J Physiol 567.

3 (2005) pp 723–735 723

Topical Review

Mechanisms of stretch-induced muscle damage in normal

and dystrophic muscle: role of ionic changes
D. G. Allen1 , N. P. Whitehead1 and E. W. Yeung2
1
School of Medical Sciences, University of Sydney F13, Sydney, NSW 2006, Australia
2
Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

Muscle damage, characterized by prolonged weakness and delayed onset of stiffness and soreness,
is common following contractions in which the muscles are stretched. Stretch-induced damage
of this sort is more pronounced in the muscular dystrophies and the profound muscle damage
observed in these conditions may involve similar pathways. It has been known for many years that
damaged muscles accumulate calcium and that elevating calcium in normal muscles simulates
many aspects of muscle damage. The changes in intracellular calcium, sodium and pH following
stretched contractions are reviewed and the various pathways which have been proposed to
allow ion entry are discussed. One possibility is that TRPC1 (transient receptor potential,
canonical), a protein which seems to form both a stretch-activated channel and a store-operated
channel, is the main source of Ca2 + entry. The mechanisms by which the changes in intra-
cellular ions contribute to reduced force production, to increased protein breakdown and to
increased membrane permeability are considered. A hypothetical scheme for muscle damage
which incorporates these ideas is presented.
(Received 29 May 2005; accepted after revision 5 July 2005; first published online 7 July 2005)
Corresponding author D. G. Allen: School of Medical Sciences, University of Sydney F13, Sydney, NSW 2006, Australia.
Email: davida@physiol.usyd.edu.au

Any period of intense or prolonged muscle activity can The weakness and histological changes of muscle damage
cause a decline in performance which can be measured as are normally gradually repaired over several weeks and
muscle weakness. By definition, if the weakness is largely can involve the regeneration of damaged skeletal muscle
reversible in minutes or hours, it is described as fatigue. If fibres from satellite cells (Hawke & Garry, 2001). While
the weakness is slowly or poorly reversible and associated small numbers of stretched contractions can cause muscle
with structural changes within the muscle, it is described damage, it is probable that much larger numbers of
as muscle damage. Muscle fatigue arises principally within isometric or shortening contractions can also cause muscle
the muscle and is thought to be caused by accumulation damage (Jones et al. 1983; Gissel & Clausen, 2001).
or depletion, either intracellular or extracellular, of various The muscle damage which follows stretched
metabolites and ions (for review see Westerblad et al. 1991; contractions has important roles in exercise training
Fitts, 1994; Allen et al. 1995). Muscle damage is particularly (Colliander & Tesch, 1990) and sports injuries (Proske
pronounced in muscles which are stretched during et al. 2004) and seems to stimulate a particular type of
contraction (eccentric or stretched contractions), for muscle redevelopment with new sarcomeres in series
instance the quadriceps group during downhill running. (Lynn & Morgan, 1994; Yu et al. 2003). Stretch-induced
Characteristically, stretch-induced muscle damage causes muscle damage is also more severe in older animals
an immediate weakness which can take a number of days to (Brooks & Faulkner, 1996) and may have a role in
recover. In addition the affected muscles develop soreness, the decline of muscle function seen in the elderly.
swelling and stiffness which typically become apparent There is also considerable evidence that repeated and
1–2 days after the initiating contractions. Sore and stiff poorly reversed muscle damage plays a part in the
muscles the day after unaccustomed and excessive exercise profound muscle degeneration which characterizes
will be familiar to physically active individuals and this muscular dystrophy. For instance, stretch-induced muscle
condition has been named delayed-onset muscle soreness damage is considerably more severe in the mdx mouse,
(DOMS). Simultaneously there is often substantial release which lacks dystrophin and is a model of Duchenne
of soluble muscle proteins, such as creatine kinase, and muscular dystrophy (Head et al. 1992; Petrof et al.
inflammatory cells are attracted to the damaged muscle. 1993). Furthermore, transfection of dystrophin into

C The Physiological Society 2005 DOI: 10.1113/jphysiol.2005.091694
724 D. G. Allen and others J Physiol 567.3

