraction (E–C) coupling occurred in eccentrically

damaged mammalian muscle and this was subsequently

confirmed by direct measurements of intracellular calcium

(Balnave & Allen, 1995; Ingalls et al. 1998). It would be

expected that T-tubules and sarcoplasmic reticulum (SR)

would be susceptible to damage in the overstretched

regions and this may be the basis for the E–C coupling

damage. It is already known that T-tubular vacuoles can

occur in a range of muscle interventions involving osmotic

loads (Krolenko et al. 1998; Khan et al. 2000; Lännergren etal. 2000). Of particular interest is a report that gross

structural damage to a muscle by cutting it in half leads to

rapid and profuse development of vacuoles associated

with the T-tubules near the site of damage (Casademont etal. 1988). Very recently it has been shown in electron

microscopy (EM) studies that the T-tubules are abnormal

in eccentrically stretched muscles (Takekura et al. 2001).

However, there are no previous reports of vacuole

development in association with eccentric muscle damage.

There is also considerable evidence of surface membrane

damage following eccentric contractions. For instance,

some studies have shown reductions in the membrane

potential immediately after eccentric damage (McBride etal. 2000), although others have not (Warren et al. 1993).

We have recently shown that pH regulation is abnormal

immediately after eccentric damage; since transporters in

the surface membrane, such as the Na+–H+ exchanger and

the lactate transporter, dominate pH regulation, this

suggests some loss of membrane function (Yeung et al.2002). Substantial increases in the plasma protein levels of

soluble muscle proteins such as creatine kinase may be

observed several days after eccentric damage (Jones et al.1986), although this may be more indicative of the start of

cell breakdown than early membrane damage.

In this study, T-tubule morphology and function were

compared between fibres which had not contracted or had

undergone only isometric contractions, and fibres which

had undergone eccentric contractions. The morphology of

the T-tubules was examined using confocal microscopy

and an extracellular fluorescent dye, sulforhodamine B.

In addition the accessibility of the T-tubules from the

extracellular space was evaluated from the rate of washout

of sulforhodamine B from the T-tubules. Finally, the role

of the Na+,K+-ATPase in the development of T-tubule

vacuoles was evaluated by performing eccentric contractions

in the presence of ouabain.

METHODSSingle fibre dissection and mountingAdult, male mice were killed by rapid neck disarticulation andsingle muscle fibres were dissected from the flexor brevis musclesas previously described (Lännergren & Westerblad, 1987). Theseprocedures were approved by the Animal Ethical Committee ofthe University of Sydney. The isolated fibres were mountedbetween an Akers AE801 force transducer (SensoNor, Horten,Norway) and the arm of a motor (Model 300H, CambridgeTechnology, Cambridge, MA, USA). The motor allowed knownlength changes to be imposed on the muscle fibre. The fibres werestimulated with platinum-plate electrodes using pulses of 0.5 msduration at an intensity of ~1.2 w threshold. All tetanic contractionswere 400 ms in duration and the standard stimulation frequencywas 100 Hz.

SolutionsThe dissection was performed in a solution of the followingcomposition (m): 136.5 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4NaH2PO4 and 11.9 NaHCO3 (pH 8.0). During the experiment, thefibres were superfused at 2.2 ml min_1 in the following standardsolution (m): 121 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4,24 NaHCO3 and 5.5 glucose, with 0.2 % (v/v) fetal calf serum(Gibco). The solution was bubbled with 95 % O2–5 % CO2 (pH 7.4).In some experiments, 1 m ouabain (Sigma-Aldrich) was addedto inhibit the activity of Na+,K+-ATPase. Ouabain was dissolved indimethyl sulfoxide (DMSO) to give a concentration of 100 m.This stock solution was stored at a temperature of _20 °C,protected from light, and diluted into the standard solutionimmediately before use. All experiments were performed at roomtemperature (22–24 °C).

Eccentric contraction protocolThe experimental protocol used was similar to those describedpreviously (Balnave & Allen, 1995; Yeung et al. 2002). The basicprotocol (eccentric series) involved superimposing a stretch oneach of a series of 10 tetani. It is important to distinguish betweenthe force deficit caused by fatigue and that associated witheccentric contractions (Morgan & Allen, 1999) and for this reasonan isometric (control) series of tetani was also performed. We alsowished to establish that the force–frequency behaviour and theforce–length properties of the fibre were unaffected by the controlseries, but modified by the eccentric series. Thus the fullexperimental protocol was as follows. (i) The force–lengthrelation was established using tetani separated by 1 min andchanging length in 40 mm steps. The fibre was adjusted to Lo (thelength giving maximum force). (ii) Tetani at 10, 20, 30, 50, 70 and100 Hz were produced with 1 min of rest between each tetanus(force–frequency relation). (iii) Ten isometric tetani at 4 sintervals were given (the control series). (iv) After 10 min, theforce–length relation was redetermined. (v) The force–frequencyrelation was redetermined. (vi) Ten eccentric tetani were given at4 s intervals. The length change was either 25 % or 40 % of Lo. Thestretch was imposed over 100 ms starting after 200 ms of thetetanus. The fibre was shortened back to the Lo over 100 msstarting 200 ms after the end of the tetanus. (vii) After 10 min theforce–length relation was re-established and the fibre length

