The Role of Ca2+ in Muscle Cell Damage

HANNE GISSEL
Institute of Physiology and Biophysics, University of Aarhus,
DK-8000 Århus C, Denmark

ABSTRACT: Skeletal muscle is the largest single organ of the body. Skeletal
muscle damage may lead to loss of muscle function, and widespread muscle
damage may have serious systemic implications due to leakage of intracellular
constituents to the circulation. Ca2+ acts as a second messenger in all muscle
and may activate a whole range of processes ranging from activation of con-
traction to degradation of the muscle cell. It is therefore of vital importance for
the muscle cell to control [Ca2+] in the cytoplasm ([Ca2+]c). If the permeability
of the sarcolemma for Ca2+ is increased, the muscle cell may suffer Ca2+ over-
load, defined as an inability to control [Ca2+]c. This could lead to the activation
of calpains, resulting in proteolysis of cellular constituents, activation of phos-
pholipase A2 (PLA2), affecting membrane integrity, an increased production of
reactive oxygen species (ROS), causing lipid peroxidation, and possibly mito-
chondrial Ca2+ overload, all of which may further worsen the damage in a self-
reinforcing process. An increased influx of Ca2+ leading to Ca2+ overload in
muscle may occur in a range of situations such as exercise, mechanical and
electrical trauma, prolonged ischemia, Duchenne muscular dystrophy, and
cachexia. Counteractions include membrane stabilizing agents, Ca2+ channel
blockers, calpain inhibitors, PLA2 inhibitors, and ROS scavengers.

KEYWORDS: calpain; phospholipase A2; reactive oxygen species; exercise;

Duchenne muscular dystrophy; surfactant; skeletal muscle

INTRODUCTION

Skeletal muscle is the largest single organ of the body, constituting about 40% of
the mass of the average human body. Skeletal muscle is formed of long multinucle-
ate, cylindrical cells called muscle fibers. Muscle damage may lead to loss of func-
tion, and widespread damage to muscle may have serious systemic implications due
to leakage of intracellular constituents to the circulation.
Ca2+ plays a very important role in skeletal muscle. All muscles use Ca2+ as their
main regulatory and signaling molecule, and the function of all muscle types is con-
trolled by Ca2+ as a second messenger.
In this review I will focus on the role of Ca2+ in the healthy muscle cell and in the
development of skeletal muscle damage. I will present a model placing an increased
influx of Ca2+ across the cellular membrane as the initiating factor leading to skele-

Address for correspondence: Hanne Gissel, Institute of Physiology and Biophysics, University
of Aarhus, Ole Worms Alle 1160, DK-8000 Århus C, Denmark. Voice: +45-8942-2820; fax:
+45-8612-9065.
[email protected]

Ann. N.Y. Acad. Sci. 1066: 166–180 (2005). © 2005 New York Academy of Sciences.
doi: 10.1196/annals.1363.013

166
GISSEL: CA2+ AND MUSCLE DAMAGE 167

tal muscle damage and propose a range of clinical situations where this may be the
case. Finally I will evaluate possible countermeasures to prevent or reduce skeletal
muscle damage.

CA2+ IN THE HEALTHY MUSCLE CELL

The concentration of free Ca2+ in the cytosol ([Ca2+]c) is maintained at ~50 nM

in the resting state.1 This is in sharp contrast to the concentration of Ca2+ in the ex-
tracellular fluid ([Ca2+]o), which is around 1 mM, creating an enormous chemical
gradient for Ca2+ across the cellular membrane. Despite a very low permeability of
the sarcolemma for Ca2+ there is continuous passive diffusion of Ca2+ into the mus-
cle cells owing to the large electrochemical gradient across the membrane.2 In order
to maintain the low resting [Ca2+]c the muscle cell must actively remove Ca2+ from
the cytosol by the expenditure of ATP using the Ca2+-ATPases in the sarcoplasmic
reticulum (SR) and sarcolemmal/T-tubular membranes.2–4 During a contraction re-
laxation cycle there is a large flux of Ca2+ ions from the SR to the contractile fila-
ments and back, causing relatively large (up to 100-fold locally) but short-lived
changes in [Ca2+]c. Excitation is also associated with a small influx of Ca2+ across
the cellular membrane (FIG. 1).5