mdx muscle causes a reduction in the magnitude of exercise which included stretched contractions caused a
stretch-induced damage, showing that the absence of substantial increase in muscle Ca2+ which accumulated in
dystrophin exacerbates muscle damage (Deconinck et al. the mitochondria.
1996; DelloRusso et al. 2002). Muscular dystrophy also Intact single fibres can be stretched during contractions
involves ionic disturbances which appear to have a central and resulting changes in force and [Ca2+ ]i determined
role in the development of the muscle pathology (Turner (Balnave & Allen, 1996). A relatively severe protocol
et al. 1988; Gillis, 1999; Yeung et al. 2003b, 2005). For of stretched contractions reduced tetanic [Ca2+ ]i to
all these reasons interest in the ionic changes caused by about 50% of the pre-stretch control; in the presence
stretch-induced damage is increasing rapidly at present. of 10 mm caffeine this tetanic [Ca2+ ]i could be returned
The earliest structural change following stretch-induced to near the pre-stretch value and tetanic force showed
muscle damage is sarcomere inhomogeneities a 10% recovery. These data demonstrate that part of
characterized by a patchy distribution of over- and the weakness of stretched muscle is caused by reduced
understretched sarcomeres and Z-line irregularities sarcoplasmic reticulum (SR) Ca2+ release. Presumably
(Fridén et al. 1981). These morphological changes can be much of the remaining force deficit is caused by the
observed within the first stretched tetanus (Brown & Hill, mechanical consequences of sarcomere inhomogeneity
1991; Talbot & Morgan, 1996) and are thought to result discussed above. Several studies have shown that resting
from the instability of sarcomeres on the descending [Ca2+ ]i shows a small increase seen only after stretched
limb of the tension–length relation (Morgan, 1990). A contractions and not after isometric contractions (Balnave
recent review of stretch-induced muscle damage focused & Allen, 1995; Ingalls et al. 1998).
mainly on the mechanical consequences of stretched The finding of elevated resting [Ca2+ ]i was confirmed
contractions (Proske & Morgan, 2001). In the present in a study performed on mice following downhill running
review we are concerned with the pathways which lead to (Lynch et al. 1997). The measurements were made on
muscle damage and, particularly, the role of ionic changes surface fibres of whole muscles at 24 h and 48 h after the
in triggering damage. run. Increases in resting [Ca2+ ]i were only noted at 48 h,
a time at which histology showed many damaged cells
infiltrated by inflammatory cells. Thus the elevated resting
[Ca2+ ]i in this study may well be a late consequence of an
Ionic changes following stretched contractions earlier damage pathway.
Calcium. There has been a long interest in the Recent studies from our own laboratory have made
role of calcium in muscle damage. Early studies by some progress in identifying pathways by which Ca2+
Duncan (1978) showed that treatment of muscles with enters the cell following stretched contractions. These
calcium ionophores, which increase intracellular calcium experiments have involved mdx mouse muscle, chosen
([Ca2+ ]i ), cause muscle damage with initial contracture, because its sensitivity to stretch-induced damage is
swelling of mitochondria, loss of soluble muscle proteins enhanced (Head et al. 1992; Petrof et al. 1993). We
and, eventually, degeneration of myofibrils. Duncan was measured the [Ca2+ ]i in single fibres following a
particularly interested in the idea that Ca2+ -activated protocol of 10 contractions (Yeung et al. 2005). Isometric
damage might have a central role in the muscle damage contractions produced no detectable increase in resting
observed in muscular dystrophy. Three outstanding issues [Ca2+ ]i but following 10 stretched contractions resting
dominate this field up to the present day. (i) What is the [Ca2+ ]i increased over 20–30 min. Over the same period
route of Ca2+ entry? (ii) Is the rise of Ca2+ simply a function tetanic [Ca2+ ]i declined; both results are similar to earlier
of passive Ca2+ entry along its electrochemical gradient results in wild-type fibres. The rise in resting [Ca2+ ]i could
following membrane damage by some other route or is it be prevented by streptomycin, Gd3+ or the spider venom
a primary contributor to the damage pathway? (iii) What toxin GsMTx4, each of which block stretch-activated
are the pathways by which elevated [Ca2+ ]i causes muscle channels (Hamill & McBride, 1996; Suchyna et al. 2000).
damage? Table 1 shows tabulated information regarding In addition, removal of extracellular calcium prevented
the main pathways for Ca2+ entry which have been the rise in resting [Ca2+ ]i confirming that it is caused by
proposed. influx of Ca2+ . These results suggest that stretch-activated
The first study of intracellular calcium in muscle channels (SACs) have a role in Ca2+ entry following
as a consequence of stretched contractions used a stretched contractions.
2 h period of downhill walking of rats (Duan et al.
1990). Muscles were isolated both immediately and Sodium. A series of stretched contractions also causes
2 days after the exercise, mitochondria extracted and an increase in [Na+ ]i in both wild-type mouse muscle
Ca2+ determined. Mitochondrial Ca2+ was increased fibres and mdx muscle fibres (Yeung et al. 2003a,b). The
about 3-fold immediately after exercise and about rise in [Na+ ]i appears to follow the stretched contractions
6-fold 2 days later. Thus these experiments showed that and takes 5–10 min to reach a peak. The rise in [Na+ ]i is

C The Physiological Society 2005


Table 1. Properties of pathways allowing ion entry during damage in skeletal muscle
Stretch-activated Growth factor-regulated Store-operated channel Prolonged exercise-
Membrane tears Ca2+ leak channel channel(SAC) channel (GRC) (SOC) induced pathway
J Physiol 567.3

Channel permeability — 10 pS (96 mM Ca2+ 13 pS (110 mM Ca2+ 8 pS (110 mM Ca2+ 8 pS (110 mM Ca2+ —
(Ca2+ ) in pipette) [3] in pipette) [4] in pipette) [15] in pipette) [21], 11 pS [20]

Voltage sensitivity — No [3] Activity increased by No [15] No [21] —

depolarization [4]

C The Physiological Society 2005

Permeable ions All ions Ba2+ , Ca2+ [3], Li+ , Na+ , Rb+ , Na+ , K+ , Ca2+ , Ba2+ [15] Na+ , Cs+ , K+ , Ca2+ , Ba2+ [20]; Na+ , Ca2+ [6]
Mn2+ [8],not Cs+ ,Ca2+ , Ba2+ , K+ [4] Mn2+ [19, 21]
Na+ [3,18]

Chemical activators — Bay K 8644, Amphipaths, lipids [7] — — Veratridine [6]

nifedipine [3]
Chemical inhibitors — AN 1043 [1, 8] Gd3+ [23] Gd3+ [15] La3+ [21]; Gd3+ , La3+ , Ni2+ [19] Tetrodotoxin inhibits
Streptomycin [22] Ruthenium red [9] the veratridine-
GsMTx [17] SKF96365 [9] induced
component [6]
Nifedipine, minor
effect [6]. Gd3+ ,
Ni2+ , no effect [6].
Permeable to large molecules Yes [13] No No No No Yes [6, 14]

Stretch-activated Appear after Channel activity Increased or Increased activity by pipette Increased channel activity by —
stretched sometimes enhanced decreased channel suction [15] pipette suction [20]
contractions [13] by pipette suction [3] activity by pipette
suction [4, 5].