E. W. Yeung and others582 J. Physiol. 540.2

kept at the original Lo. (viii) The force–frequency relation wasredetermined. (ix) The fibre was then stretched to the newoptimum length (post eccentric Lo). (x) The force–frequencyrelation was redetermined. (xi) Finally, the fibre was removedfrom the motor and positioned on the coverslip of a new chamberused for confocal microscopy.

Confocal microscopyTo visualize the morphology of the T-tubular system, the fibre wasexamined with an inverted confocal microscope (Leica TCS SL,Heidelberg, Germany), using a w 63, NA 1.2 water immersionobjective lens. After the contraction protocol, the muscle fibre wastransferred into a perfusion chamber. The two tendons of the fibrewere fixed to the coverslip with silicone grease. The perfusionchamber was narrow so that the washout of extracellular spaceafter a solution change was reasonably fast (see Fig. 4A). T-tubularmorphology was examined by adding 0.5 m sulforhodamine B(Molecular Probes, OR, USA) to the perfusate which diffusedinto the T-system (Endo, 1966; Lännergren et al. 2000).Sulforhodamine B fluorescence was excited using the 543 nm lineof a helium–neon laser operated at 50 % maximum power andemitted light was collected between 570 and 610 nm. Theresolution of the microscope in this configuration was estimatedusing 170 nm diameter green fluorescent beads (PS-Speck,Molecular Probes). The half-maximal width of the resultingfluorescence signal was 0.35 mm in the horizontal direction and0.9 mm in the vertical direction.

Confocal microscopy was performed on four unstimulated fibresand on three fibres that underwent only the control series ofisometric tetani. Seven fibres underwent the full protocoldescribed above. The entire length of the muscle fibre andmultiple depths were examined for morphological alterationsand/or presence of vacuolation, but because of dead fibres andconnective tissue the ends of the fibre were harder to visualize.Images are presented in two formats. In some figures, e.g. Fig. 2,sulforhodamine B was present in the extracellular space andproduced a very large fluorescent signal that caused the detectorto saturate (indicated by a blue colour). Even in a confocalmicroscope some fractions of the fluorescence above and belowthe fibre contributed to fibre fluorescence and this reduced theresolution of fluorescent structures within the fibre. For thisreason we also show figures, e.g. Fig. 5, collected 1 min afterextracellular sulforhodamine B had been washed out of theextracellular space. Images were scanned at a resolution of512 w 512 pixels.

In addition to studying the morphological alterations of theT-tubules, a second objective of our study was to examine thefunction of the T-tubules following eccentric contractions.Water-soluble dyes, such as sulforhodamine B (MW 559), diffusein and out of the T-tubular system with a time course in the rangeof 4–10 s (Endo, 1966). It is thus possible to examine theaccessibility of the T-tubules by measuring the rate of exit of thefluorescent dye. In order to achieve this, fibres were superfusedwith the standard solution containing sulforhodamine B for15–20 min before observations. The dye-containing solution wasthen removed and washed out with standard solution for at least20 min. Confocal images of the fibres were obtained every 10 s forthe first minute, every minute for the next 4 min and then at 5 minintervals over 20 min. The rate of loss of sulforhodamine B fromthe T-system was estimated from the decline of fluorescence,which was analysed using NIH Image (Scion Corporation, MD,USA). The average fluorescence intensity was determined in a

window of roughly half the diameter of the fibre placed overa representative region of the fibre. Before exposure tosulforhodamine B the fibres exhibited no significant fluorescence.Contributions to the fluorescence from dye outside the fibreprofile were judged to be small because the intensity of vacuoleschanged little as the extracellular dye was washed away (comparevacuoles in Fig. 3Ba and b). This procedure was repeated forseveral regions of each fibre to ensure that fluorescence intensitymeasurements were representative. When vacuoles were present amuch smaller box comparable to the size of a few vacuoles wasused and placed over the same group of vacuoles in each of a seriesof images.