FIGURE 1. Schematic diagram of a skeletal muscle cell showing the fluxes of Ca2+
during excitation. As the action potential moves along the cellular membrane and down into
the T-tubules, the electric signal is transferred to the SR, where it is converted to a chemical
signal by the release of Ca2+. The increase in [Ca2+]c activates contraction. The Ca2+ re-
leased from the SR is quickly reaccumulated into the SR by Ca2+-ATPases in the SR mem-
brane and contraction is stopped. Thus during contraction there is a large flux of Ca2+ from
the SR to the contractile filaments and back. The major storage compartment for Ca2+ is the
SR, although the mitochondria may also participate in Ca2+ regulation. Ca2+ influx can also
occur through Ca2+-conducting ion channels and Ca2+ is exported from the cell via the Ca2+-
ATPase in the cellular/T-tubular membrane or via Na+/Ca2+ exchange. (1) SR Ca2+-ATPase;
(2) SL Ca2+-ATPase; (3) Na+/Ca2+ exchanger.
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In the resting cell, approximately 5% of the total energy turnover is dedicated to

Ca2+ cycling, whereas during contraction Ca2+ cycling may consume 20–50% of the
total energy turnover.6

Transport Mechanisms
The skeletal muscle cell has several ways of clearing Ca2+ from the cytoplasm. The
most important is the SR Ca2+-ATPase, which is responsible for reuptake of Ca2+ into
the SR. It has a high affinity for Ca2+ (Km < 0.5 µM) on the cytoplasmic side and a
maximum velocity of 6.4 and 2.4 µmol/g fiber protein/min in fast and slow single hu-
man fibers, respectively.7 The sarcolemmal and T-tubular Ca2+-ATPases (SL Ca2+-
ATPase) actively export Ca2+ from the muscle cell. These are high-affinity (Km =
0.5 µM) but low-capacity systems (12–30 nmol Ca2+/mg protein/min in rabbit white
muscle)2,3 that are active in regulating [Ca2+]c especially under resting conditions.2,8
A second export mechanism is Na+-Ca2+ exchange. Na+-Ca2+ exchange has been
widely studied in cardiac muscle, where it plays a major role in Ca2+ regulation. In
skeletal muscle the capacity and quantitative importance of Na+-Ca2+ exchange is
more controversial. The existence of Na+-Ca2+ exchange in mammalian skeletal
muscle has been documented in several studies.9,10 It is a low-affinity but high ca-
pacity system. In sarcolemmal vesicles the transport rate was 5–10-fold lower and
Km for Ca2+ was an order of magnitude higher in Na+/Ca2+ exchange compared to
Ca2+-ATPase transport.3,11 Studies on frog muscle and single mouse fibers showed
that Na+/Ca2+ exchange activity was low during normal resting conditions and only
becomes measurable when [Ca2+]c is increased substantially above 80 nM.10 Thus it
was suggested that Na+-Ca2+ exchange may be active in Ca2+ removal at high
[Ca2+]c, as during fatiguing stimulation,10 and may participate in the long-term reg-
ulation of myoplasmic Ca2+.9

Internal Storage of Ca2+

The muscle cell is capable of storing large amounts of Ca2+. Two organelles are
involved in Ca2+ storage: the sarcoplasmic reticulum (SR) and the mitochondrion.