Store-depletion activation — Yes (6) — — Yes (3) —

Increased activity in mdx Yes [16] Yes [3] Yes [5] ?; increased expression in Yes [20, 21] —
Stretch-induced muscle damage

δ-sarcoglycan-deficient
hamster muscle [15]
Molecular identity — — TRPC1 [11] 44% homology to TRPV1 [10] TRPC 1 or 4 [21] —

Physiological trigger Mechanical damage Required contractions Activated by Cyclically stretching leads to Functional role not explored. Induced by repeated
prominent after and membrane stretched increased Ca2+ uptake and contractions. May be
stretched damage to trigger in contractions [24]. creatine kinase (CK) efflux partly ischaemia
contractions [13, 16] mdx muscle. Insertion Activity can persist for blocked by Gd3+ , SKF 96365, induced [6, 14].
in membrane involves 24 h [12] Ruthenium red [9]. Induced
raised [Ca2+ ]i and by by IGF-1 which causes
proteolysis [2] translocation to surface
membrane [9, 10];
translocation blocked by
Gd3+ [9]
Tabulated list of properties of pathways allowing influx of ions into damaged muscles. References: 1, Alderton & Steinhardt (2000a); 2, Alderton & Steinhardt (2000b); 3, Fong
et al. (1990); 4, Franco & Lansman (1990); 5, Franco-Obregon & Lansman (2002); 6, Gissel & Clausen (2001); 7, Hamill & McBride (1996); 8, Hopf et al. (1996); 9, Iwata et al.
(2003); 10, Kanzaki et al. (1999); 11, Maroto et al. (2005); 12, McBride et al. (2000); 13, McNeil & Khakee (1992); 14, Mikkelsen et al. (2004); 15, Nakamura et al. (2001); 16,
Petrof et al. (1993); 17, Suchyna et al. (2000); 18, Turner et al. (1991); 19, Tutdibi et al. (1999); 20, Vandebrouck et al. (2001); 21, Vandebrouck et al. (2002); 22, Winegar et al.
(1996); 23, Yang & Sachs (1989); 24, Yeung et al. (2003a).
725
726 D. G. Allen and others J Physiol 567.3

substantial (from 7 to 16 mm in wild-type fibres) and of fibres in the triceps muscle had taken up albumin
would be expected to be accompanied by osmotically from the plasma indicating that the membrane had
equivalent water which would contribute to the swelling been damaged compared to 3% in sedentary controls.
associated with stretched contractions (Foley et al. 1999). These results were obtained immediately after the 1 h
The rise in [Na+ ]i should activate the Na+ –K+ pump, period of exercise so that the time between stretched
increasing Na+ efflux which will be accompanied by contractions and measurement would have varied from
osmotically associated water. This is the presumed reason 0 to 1 h; after 24 h the number of fibres staining for
for the appearance of vacuoles attached to the T-system albumin had fallen to 5%. Another technique for assessing
following stretched contractions (Yeung et al. 2002a). membrane permeability involves the injection of the
Note that these vacuoles would constitute a region of the animals with the membrane-impermeant fluorescent dye
membrane which is distorted or stretched long after the Evans blue, and subsequently uptake of dye by damaged
precipitating stretched contractions. In the mdx mouse it cells can be observed either macroscopically (Straub
appears that the resting [Na+ ]i is elevated and this elevation et al. 1997) or microscopically (Hamer et al. 2002).
is inhibited by Gd3+ and streptomycin, which also inhibit Uptake of Evans blue has also been used to assess the
the rise of [Na+ ]i following stretched contractions (Yeung reversal of the susceptibility to muscle damage after
et al. 2003a,b). These observations suggest that SACs dystrophin expression in mdx mice (Deconinck et al.
may have an increased opening probability during normal 1996; Gregorevic et al. 2004). Alternatively impermeant
activity in the mdx mouse and there is a further increase in fluorescent dyes, such as procion orange, can be perfused
opening probability triggered by stretched contractions. over isolated muscles subjected to stretched contractions
and histological approaches used to assess the number
pH. Protons also have an inward electrochemical gradient of permeabilized fibres (Petrof et al. 1993). It is
and are actively extruded to maintain a steady state. Resting important to note that these experiments provide
intracellular pH was more acid by 0.17 pH units after a evidence of membrane permeability to large molecular
series of stretched contractions and no such effect was weight markers; whether this permeability is caused by
seen after isometric contractions. In addition removal of mechanical tears or some other mechanism remains to be
an acid load, which occurs principally on the Na+ –H+ determined.
exchanger, was slowed. These findings suggest that the Cells are capable of resealing artificially produced
Na+ –H+ exchanger is less effective although an increased membrane defects and this is a process which requires
inward leak of H+ has not been excluded (Yeung et al. the presence of extracellular Ca2+ and seems to involve
2002b). The increased [Na+ ]i would provide part of the the production of intracellular vesicles which subsequently
explanation for reduced effectiveness of the Na+ –H+ fuse, forming a patch over the membrane defect (McNeil
exchanger; in addition damage to T-tubules might isolate et al. 2000). Resealing can be inhibited by botulinum
some exchangers rendering them ineffective. Other studies or tetanus toxin suggesting that the fusion of vesicles
have looked at longer term changes in pH regulation and is analogous to exocytosis (for review see McNeil &
show that 2 days after a period of stretched contractions Steinhardt, 2003).
pH buffering and activity of the lactate exchanger were A valuable new approach to understanding membrane
reduced (Pilegaard & Asp, 1998). repair is to cause local damage to the membrane by means
of an intense laser pulse (Bansal et al. 2003). In the presence
of the dye FM 1-43, which fluoresces when in contact with
Pathways for ionic entry
cell membrane, it appears that normal fibres repair these
Membrane tears. The presence of overextended and holes in less than 1 min whereas repair in mice with absent
underextended sarcomeres in muscles damaged by dysferlin, which causes a mild type of muscular dystrophy,
stretched contractions (Fridén et al. 1981) means that is greatly slowed. Interestingly, the rate of repair in mdx
T-tubules, which are located at the A–I junction in fibres was normal.
mammalian fibres, can be severely distorted and may be These studies show clearly that membrane defects are a
damaged or torn (Allen, 2001). Mechanical damage of normal consequence of muscle activity, particularly when
this, or other types, to the surface membrane would be it involves stretched contractions. Membrane defects can
expected to allow rapid equilibration of the intracellular be repaired and, at least for some types of injury, the repair
and extracellular spaces causing increases in [Na+ ]i and can be very rapid. Inevitably such membrane defects would
[Ca2+ ]i , loss of soluble intracellular proteins, uptake of also allow equilibration of ions across the membrane.
large molecular weight markers and depolarization of the Given this evidence of membrane defects, we have
membrane potential. searched for localized regions of elevated ions which might
The existence of membrane tears was proposed by be expected to accumulate on the intracellular side of
McNeil & Khakee (1992) from experiments involving such a defect. Imaging single fibres for [Ca2+ ]i in the
downhill running by rats. They showed that about 21% 30 min period following stretched contractions failed to