Na+,K+-ATPase inhibitorWe further examined the morphology of the T-tubules aftereccentric contractions (n = 3) during exposure to 1 m ouabain,an inhibitor of Na+,K+-ATPase. The fibre was exposed to ouabainfrom 15 min before the eccentric protocol and the ouabainremained present throughout the eccentric protocol and duringthe examination of the fibre on the confocal microscope.Subsequently ouabain was washed out using standard solution forthe next 2 h and we again examined the fibre on the confocalmicroscope. Two fibres were treated in an identical manner exceptthat the experimental protocol involved isometric tetani only.

StatisticsData are quoted as means ± standard error of means (...) withthe number of experiments denoted as n. Statistical significancewas determined with Student’s paired t test for force recordingsand optimal length. The unpaired t test was used to compare therate of dye exit between fibres following isometric and eccentrictetani. The significance level was set at P < 0.05.

RESULTSEffect of the eccentric series on mechanicalperformanceThe force developed by muscle fibres following the eccentric

series (40 % stretch, n = 7) showed a large reduction to

34 ± 4 % of control (P < 0.0001), measured in a 100 Hz

isometric tetanus 10 min after the eccentric series and at

the original Lo (Fig. 1A). Force was not significantly reduced

after the isometric series (Fig. 1A). As a consequence of

eccentric contractions, there was a shift in optimum length

for force to longer muscle lengths which amounted to an

increase of Lo by a factor of 1.24 ± 0.02. There was no

significant shift in Lo after the isometric series (dashed line

in Fig. 1A). This shift in Lo following eccentric contraction

confirms earlier studies (Katz, 1939; Talbot & Morgan,

1998; Yeung et al. 2002). If the muscle was stretched to the

post-eccentric Lo the decline in force was reduced and the

developed force was 47 ± 3 % of the control conditions.

Figure 1B illustrates the force–frequency relations of the

fibres normalized to 100 Hz stimulation under each

condition. As previously demonstrated, eccentrically

damaged muscles have a steeper force–frequency relation

(Jones et al. 1989; Balnave & Allen, 1995; Yeung et al.2002). It can be seen that the pattern of force reduction was

similar regardless of whether the fibre was at the original Lo

or the post-eccentric Lo. As expected, the isometric series

Vacuoles in eccentric muscle damageJ. Physiol. 540.2 583

had no significant effect on the force–frequency relation

(dashed line in Fig. 1B). These data establish that the

muscle fibres show the previously established mechanical

criteria of eccentric muscle damage and that fatigue due to

the 10 isometric tetani was not a significant factor.

Confocal images of dye-loaded muscle fibresUnder confocal microscopy, control experiments with no

stimulation (n = 4) and those with isometric tetani series

(n = 3) show regular, well-aligned fluorescent lines running

transversely across the fibre (Fig. 2A). Only in the best

resolution images (e.g. Figs 2A and 3Aa) were we able to

partly visualize two T-tubules per sarcomere as has

previously been established in mammalian muscles

(Lännergren et al. 2000). This inability to resolve two

T-tubules per sarcomere probably results from the difficulty

of dissecting the fibres sufficiently cleanly that the fibre lies

very close to the coverslip (see discussion in Lännergren etal. 2000). In occasional images, longitudinal connections

were seen spanning two to five transverse lines.

The most striking feature after 40 % stretch was the

presence of vacuoles in six of the seven fibres examined

(Fig. 2B and C). These vacuoles were present at our earliest

observation, which was 10–20 min after the eccentric

contractions. Vacuolation did not appear throughout the

whole preparation, but was confined to a focal region of

the fibre, so it is possible that they were present in the fibre

in which we failed to identify vacuoles, but in a very

restricted region. In the seven fibres, we counted the

number of vacuoles in a 50 mm length of the fibre and a

single plane of focus in the region where vacuoles were

most prevalent. The number of vacuoles varied between 32

and 0 (11 ± 4). Because the vacuoles were filled with dye

from the extracellular space, they must have had access to

the extracellular space presumably through the T-system.

The vacuoles varied in size and shape with some ovoid in

cross-section running parallel to the length of the muscle

fibre (Fig. 2B). In other experiments, the vacuoles were

larger (up to 2 mm) and roughly spherical in shape

(Fig. 2C). Sulforhodamine B was often applied several

times and the vacuoles persisted for more than 2 h. They

showed no sign of disappearing in contrast to vacuoles

after fatiguing stimulation, which had largely resolved after

1 h (Lännergren et al. 2000). In the eccentrically damaged

fibres, the T-tubules formed a wavy line across the fibre,

the edges of the T-tubules were less clearly defined, and

in some regions increased numbers of longitudinal

connections were apparent. These features were also

present in the one fibre in which no vacuoles could be

located. There was no obvious correlation between of the

presence or number of vacuoles and the magnitude of the

force deficit; for instance the fibre in which no vacuoles were

located had a roughly similar decline in force. In an attempt

to examine whether vacuoles could develop during the

eccentric stretches, but then seal over (Fraser et al. 1998),

we performed the eccentric protocol in two fibres in the

presence of sulforhodamine B. In these experiments the

vacuoles appeared similar to other fibres; furthermore the

dye was gradually lost from all vacuoles when extracellular

dye was removed. Thus we found no evidence of sealed

vacuoles that developed during the eccentric contraction.