The Sarcoplasmic Reticulum (SR)

The major storage compartment of the muscle cell is the SR, which is tradition-
ally considered the organelle responsible for the ambiguous regulation of Ca2+. Be-
cause of the presence of the Ca2+-binding protein calsequestrin, the buffer capacity
of the SR for Ca2+ is large. Calsequestrin is capable of binding 40–50 moles of Ca2+/
mol.1 The total concentration of Ca2+ within the SR at rest has been reported to be
11 and 21 mM for slow-twitch and fast-twitch fibers, respectively, whereas the con-
centration of free Ca2+ is kept around 1 mM.12 Studies have shown that at resting
[Ca2+]c the SR of slow-twitch fibers is saturated with Ca2+, whereas the SR of fast
twitch fibers is only one-third saturated.12 This means that the buffering capacity of
the SR in slow-twitch fibers is smaller than that of the fast-twitch fibers.

Mitochondria
As early as the 1960s, it was discovered that the mitochondria were capable of
taking up large amounts of Ca2+.13 In mitochondria Ca2+ uptake occurs via the Ca2+
GISSEL: CA2+ AND MUSCLE DAMAGE 169

uniporter located in the inner membrane of the mitochondrion. The Km for the uni-
porter is between 10 and 100 µM.14 Since global increases of [Ca2+]c to this range
are usually only seen under pathologic conditions it was seriously questioned
whether Ca2+ was taken up by the mitochondria under physiological conditions. Re-
cently the mitochondria have reemerged as active players in the regulation of Ca2+.
On the basis of 3-D fluorescence imaging, it has been proposed that the mitochon-
dria are located close to Ca2+-release channels of the SR, and upon activation the
mitochondria sense highly localized increases in [Ca2+] (20–30 µM) as it is being
released. The high local [Ca2+] enables the uniporter to take up Ca2+.15 Recently it
was shown in vivo in mouse skeletal muscle that mitochondria take up Ca 2+ during
contraction and release it during relaxation. The mitochondrial Ca2+ uptake was de-
layed by a few milliseconds compared with the cytosolic rise and occurred both dur-
ing a single twitch and upon tetanic contraction.16 Thus, it has become apparent that
the mitochondria may play an important role in the regulation of Ca2+ in skeletal
muscle.

EFFECTS OF CA2+ OVERLOAD

Ca2+ overload, defined as an excessive accumulation of Ca2+ in the muscle cell,

resulting in an inability of the muscle cell to control [Ca2+]c, is a serious condition
involved in a wide range of situations leading to muscle cell damage.
As mentioned previously, Ca2+ is an important messenger in skeletal muscle, ca-
pable of activating a wide range of processes. Therefore, it is vital to the muscle cell
to tightly control [Ca2+]c, a function performed by the combined action of the SR,
the mitochondria, and the SL Ca2+-ATPase. In addition fast muscle fibers from lower
vertebrates and small mammals contain high amounts of the cytosolic Ca2+-binding
protein parvalbumin.17 In humans parvalbumin is only present in the intrafusal fibers
and is therefore not important in Ca2+ regulation in skeletal muscle.18
If the permeability of the sarcolemma for Ca2+ is increased (e.g., due to damage
or opening of Ca2+-conducting channels), a larger influx of Ca2+ will occur, increas-
ing the Ca2+ load on the cell. Because of the large storage capacity of the SR and the
mitochondria this may not necessarily present a problem for the cell, as the extra
Ca2+ can be stored. However, if the increased influx of Ca2+ persists, Ca2+ overload
of the cell may result.
Increases in [Ca2+]c may result in activation of Ca2+-activated neutral proteases
(calpains), activation of PLA2, an increased production of ROS, and/or mitochondri-
al Ca2+ overload. Each of these will be discussed in the following sections.

Calpain
It has become increasingly clear over the past years that calpains play an impor-
tant regulatory role in cellular functions. Three types of calpains are of interest in
skeletal muscle: calpain 1 (µ-calpain), calpain 2 (m-calpain), and calpain 3 (p94).
The latter is almost exclusively expressed in skeletal muscle19 and the amount of
mRNA for this calpain is 10 times higher than mRNA for calpains 1 and 2.20
The three calpains differ in their sensitivity to Ca2+. The threshold for initial ac-
tivation is lowest for calpain 3 (approximately 0.5 µM),21 while for calpain 1 it is
170 ANNALS NEW YORK ACADEMY OF SCIENCES