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J Physiol 567.3 Stretch-induced muscle damage 727

identify any such areas (Balnave et al. 1997). This might be these channels turn on or off within 1 s when pressure is
because Ca2+ entry is rapidly sequestered in the SR and/or applied or removed (Yeung et al. 2005). These channels are
mitochondria or because the defects were too few or too blocked by Gd3+ at 10–20 µm and by aminoglycoside anti-
small to be detected. To eliminate the possibility that SR or biotics such as streptomycin at 100–200 µm (for review
mitochondrial uptake disguised entry, we imaged [Na+ ]i in see Hamill & McBride, 1996). Neither of these agents is
fibres during the period when [Na+ ]i was rising but again specific for SACs but the spider venom toxin GsMTx4 is
failed to identify localized areas of elevated [Na+ ]i (Yeung both more potent (effective at 5–10 µm) and appears to
et al. 2003a). These data, combined with the observation be more specific (Suchyna et al. 2000). These channels are
that blockers of stretch-activated channels prevent the more prevalent in neonatal tissues and myotubes grown
early rise in [Na+ ]i and [Ca2+ ]i , suggest that the early ion from satellite cells and appear to decline in frequency
entry is through channels rather than membrane defects. as the skeletal muscle becomes more mature (Haws &
Lansman, 1991). Also, these channels seem to be expressed
more frequently in mdx mouse muscle (Haws & Lansman,
Leak channels. Calcium-specific leak channels were first
1991) and human Duchenne muscular dystrophy
described by Fong et al. (1990) in mouse and human
myotubes (Vandebrouck et al. 2001).
muscle fibres. These channels have a permeability of
Do these channels contribute to ion entry after
∼10 pS, open spontaneously at rest, are not voltage
stretched contractions? There is no direct patch clamp
dependent and allow entry of Ca2+ , Ba2+ and Mn2+ but
data establishing that these channels show increased
not Na+ . Their open probability is higher in mdx muscle
opening after stretched contractions and if muscle stretch
and muscles from humans with Duchenne muscular
is comparable to negative pressure in the patch clamp, then
dystrophy (Fong et al. 1990). Paradoxically, their opening
channel activity should revert to normal within seconds of
probability was increased by the L-type Ca2+ channel
the stretch. However, the [Ca2+ ]i and [Na+ ]i increase over
blocker nifedipine but blocked by the nifedipine analogue
10–20 min following stretched contractions and if SACs
AN 1043 (Alderton & Steinhardt, 2000a). This channel
are involved their opening would need to be controlled
is activated by store depletion using cyclopriazonic acid
by a slow process which was a consequence of stretched
judged both by patch-clamp estimates of channel open
contractions. One possibility is that cytoskeletal elements
time and by the rate of Mn2+ influx and quenching
are damaged by the stretched contraction (Lieber et al.
of fura-2 fluoresence (Hopf et al. 1996). The function
1996) and then influence the opening of SACs (Guharay &
of these channels in normal muscle is unclear because
Sachs, 1984). As noted above, the various blockers of SACs
contractile activity does not appear to influence their
are capable of preventing the rise of [Ca2+ ]i and [Na+ ]i
activity (Alderton & Steinhardt, 2000a) but in dystrophic
which provides strong evidence that the influx is through
muscle these channels appear to develop around the
these channels (Yeung et al. 2003b, 2005).
site of artificially induced membrane damage and the
Further evidence supporting a role of SACs following
appearance of these channels was inhibited by the calpain
stretched contractions arises from measurements of the
inhibitor leupeptin (McCarter & Steinhardt, 2000). Thus
membrane potential. McBride et al. (2000) showed that
the sequence of events proposed to lead to damage
after stretched contractions the membrane potential was
in dystrophic muscle is that membrane tears lead to
depolarized by about 10 mV for at least 24 h. This
localized Ca2+ entry which instigates the repair of the
depolarization could arise from increased permeability to
defect. Activation of proteolysis is thought essential for
Ca2+ and Na+ either through SACs or membrane tears
the activation of Ca2+ leak channels which lead to further
but this depolarization was partly prevented by either
Ca2+ entry and Ca2+ -activated proteolysis which is the
oral streptomycin, fed to the animals for several days
final common pathway of damage (Alderton & Steinhardt,
beforehand, or by Gd3+ , added to the muscle bath at the
2000b).
time of measurement. Thus SAC blockers prevent some of
the depolarization which strongly suggests that SACs are
Stretch-activated channels. SACs were first described responsible for one component of the depolarization and
in fetal skeletal muscle by Guharay & Sachs (1984). the increased ion permeability which underlies it.
Characteristically these channels increase their opening A very important recent development is the first
probability when negative pressure is applied to the patch molecular identification of SACs (Maroto et al. 2005).
pipette. The channels, observed in adult skeletal muscle These authors studied a mechanosensitive channel
by several groups (Winegar et al. 1996; Vandebrouck et al. (MscCa) in frog oocytes with characteristics which are
2001), are non-selective cation channels permeable to Na+ , very similar to the SACs identified in mammalian muscle.
K+ , Ca2+ and Ba2+ and have a conductance of 13 pS Proteins extracted from oocytes were run on HPLC and
when the pipette is filled with 110 mm Ca2+ (Franco & protein peaks could be extracted and reconstititued in
Lansman, 1990). At rest their open probability is low liposomes to give channels with identical characteristics.
but it is increased steeply by reduced pressure. Typically This protein had a molecular weight of 80 kDa and bound