Milder stretches of 25 % of Lo did not lead to the

production of vacuoles though force was reduced to

65 ± 10 % of control (n = 2).

E. W. Yeung and others584 J. Physiol. 540.2

Figure 1. Changes in mechanical properties of musclefollowing eccentric contractionsA, the length–force relations at various stages of the experimentalprotocol (n = 8). 0, control; 9, after the isometric series; and 1,after the eccentric series. The curves fitted to each set of data pointsare Gaussian distributions. B, force–frequency relations at variousstages in the protocol. Each plot shows relative force normalized to100 Hz force vs. different frequencies of stimulation (n = 8). 9,control; 0, post-isometric contractions; 1, post eccentriccontractions at the original Lo; and 6, after adjusting to newoptimal length. * Statistically significant difference from controlsituation (P < 0.05).

Rate of fluorescence washout from muscle fibresThe time course of sulforhodamine B washout gives an

estimate of the accessibility of the T-system to the

extracellular space (Endo, 1966). Figure 3 illustrates

representative images obtained during such experiments.

Figure 3Aa is a control image in the presence of

sulforhodamine B in a fibre that had the isometric series of

contractions only. Figure 3Ab and c show images after 1

and 20 min washout. Note that the major part of the dye

had left the T-tubules at 1 min, but faint T-tubules of

roughly similar intensity are present in the images both at

1 min and 20 min. Figure 3Ba–d shows an equivalent

series from an eccentrically damaged fibre. Note the

prominent vacuoles in the image in the presence of

sulforhodamine B (Fig. 3Ba) and that the T-tubules are

less clear than in the equivalent control image. After a

1 min washout, the intensity of dye in the vacuoles was

little changed, but most dye had left the T-tubules. After

20 min and even 2 h, vacuoles still contained some dye and

faintly stained T-tubules were still detectable.

These issues are explored more quantitatively in the graphs

shown in Fig. 4. In Fig. 4A the filled circles show the

washout of the extracellular space, which was reduced to

~5 % at 1 min. The open circles show the rate of decline of

fluorescence in T-tubules in fibres following isometric

tetani, which was roughly exponential with a half-time of

18 ± 1 s. As expected from the work of Endo (1966), the

main decline lags behind the extracellular space by 10–20 s

only, but there is an irreducible tail of fluorescence, which

is about 20 % of the maximum and shows only a minor

decline over 20 min. We are uncertain of the origin of this

fluorescence, but the most likely possibility is that of a

small fraction the dye, possibly caused by a small degree of

lipid solubility, remains bound to the membranes of the

T-system and dissociates very slowly. Following eccentric

contractions, the T-system (vacuoles + T-tubules) (n = 7)

showed significant slowing of washout rate when compared

with those fibres after isometric contractions (P < 0.01).

Note that in these data, which include both T-tubules and

vacuoles, there seems to be two phases in the decline, with

a rapid phase over the first minute and then a slower phase

over at least 20 min.

To determine if the efflux rate of the dye differs from

various structures, we first examined the decline of

fluorescence in vacuoles (Fig. 4B, filled circles, n = 5). It is

clear that the efflux rate is very slow and seems to follow

a roughly exponential time course with a half-time of

6.3 ± 2.4 min. We also examined the efflux from T-tubules

of fibres that had contracted eccentrically, but did not

show vacuoles in the field of view (Fig. 4B, filled triangles,

n = 4). These four fibres included the one eccentrically

damaged fibre that did not show vacuoles, two fibres from

experiments with 25 % stretch that also failed to exhibit

vacuoles, and one fibre that showed vacuoles, but the

measurements were made from an area that did not

exhibit vacuoles. None of these data were significantly

different from each other and they have been combined

in Fig. 4B. The dye leaves the eccentrically damaged

T-tubules much faster than vacuoles and the decline is

roughly exponential with a half-time of 39 ± 4 s. Finally, in

Vacuoles in eccentric muscle damageJ. Physiol. 540.2 585

Figure 2. Confocal images of the muscle fibres in thepresence of extracellular sulforhodamine BA, confocal image of a fibre following isometric contractionshowing well-aligned fluorescent patterns of the T-tubules. In allthe confocal images shown, increasing fluorescence intensity isindicated by the following colour order: black, dark red, yellow,white. Saturation of the detector occurs with dye outside the fibreand is indicated by the blue colour. B and C, fibre that had beenstretched by 40 % Lo showing vacuolation of the T-system. B showsan example of the ovoid-shaped vacuoles appearing longitudinallyover the fibre and C shows roughly spherical vacuoles locatedrandomly throughout the fibre. Scale bar, 10 mm.