3 µM and for calpain 2 it is 400 µM. However, when the protein is autolyzed, pro-
teolytic activity occurs at [Ca2+] of 0.5–2 µM for calpain 1 and 50–150 µM for
calpain 2.19 Recent evidence suggests that autolysis is necessary for activation of
calpain 3.22
Calpains 1 and 3 are expected to be activated at physiological [Ca2+] and are
thought to be important in the regulatory function of the cell. Calpain 2, with the
higher Ca2+ requirement, is thought to be involved in fiber degeneration.23
Calpain cleaves a variety of protein substrates including cytoskeletal, myofibril-
lar, and membrane proteins. In particular titin (α-connectin), a large elastic protein
that tethers the thick filament at the center of the sarcomere, by anchoring it to the
Z-line, is readily proteolyzed by calpain.19 Calpain is closely associated with the I
and the Z band regions and calpain-mediated degradation is thought to contribute to
the changes in muscle structure (Z-line streaming) and function (EC uncoupling)
that occur immediately after exercise.24–26 Increases in calpain activity in muscle
has been observed during prolonged running,26,27 following chronic low-frequency
stimulation,23 in sepsis28 and in muscle from mdx mice (the murine model for
Duchennes muscular dystrophy).29

PLA2

PLA2 may be activated by elevated [Ca2+]c.30 The activated PLA2 will attack mi-
tochondrial and other membrane phospholipids, giving rise to lysophospholipids and
free fatty acids. Lysophospholipids will disrupt membrane lipid organization and
free fatty acids may have a detergent action, causing membrane damage.31 Among
the free fatty acids liberated is arachidonic acid, which may promote ROS produc-
tion by the mitochondria (see the next section). There is evidence that increased
[Ca2+]c activates PLA2, based on the observation that enzyme efflux from experi-
mentally damaged muscles (suffering Ca2+ overload) can be blocked by PLA2 inhib-
itors.31,32 NDGA (a lipo-oxygenase inhibitor) completely inhibited CK efflux from
skeletal muscle following treatment with the Ca2+ ionophore A23187 or DNP (dini-
trophenol, poisoning of the mitochondria) showing that PLA2 activation, and in par-
ticular the lipooxygenase pathway, affects cellular membrane integrity.33

Reactive Oxygen Species

The mitochodria of skeletal muscle cells continuously generate ROS. Increased

Ca2+ load on the muscle cell may increase superoxide anion synthesis. Enzymatic
synthesis by xanthine oxidase is stimulated by increased [Ca2+]c.34 Furthermore, it
has been shown that mitochondrial H2O2 production is stimulated by physiological
levels of ADP and calcium, possibly via a PLA2-mediated pathway.35
Oxidative stress may disrupt the normal Ca2+ handling kinetics in muscle cells by
disturbing SR function36 and may be a causative factor in opening of the mitochon-
drial permeability transition pore (mPTP), especially in combination with high mito-
chondrial Ca2+ content ([Ca2+]m) (see the next section).37,38 Oxidative stress leads
to lipid peroxidation, affecting membrane structures in the cell, which has been
shown following exhaustive exercise in both human and animal studies.39–41
GISSEL: CA2+ AND MUSCLE DAMAGE 171

Mitochondrial Ca2+ Overload

In the healthy cell, mitochondria may assist in refilling SR Ca2+ stores by inter-
action with store-operated Ca2+ channels;42 furthermore the mitochondria may act
as Ca2+ buffers, rapidly taking up surplus Ca2+ and slowly releasing it again to avoid
detrimental oscillations in [Ca2+]c. If the muscle cell faces increased influx of Ca2+
from the outside, the mitochondria will accumulate Ca2+.
It is by now well established that small increases in mitochondrial Ca2+ content
([Ca2+]m) stimulate ATP synthesis. Uptake of Ca2+ during activity may therefore
serve as an important signal to ensure that energy production matches energy expen-
diture. However, larger accumulations may have detrimental effects. As mentioned
above, increased Ca2+ load of the mitochondria will increase ROS production, and
with it, the risk of increasing nonspecific inner membrane permeabilization.43 In-
creases in [Ca2+]m also increase the probability of opening of the permeabilization
transition (mPTP) pore.37 The mPTP is a large conductance pore spanning both the
inner and outer membrane of the mitochondria. The pore opens most clearly under
specific and usually pathologic conditions. Opening leads to depolarization of the
mitochondrial membrane potential, effectively shutting down ATP production and
resulting in a massive efflux of Ca2+ from the mitochondria, causing further rises in
[Ca2+]c and initiating a vicious cycle leading to apoptosis or necrosis.37,38