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728 D. G. Allen and others J Physiol 567.3

antibodies to TRPC1. Human TRPC1 was expressed in quite similar permeabilities (Table 1). Another similarity
Chinese hamster ovary (CHO-K1) cells, chosen for low is that both the leak channel and the GRC have been
endogenous expression of MscCa, and produced a 10-fold reported to be both store operated and stretch activated
increase in channel activity with similar properties to the (see references in Table 1). Furthermore, both the muscle
frog channel. In CHO-K1 cells TRPC1-induced channel SOC and SAC appear to be encoded by either the TRPC1
activity was inhibited by 3 µm Gd3+ . TRPC1, which or 4 gene (Vandebrouck et al. 2002). Given the recent
is widely expressed in tissues including skeletal muscle proposal that the vertebrate SAC is encoded by the TRPC1
(Vandebrouck et al. 2002), has previously most frequently gene (Maroto et al. 2005) it now seems likely that the
been characterized as a store-operated channel (Beech et al. muscle SAC and SOC are closely related and composed
2004). Thus it now seems likely that the SAC described in of TRPC1 protein. Although the functional properties of
muscle is encoded by TRPC1. the GRCs appear very similar to SACs, their molecular
identity showed the closest homology to TRPV1 (Kanzaki
et al. 1999; Iwata et al. 2003). One clear distinction between
Growth factor-regulated channels. These channels,
the various channels is that the Ca2+ leak channel is the
originally described as CD20 in lymphocytes, are
only channel reported as not permeable to Na+ (Fong
Ca2+ -permeable channels activated by insulin-like growth
et al. 1990). Given these differences it seems unlikely that
factor (IGF-1) (Kanzaki et al. 1997). Later they were
the molecular composition of all the channels is identical.
renamed growth factor-regulated channels (GRCs) and
One possibility is that the various channels described are
shown to have considerable homology with the TRPV
all TRP channels but exhibit the heterogeneity of subunit
family (Kanzaki et al. 1999). These channels may underlie
composition which is a feature of TRP channels (Beech
store-operated channel behaviour (Ju et al. 2003) but
et al. 2004).
also appear to be stretch activated (Nakamura et al.
2001) with properties similar to the stretch-activated
channel described in wild-type and mdx mice (Franco Muscle damage following repeated isometric muscle
& Lansman, 1990). Iwata et al. (2003) showed that contractions. Early work established that repeated
in the δ-sarcoglycan-deficient Syrian hamster, which isometric contractions could cause damage characterized
has a cardiomyopathy and muscular dystrophy, these by irreversible loss of force and release of soluble intra-
channels are overexpressed and can be activated by cellular enzymes (Jones et al. 1983). This type of damage
stretch, allowing Ca2+ entry which appears to trigger loss was accelerated in anoxia, could be produced in resting
of creatine kinase. muscles by metabolic inhibitors, required extracellular
calcium and could be produced by elevating [Ca2+ ]i with
Store-operated channels. Store-operated channels a calcium ionophore (Jones et al. 1984; Duncan & Jackson,
(SOCs) have been described a number of times in skeletal 1987). Thus it was thought that metabolic depletion caused
muscle though their function is unclear (Kurebayashi a rise in [Ca2+ ]i involving entry of extracellular Ca2+ and
& Ogawa, 2001; Launikonis et al. 2003). Tutdibi et al. that Ca2+ -activated intracellular pathways were critical to
(1999) used the Mn2+ quench method to identify SOCs damage.
in muscle and showed that their activity was about twice Gissel & Clausen have developed a model of damage
as high in mdx compared to wild-type and was blocked in which isometric rat extensor digitorum longus (EDL)
by Gd3+ , La3+ and Ni2+ . The distinction between SOCs muscles are subjected to prolonged stimulation. In some
and SACs in skeletal muscle has been called into question studies 1 Hz twitches were repeated for 4 h (Gissel &
by Vandebrouck et al. (2002) who showed that a channel Clausen, 2003); in others tetani (10 s at 40 Hz followed
apparently identical to the muscle SAC was opened by by 30 s rest) continued for 1 h (Mikkelsen et al. 2004).
store depletion. They showed that both wild-type and Developed force was only about 5% of the original
mdx fibres expressed TRPC1, 4 and 6 in the membrane level after 1 h stimulation and there was virtually no
fraction and that antisense mRNA for TRPC1 and 4 was recovery. These muscles show damage judged by gradually
able to reduce the activity of spontaneous Ca2+ channels increasing Ca2+ uptake and gradually increasing lactate
in mdx fibres while having no effect on voltage-dependent dehydrogenase release. There were also changes in cell
Na+ channels. This study suggests that both SAC and Na+ , K+ and sucrose spaces which were compatible with a
SOC activity could arise from a single channel encoded small fraction of the cells becoming damaged and therefore
by the TRPC1 or 4 gene. permeable to these substances. Other studies from this
The above sections describe four channels leading to laboratory (Gissel & Clausen, 2003) have shown that
ionic entry associated with stretched contractions. This electroporation or application of the calcium ionophore
raises the question as to whether some of these channels can also simulate the increase in LDH release only
are really the same? There are a number of intriguing if extracellular Ca2+ was present suggesting that Ca2+
similarities between the various channels identified but entry from the extracellular space is a key aspect of the
also significant differences. The various channels all have subsequent damage.