E. W. Yeung and others586 J. Physiol. 540.2

Figure 3. Confocal images showing washout of sulforhodamine B Aa, fibre that had been subjected to 10 isometric tetani and was being perfused with sulforhodamine B. Aband c are after 1 and 20 min washout with standard solution. After the removal of the sulforhodamine B, thefluorescence density of the fibre reduces rapidly in the first minute. Ba, fibre that has been subjected toeccentric tetani of 40 % Lo in the presence of extracellular sulforhodamine B. Bright fluorescent vacuolesappeared longitudinally along the muscle fibre. Bb, c and d correspond to the time course of washout periodof 1 min, 20 min and 2 h, respectively. Scale bars, 10 mm.

Fig. 4B the open circles show the decline of fluorescence

from T-tubules of isometrically contracted fibres, which is

clearly faster (same data as Fig. 4A).

These data also show that the steady-state T-tubule-

fluorescence is greater in eccentrically stretched fibres than

in control fibres. Possible explanations will be considered

in the Discussion.

Effects of Na+,K+-ATPase inhibitor on the formationof vacuolesCasademont et al. (1988) showed that gross damage to

muscle fibres caused the production of vacuoles which

could be inhibited with 1 m ouabain. We therefore

sought to determine whether the production of vacuoles

following eccentric damage was also inhibited by ouabain.

In three fibres the tetanic contraction and the force–

frequency relation were not significantly affected by the

addition of 1 m ouabain and the decline of force following

eccentric contractions in the presence of ouabain was to

28 ± 8 % of the control (not significantly different from

the eccentric protocol in the absence of ouabain). In these

three fibres exposed to ouabain throughout the eccentric

protocol, no vacuoles could be detected for a period of

30 min after the eccentric protocol (Fig. 5Ba). This

number of vacuoles is significantly smaller than in the

seven fibres studied in the absence of ouabain (Mann-

Whitney rank sum test, P < 0.05). The ouabain was then

washed out and after 45–60 min small vacuoles began

to appear (Fig. 5Bb and c). The extent of vacuolation

progressively increased over the next 60 min (Fig. 5Bd).

Bright fluorescent vacuoles appeared in large numbers and

mostly formed along the longitudinal axis in all three fibres

observed. In one experiment, ouabain was reapplied in an

attempt to examine whether the vacuoles were reversible.

However in this fibre vacuoles persisted for another

60 min of observation, suggesting that once formed, the

vacuoles were stable and did not require a continuous

influx of Na+ and accompanying H2O for their maintenance.

Experiments on two fibres subjected to isometric

contractions did not show any vacuoles either in the

presence of ouabain (Fig. 5Aa) or after the removal of

ouabain (Fig. 5Ab)).

DISCUSSIONIn this study we used an established model of eccentric

damage to single mouse muscle fibres (Balnave & Allen,

1995; Yeung et al. 2002). We used a stretch of 40 % Lo,

which is within the physiological range (Brooks et al.1995). The fibres remained excitable and produced force,

but showed three characteristic signs of eccentric damage:

(i) reduced force, (ii) a shift in the peak of the force–length

relation (Wood et al. 1993; Talbot & Morgan, 1998; Yeung

et al. 2002) and (iii) reduced force at low frequencies of

stimulation compared to high frequencies (Jones et al.1989; Balnave & Allen, 1995; Yeung et al. 2002). The

design of the study established that these changes were a

consequence of the eccentric component of the contraction

and not simply the fatigue that might result from repeated

isometric tetani. The main novel findings of this study

were that eccentric muscle damage caused the formation

of vacuoles connected to the T-tubules and that the

diffusion of molecules into and out of both the T-system

and the vacuoles was slowed. We also showed that the

formation of vacuoles was dependent on the activity of

the sodium pump. These new results provide a more

functional understanding of the damage to T-tubules and

extend the structural findings of a recent EM study

(Takekura et al. 2001).

Vacuoles in eccentric muscle damageJ. Physiol. 540.2 587

Figure 4. Fluorescence intensity during dye washout A, measurements from large areas of the fibre. 9, eccentric series(n = 7); 1, isometric series (n = 4); 0, background fluorescencetracing of the extracellular dye (n = 11). Error bars represent ...values. B, the fluorescence intensity of vacuoles in eccentricallydamaged fibres (0) (n = 5), T-tubules from eccentrically damagedfibres without vacuoles and T-tubules from 25 % eccentricstretches (8) (n = 4) and T-tubules from isometric-only series (1)(n = 4).

E. W. Yeung and others588 J. Physiol. 540.2

Figure 5. Confocal images of muscle fibres in ouabain experimentsA, fluorescence images following isometric (control) series. Aa, 20 min after isometric contractions, ouabainpresent throughout. Ab, after ouabain had been washed out for 60 min. No vacuoles were observed at anyperiod. B, fluorescent images following the eccentric series. Ba, 20 min after the eccentric series, ouabainpresent throughout, no vacuoles observed. Bb, after 40 min washout, hints of vacuoles began to appear. Bcand d correspond to 60 and 80 min after the removal of ouabain. Extensive vacuoles appear over this period.Scale bars, 10 mm. All images in this figure were collected 1 min after washout of sulforhodamine B, whichwas present for 10–20 min before washout.

The study by Takekura et al. (2001) involved rats that

performed a series of downhill runs over 90 min. The

animals were killed at intervals up to 10 days after the

termination of exercise. Identified fast or slow fibres were

studied with high voltage EM and a staining technique that

identified T-tubules or SR. Four changes in the T-system

were identified: (i) increase in the longitudinal elements of

the T-tubules, (ii) changes in the organization of triads,

(iii) the development of calveolar clusters and (iv) the

appearance of multiple connections between two and three

T-tubules and three and four terminal cisternae (pentads

and heptads). The increase in longitudinal T-tubules was

greatest at 3 days and, although we used objective methods

to search for them in our fibres, we could not detect

increased longitudinal T-tubules in our time frame

(0.5–2 h). The calveolar clusters are interesting and were

apparent immediately after exercise but only in slow fibres.

Nevertheless it seems possible that some vacuoles and

caveolar clusters might be similar or identical structures.

Mechanism of production of vacuoles in skeletalmuscleVacuoles have previously been described in a range of

situations including glycerol removal (Krolenko et al.1998; Khan et al. 2000), muscle fatigue (Lännergren etal. 2000) and gross membrane damage (Casademont et al.1988). An extensive review of the mechanism of production

and the significance of skeletal muscle vacuoles has recently

been published (Krolenko & Lucy, 2001). However vacuoles

have not previously been described as part of eccentric

damage, although with the benefit of hindsight, structures

that might be vacuoles are visible in the study by Warrren etal. (1995) (see their Fig. 6).

Lännergren et al. (2000) showed that vacuoles could

develop during the recovery from repeated isometric

tetani in amphibian muscle fibres. Thus it is important to

establish that the vacuoles we observed in the present

experiments were a consequence of the eccentric damage

and not caused by some aspect of the repeated tetani.

Firstly, like Lännergren et al. (2000), we did not observe

vacuoles in mouse fibres after repeated isometric tetani.

Secondly, vacuoles were localized following eccentric

damage, whereas they were widespread in fatigued

amphibian fibres. Thirdly, vacuoles in association with

fatigue disappeared over the course of an hour or so,

whereas those associated with eccentric damage seemed

quite stable over several hours. Thus the vacuoles described

in the present study have different characteristics to those

observed after repeated contractions. It seems that the

vacuoles we observed are specific to eccentric damage.

In all situations where vacuoles have been described, the

muscle is subject to an abnormal osmotic load (Krolenko

& Lucy, 2001). For instance when glycerol is removed from

the extracellular space after a period of exposure, the muscle

Vacuoles in eccentric muscle damageJ. Physiol. 540.2 589

Figure 6. Hypothesis for mechanism and consequences of T-tubular rupture in eccentricmuscle damageA, diagram illustrating sarcomere inhomogeneities in an eccentrically damaged fibre. Sarcomeres 1 and 4 areof normal length, sarcomere 2 is shorter than average while sarcomere 3 is longer. T-tubules in mammalianmuscle are at the overlap of thick and thin filaments and are assumed to be subject to shearing stress(indicated by dashed lines). Where the shear stress is greatest (between sarcomeres 2 and 3), T-tubules areassumed to rupture. B, illustration of some hypotheses of the consequences of eccentric damage followingrupture of T-tubules; AP, action potential. See text for further discussion.

swells as water enters to equilibrate intracellular and

extracellular osmolarity. Subsequently the fibre slowly

returns to its normal volume as glycerol and accompanying

water leave the cell. Vacuoles (and detubulation) occur

during this latter phase and it is thought that the volume

load of water and glycerol leaving the cell via the T-tubules

exceeds their capacity causing them to swell and form

vacuoles. Similarly, during fatigue it is postulated that the

load of lactic acid and accompanying water leaving the cell

in the recovery period causes vacuole development

(Lännergren et al. 2000). It is less clear whether the volume

load simply exceeds the normal flow of the T-system or

whether the osmotic forces also lead to compression of

some T-tubules and reduce their fluid transport capability.