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J Physiol 567.3 Stretch-induced muscle damage 729

The rat EDL muscle is around 2 mm in diameter so that following stretched contractions (Warren et al. 1993). If
in vitro preparations will inevitably develop a hypoxic core depolarization occurs, it would cause inactivation of Na+
during intense activity; thus part of the damage in these channels and reduce the amplitude of the action potential
studies may represent ischaemia and/or hypoxia affecting and hence Ca2+ release. The rise in [Na+ ]i described above
in particular the central cells. This idea is supported by would also reduce the amplitude of the action potential
studies showing that the hypoxia is capable of simulating and Ca2+ release. The acidosis following stretched
the damage observed by raising [Ca2+ ]i in muscle cells and contractions could also contribute to reduced force since
that it is the central cells in a muscle which exhibit the most acidosis has been shown to reduce both the Ca2+ sensitivity
striking signs of damage (Bannister & Publicover, 1995). and the maximum Ca2+ -activated force of the myofibrillar
It is not yet clear whether these studies have identified proteins (Fabiato & Fabiato, 1978). However, these pH
a damage pathway caused by isometric contractions or effects are probably relatively small at 37◦ C (Pate et al.
whether ischaemia, which may not be a feature of fatiguing 1995).
contractions in vivo, is the cause of the damage. There are many suggestions that elevated resting [Ca2+ ]i
can affect the excitation–contraction coupling process (for
review see Westerblad et al. 2000). Typically, situations
which caused an elevation of resting [Ca2+ ]i produce
Consequences of ionic changes
a prolonged reduction in tetanic [Ca2+ ]i which reduces
Reduced force production. The causes of the early decline force particularly at low stimulation frequencies where the
of force after stretched contractions fall into several force is most sensitive to changes in [Ca2+ ]i (Westerblad
categories. Muscles subjected to stretched contractions et al. 1993; Chin & Allen, 1996). Probably the clearest
exhibit regions of over- and understretched sarcomeres evidence arises from skinned fibres with intact T-tubular
(Fridén et al. 1981) and these cause a shift in the peak and SR connections in which elevation of resting [Ca2+ ]i ,
of the tension–length relation to longer lengths (Talbot for example to 2.5 µm for 1 min, leads to a substantial
& Morgan, 1998). Because of the shift of the peak of reduction in the ability of T-tubular depolarization to
the tension–length relation, force at the original optimum trigger SR Ca2+ release (Lamb et al. 1995). Under these
length declines and there is a recovery of force as the muscle circumstances caffeine is still capable of releasing the SR
is stretched to the new optimum length (Wood et al. 1993; Ca2+ content indicating that the Ca2+ -sensitive step lies
Brockett et al. 2001). Thus the degree of recovery of force as between the T-tubule and the SR. Recent work from the
it is stretched to the new optimum length can be regarded same laboratory (Verburg et al. 2005) has shown that
as indicating the degree of damage arising from this cause skinned muscles contain calpain 3, which is activated by
(for review see Proske & Morgan, 2001). the rise of [Ca2+ ]i , and is capable of cleaving titin as well as
A second cause of the early decline of force is that disrupting SR Ca2+ release. The likelihood that calpain 3
excitation–contraction coupling is impaired. Armstrong’s underlies the Ca2+ -dependent uncoupling is suggested by
group were the first to specifically suggest that changes in recent work showing that calpain 3 can be activated by sub-
excitation–contraction coupling might have an important micromolar [Ca2+ ]i (Branca et al. 1999) and by showing
role in the muscle weakness after stretched contractions that leupeptin, a calpain 1, 2 and 3 inhibitor, blocked
(Warren et al. 1993). They reached this conclusion from uncoupling while calpastatin, which inhibits calpain 1
the observation that while 10 or 20 stretched contractions and 2 but not calpain 3 (Ono et al. 2004), did not
caused a substantial reduction in tetanic tension, the prevent uncoupling. Further evidence for the rise in resting
caffeine contractures were little affected. Assuming that [Ca2+ ]i having a role in reduced tetanic [Ca2+ ]i comes
caffeine caused a direct release of Ca2+ from the SR, they from a recent study of mdx fibres in which either removal
deduced that in stretched contractions failure of SR Ca2+ of extracellular Ca2+ or blocking SACs partially reversed
release was an important contributor. This conclusion was the reduction in tetanic [Ca2+ ]i (Yeung et al. 2005).
confirmed when it was shown that tetanic [Ca2+ ]i declined On the other hand a study of the stretch-induced force
in single fibres subjected to stretched contractions (Balnave deficit in wild-type mouse EDL found that a cocktail of
& Allen, 1995). Some of the possible pathways leading to protease inhibitors (including leupeptin) was not capable
reduced force following stretched contractions are shown of reversing the force deficit (Warren et al. 2002). This
as a flow chart in Fig. 1. study does not support a role for calpain 3 but access of
There is a range of possible causes of the reduced Ca2+ the protease inhibitors to the centre of an isolated muscle
release following stretched contractions. One possibility is one possible concern.
is the membrane depolarization described by McBride Yet another possible cause of reduced force after
et al. (2000). This depolarization was blocked by stretched contractions arises from recent work showing
Gd3+ or streptomycin suggesting that it arose from that reactive oxygen species (ROS) produced by tetanic
increased activity in stretch-activated channels. In contrast contractions can reduce the myofibrillar Ca2+ sensitivity
another study found no evidence of depolarization (Moopanar & Allen, 2005). Previous studies have shown