What might cause an osmotic load in association with

eccentric muscle damage? Key observations were made by

Casademont et al. (1988) who showed that when a muscle

was cut in half, very large numbers of vacuoles developed

that were attached to the T-tubules close to the cut region.

They showed that vacuole production could be limited by

a range of procedures including removing Na+ from the

extracellular fluid and addition of a sodium pump

inhibitor. They argued that following surface membrane

damage, Na+ entered the myoplasm from the extracellular

space. This stimulated the Na+ pump which would then

pump the excess Na+ into the extracellular space of the

T-tubules. The volume flow of H2O which accompanies

the Na+ was proposed to exceed the flow capability of the

T-tubules and to cause the vacuole production. We propose

that in eccentric damage, T-tubules suffer shearing damage

when adjacent myofilaments show disparate degrees of

stretch (Fig. 6A). Once disruption of a T-tubule occurs

there will be leakage of extracellular contents into the

myoplasm and vice versa. One important consequence of

this process will be an increase in [Na+]i and [Ca2+]i close to

the end of the disrupted T-tubule. Presumably disrupted

T-tubules either reconnect or seal over and remain

disconnected; however, we have no information on the time

course or frequency of these two processes. We propose

that this localized increase in [Na+]i and [Ca2+]i initiates

two consequences of the initial T-tubular disruption as

indicated in Fig. 6B.

The main evidence in favour of our proposal is extensive

earlier work by Casademont et al. (1988) in a situation of

indisputable membrane damage and our demonstration

that ouabain can completely prevent the development of

vacuoles. It might be argued that the failure to observe

vacuoles in ouabain is simply due to chance, since not all

eccentrically stretched fibres exhibited vacuoles, but this

explanation is eliminated by the subsequent observation of

extensive vacuole development as the ouabain was washed

out of the preparation. The increased frequency of vacuole

development after ouabain is consistent with an increase

in intracellular sodium when the Na+ pump is inhibited.

The slow time course of the appearance of vacuoles as

ouabain is washed off (which is slower than the appearance

of vacuoles after eccentric contractions) is consistent with

the slow washout of ouabain from muscles (Nielsen &

Clausen, 1996). The gradual increase in background fibre

fluorescence as ouabain is washed off (compare Fig. 5Bb to

d) probably represents a slow increase in the number of

vacuoles and in the number of blocked T-tubules so that

sulforhodamine leaves the fibre more slowly with each

subsequent washout.

Our hypothesis for the formation of vacuoles requires the

presence of Na+ pumps in the T-tubules. Early studies

showed that Na+ pumps are present at a reduced density in

the T-tubules (Venosa & Horowicz, 1981). More recent

studies have confirmed the original observation and, in

addition, demonstrate that the distribution of Na+ pump

isoforms is different in the T-tubule compared to the

surface membrane (Williams et al. 2001).

Slowed diffusion in and out of the T-systemIt is established that molecules enter and leave the

T-system with a time course that is only moderately

greater than that expected for free diffusion (Hodgkin &

Horowicz, 1960). In keeping with this, sulforhodamine B

enters and leaves the T-system with a half-time of between

5 and 20 s depending on the diameter of the fibre (Endo,

1996); we confirmed this finding in normal fibres which

had a half-time for removal of sulforhodamine B of

18 ± 1 s. In contrast, removal of sulforhodamine B from

the vacuoles is greatly slowed with a half-time of around

6 min. It is clear from these studies that a substantial

barrier to diffusion is located at the point of connection of

the vacuoles with the T-system.

Of special interest is our observation that, in eccentrically

damaged fibres that did not exhibit vacuoles, the rate of

diffusion of sulforhodamine from the preparation was

significantly slowed to 39 ± 4 s. This is important because

it establishes that there is some change in T-tubular

properties in eccentrically damaged fibres even in the

absence of vacuoles. Furthermore it suggests that at least

some of the barrier to diffusion lies distributed in the

T-system. One possibility is that the T-system is compressed

by the osmotic changes reducing both volume flow and

diffusion. Another possibility is that the rupture of T-tubules

and subsequent sealing over make the diffusion pathway

more restricted and/or more tortuous.