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that stretched contractions can reduce Ca2+ sensitivity (calpain 1) and m-calpain (calpain 2) and, in addition,
(Balnave & Allen, 1996) and there are also suggestions calpain 3 which is relatively specific to skeletal muscle
that ROS production is increased by stretched contractions (Goll et al. 2003). Calpains 1 and 2 are activated by
(Best et al. 1999) although only after a 24 h delay. [Ca2+ ]i in the micromolar and millimolar range and thus
would not be expected to be activated under physiological
Protein breakdown. Intense exercise leads to increased conditions unless additional, as yet unidentified, factors
protein breakdown both in the whole animal and in contribute to their activation. Calpain 3, while initially
isolated muscles (Belcastro et al. 1998). Although thought to be Ca2+ insensitive, has recently been shown to
lysosomal and ubiquitin–proteasome pathways are be extremely [Ca2+ ]i sensitive in the nanomolar range but
involved, there is a consensus that Ca2+ -activated proteases is normally maintained inactive while bound to specific
or calpains are one of the pathways involved (Belcastro sites in muscle, notably titin (Branca et al. 1999). The
et al. 1998). Muscle contains the ubiquitous µ-calpain calpains are cysteine proteases and cleave only a limited

stretched contractions
Ca2+ activated proteolysis
stretch activated
store depletion

opening of TRP channels

[Ca2+]i

mitochondrial
calpain activity phospholipase activity
Ca2+ uptake

ROS damage to SR
proteins

lipid
peroxidation
membrane
permeability
myofibrillar
reduced
Ca2+ sensitivity
membrane
potential

tetanic [Ca2+]i

force CK loss

Figure 1. Pathways involved in stretch-induced muscle damage

Possible intracellular pathways by which stretched contractions cause reduced force production and increased
membrane permeability. Dashed box indicates hypothetical mechanisms which may be involved in activating
channels for Ca2+ entry. Dashed arrow indicates positive feedback pathway that would occur when increased
membrane permeability causes elevated [Ca2+ ]i . For further details see text. ROS, reactive oxygen species; creatine
kinase (CK).

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J Physiol 567.3 Stretch-induced muscle damage 731