Another interesting observation is the presence of

fluorescence in the T-tubules of normal fibres after 20 min

and the increase in this intensity in eccentrically damaged

fibres. We argued earlier that the fluorescence in normal

fibres could represent binding of the dye to the membrane.

The increased fluorescence following eccentric damage

could arise (i) because the surface area of T-system has

increased, (ii) because the diffusion pathway is more

E. W. Yeung and others590 J. Physiol. 540.2

tortuous, particularly if many T-tubules near the surface

membrane were disrupted, (iii) because some T-tubules

have formed sealed sections from which the dye cannot

escape or (iv) because some dye has entered the myoplasm

and is trapped there. Some of these possibilities could be

distinguished if higher resolution images of T-tubular

staining were available or EM studies of the T-tubules at

the appropriate time were made.

Consequences of T-tubular damage for eccentricmuscle damageThe observation of vacuoles is the most compelling

evidence of T-tubular damage, but do vacuoles necessarily

affect muscle function? In muscle fatigue, vacuoles are a

prominent feature of the recovery period in amphibian

fibres, but they correlate poorly with the degree of

functional recovery (Lännergren et al. 2000). Similarly in

our experiments all the features of eccentric damage were

present in 1/7 fibres subjected to 40 % stretch and 2/2

fibres subjected to 25 % stretch and yet no vacuoles could

be found. In addition, the distribution of vacuoles was

often quite localized while the sarcomere inhomogeneities

and ionic changes appeared to be much more widely

distributed (Balnave et al. 1997). Furthermore, the degree

of reduction of force was similar in the experiments in the

presence of ouabain despite the fact that no vacuoles were

detected. These features lead us to the view that the

fundamental T-tubular defect is not the presence of

vacuoles, but a more minor degree of damage to the

T-tubules that is not detectable with our present images.

However we do have evidence for this abnormality based

on the slower diffusion of sulforhodamine B from the

T-tubules of eccentrically damaged fibres that did not

exhibit vacuoles. Thus we propose that shearing damage to

T-tubules at multiple sites in the muscle, triggered by the

sarcomere inhomogeneities, is the primary T-tubular

defect (Fig. 6A). Vacuoles presumably develop in sites

where this primary damage is so great that the osmotic load

of pumping out the consequent rise in Na+ is sufficiently

great to produce vacuoles (central column of Fig. 6B).

Figure 6B illustrates how we propose that the shearing

damage to T-tubules contributes to other aspects of

eccentric damage. The left hand pathway illustrates some

of the consequences of Ca2+ influx from the extracellular

space. A rise in resting [Ca2+]i has been noted in several

studies (Balnave & Allen, 1995; Ingalls et al. 1998) though

focal rises, which would be predicted by our hypothesis,

have not been detected (Balnave et al. 1997), perhaps

because they are very transient. Several studies have shown

that a rise of the time-averaged [Ca2+]i in a muscle can

inhibit SR Ca2+ release (Lamb et al. 1995; Bruton et al.1996; Chin & Allen, 1996) so the rise in resting [Ca2+]i

could conceivably cause the reduced Ca2+ transients that

have been observed in eccentrically damaged muscles

(Balnave & Allen, 1995; Ingalls et al. 1998). It is also widely

thought that rises in [Ca2+]i may contribute to activation of

proteases and phospholipases and to the ensuing cell

damage and inflammation (Belcastro et al. 1998).

The right hand pathway illustrates some of the possible

consequences of the ruptured or compressed T-tubules. It

is possible that inward conduction of the action potential

would be affected and this is another possible mechanism

for the reduced SR Ca2+ release, which is characteristic

of eccentrically damaged mammalian muscle (references

above). This component of damage could be the cause of

the slowed diffusion of sulforhodamine B, and if removal of

protons is also slowed, it may explain the impaired pH

regulation that we have recently described (Yeung et al. 2002).

ConclusionThese observations of damage and functional changes in

the T-tubular system offer new insights into some of the

early changes in eccentric damage. The formation of

vacuoles, which can be inhibited by ouabain, is strong

evidence of intracellular Na+ loading, presumably through

damaged T-tubules. The ionic changes secondary to

T-tubular damage and the reduced exchange of ions,

metabolites and fluid across the T-tubular network are

capable of explaining a range of phenomena that occur in

eccentrically damaged muscle.

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AcknowledgementsThe confocal microscope used in these studies was funded from aCollaborative Research Grant with Pfizer, research funds from theUniversity of Sydney and an equipment grant from the NationalHealth and Medical Research Council of Australia. The workdescribed will be submitted to the University of Hong Kong byElla W. Yeung as part of her doctoral thesis.

E. W. Yeung and others592 J. Physiol. 540.2