range of intracellular proteins including titin, nebulin, idea that membrane tears occur as a result of contractile
desmin, troponin, tropomyosin and many kinases and activity and especially stretched contractions has been
signalling molecules. Thus their physiological role, while advocated (McNeil & Khakee, 1992; Petrof et al. 1993).
still uncertain, appears to involve remodelling of cells, It is clear that the membrane permeability is greater
cell fusion and the proteolytic degradation of various in mdx muscle and this has been attributed to greater
signalling molecules (Goll et al. 2003). The recent study membrane ‘fragility’ in the absence of dystrophin and/or
of skinned toad muscle described above (Verburg et al. the dystroglycan-related complex (Petrof et al. 1993).
2005) provides more direct evidence that calpain 3 could Support for the role of dystrophin comes from expression
have a role in damage to titin and excitation–contraction of dystrophin in mdx muscle and the finding that
coupling when quite moderate rises in [Ca2+ ]i occur. membrane permeability is thereby reduced (Deconinck
It is likely that calpains contribute to muscle damage et al. 1996). However, against this hypothesis is the failure
following stretched contractions (for review see Belcastro to measure increased ‘fragility’ of the membrane in
et al. 1998). In support of this concept is the rise mdx mice (Hutter et al. 1991) and the failure to observe
in resting [Ca2+ ]i following stretched contactions (see localized areas of elevated ion concentrations at the
earlier section), the increased activity of calpain following putative sites of tears (Balnave et al. 1997; Yeung et al.
prolonged treadmill running (Belcastro, 1993), and the 2003a). An alternative hypothesis is that the increase in
rapid breakdown of the cytoskeletal protein desmin, which membrane permeability is a secondary consequence of
is a target of calpain, following stretched contractions Ca2+ entry and this possibility is supported by studies
(Lieber et al. 1996). It has also been shown that the calpain which show that elevating [Ca2+ ]i causes increased
inhibitor leupeptin can reduce the proteolysis of proteins membrane permeability (Duncan & Jackson, 1987; Howl
induced by elevating intracellular calcium (Zeman et al. & Publicover, 1990; Gissel & Clausen, 2003).
1985). We recently re-examined this issue in isolated mdx
The role of calpains in dystrophic muscle damage muscles using uptake of procion orange as the marker
has also received considerable attention. Early studies by of increased membrane permeability (N. P. Whitehead,
Turner et al. (1988) showed that [Ca2+ ]i was elevated M. Streamer & D. G. Allen, unpublished observations).
in mdx muscles and that proteolysis was increased Stretched contractions were imposed and the percentage
but could be decreased by lowering extracellular (and of permeabilized cells determined histologically. In the
presumably intracellular) calcium. Two studies have absence of stretches 1–2% of fibres were permeabilized.
directly tested the role of calpain activation in muscular Immediately after the stretched contractions, when one
dystrophy by the use of calpain inhibitors. Badalamente might expect that mechanical tears would be most
& Stracher (2000) injected the calpain inhibitor leupeptin apparent, 5% of cells were permeabilized. However, the
into the limbs of mdx mice for 30 days. This treatment number of permeabilized cells increased progressively
reduced the calpain activity of limb muscles which to 12–15% by 2 h. This suggests that there is an early
also showed reduced central nuclei and larger muscle component of increased permeability, which might be
fibres suggesting that fibre damage and regeneration had caused by tears or other rapid processes, followed by a
been reduced by the treatment. Spencer & Mellgren slower process. This conclusion was reinforced by using
(2002) crossed mdx mice with a transgenic mouse over- either streptomycin or GsMTx4 to block SACs and both
expressing calpastatin, an endogenous calpain inhibitor of these treatments reduced the number of permeabilized
which inhibits calpain 1 and 2 but not 3. The mdx cells at 1 h to around 5%. Since streptomycin and GsMTx4
mice which overexpressed calpastatin showed reductions prevent the rise of [Ca2+ ]i after stretched contractions
in histological signs of muscle damage and reduced (Yeung et al. 2005) this suggests that a Ca2+ -dependent
regeneration of fibres. However, membrane damage mechanism is involved in the slow increase in permeability.
assessed by procion orange uptake and creatine kinase By what mechanism does elevated [Ca2+ ]i cause
release was unaffected. These two studies support a role increased membrane permeability? One possibility is that
for calpains in the muscle damage in mdx mice but the elevated [Ca2+ ]i causes increased ROS production by
exact pathway is unclear and it seems that the membrane mitochondria (for review see Brookes et al. 2004) and that
damage might involve a different pathway or perhaps peroxidation of membrane lipids then causes membrane
involve calpain 3 rather specifically. defects to develop, perhaps by changes in membrane
fluidity (Mason et al. 1997; Child et al. 1998). The
Increased membrane permeability. A characteristic importance of ROS was suggested by the effectiveness of
feature of stretch-induced muscle damage is an increase in deferioxamine, dithiothreitol and reduced glutathione in
membrane permeability judged by loss of creatine kinase reducing [Ca2+ ]i -induced membrane permeability (Howl
and entry of membrane-impermeant dyes into muscle & Publicover, 1990). There is also evidence that the
cells (McNeil & Khakee, 1992). However, the mechanism elevated [Ca2+ ]i activates phospholipases, particularly
involved remains the subject of continuing debate. The phospholipase A2, based on the observation that

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732 D. G. Allen and others J Physiol 567.3

inhibition of phospholipase A2 reduces [Ca2+ ]i -induced Balnave CD & Allen DG (1996). The effect of muscle length on
membrane permeability (Duncan & Jackson, 1987; intracellular calcium and force in single fibres from mouse
Howl & Publicover, 1990). Phospholipases lead to the skeletal muscle. J Physiol 492, 705–713.
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sarcomere length and [Ca2+ ]i in single fibres from mouse
of which can disrupt membrane structure.
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membrane permeability. For instance leupeptin did and hypoxia in the induction of sarcolemmal damage in
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Yeung EW, Ballard HJ, Bourreau JP & Allen DG (2003a). We thank the National Health and Medical Research Council and
Intracellular sodium in mammalian muscle fibers after the Australian Research Council for financial support. N.P.W.
eccentric contractions. J App Physiol 94, 2475–2482. holds a Rolf Edgar Lake Fellowship of the Faculty of Medicine,
Yeung EW, Balnave CD, Ballard HJ, Bourreau JP & Allen DG University of Sydney. E.W.Y. acknowledges support from Internal
(2002a). Development of T-tubular vacuoles in eccentrically Competitive Research Grants (A-PE65 and A-PF31) from Hong
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C The Physiological Society 2005