The Vicious Cycle

The vicious cycle model describes how the above-mentioned factors act in con-
cert, creating a self-reinforcing cycle that leads to muscle cell necrosis/apoptosis
(FIG. 2).
An increase in Ca2+ influx across the cellular membrane is observed in many sit-
uations (some of which I will discuss in detail later). Influx of Ca2+ will lead to lo-
cal increases in [Ca2+] in the subsarcolemmal compartments, which may lead to
local activation of calpain. Some of the Ca2+ will be exported again by the SL Ca2+-
ATPase and some will be taken up by the SR via the SR Ca2+-ATPase and stored.
The SR is capable of storing quite large quantities of Ca2+. However, buffer capac-
ity differs between different fiber types. The SR of fast-twitch fibers is capable of
increasing their Ca2+ content threefold, whereas the SR of slow-twitch fibers is al-
most saturated at resting [Ca2+]c. Thus in the fast-twitch fibers, the SR represents a
large buffer capacity, whereas slow-twitch fibers most likely rely on their more
abundant mitochondria for Ca2+ buffering. In addition fast fibers from lower verte-
brates and small mammals contain high concentrations of parvalbumin,1 which may
act as a significant buffer for Ca2+.
The mitochondria will participate actively in the Ca2+ handling, particularly at
the time of SR saturation. Mitochondria are capable of storing large amounts of
Ca2+; however, if the storage capacity is exceeded, detrimental consequences may
occur. Increased Ca2+ load leads to increased production of ROS, causing peroxida-
tion of membrane lipids, and possibly decreases in ATP synthesis. Mitochondrial
Ca2+ overload also increases the risk of opening of the mPTP, which will ultimately
lead to apoptosis or necrosis. Local increases in [Ca2+]c may lead to PLA2 activa-
tion, resulting in degradation of the cellular membrane as well as the membranes of
organelles. This leads to an increased production of arachidonic acid leading to fur-
172 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. The “vicious cycle” model of how an initial event or condition leading to
an increased influx of Ca2+ may result in muscle cell apoptosis or necrosis.

ther increases in production of ROS in the mitochondria. Finally, activation of

calpain is expected, leading to degradation of cytoskeletal, myofibrillar, and mem-
brane proteins. These processes ultimately lead to an increased permeability of the
cellular membrane, causing further influx of Ca2+, and the vicious cycle is activated.
The muscle cell has a high capacity for repairing damages to the membrane,44
and therefore if the degradation process is stopped in time, the vicious cycle may be
halted and the muscle cell may recover. However, a “point of no return” exists, be-
yond which it is not possible for the cell to regain control of cellular Ca2+, eventually
leading to the destruction of the cell.

CA2+ OVERLOAD SCENARIOS

Ca2+ overload occurs in a wide range of situations, and even though many of
these situations seems to differ, a common denominator across the following exam-
ples is an initial increase in Ca2+ influx leading to muscle Ca2+ overload and muscle
damage.
One of the serious consequences of muscle Ca2+ overload is rhabdomyolysis, a
condition that is characterized by muscle tissue breakdown, resulting in the release
of cytosolic components into the blood circulation. The degree of rhabdomyolysis
ranges from an asymptomatic illness with elevation in the plasma CK level to a life-
threatening condition associated with extreme elevations in plasma CK, electrolyte
imbalances, acute renal failure, and disseminated intravascular coagulation.45 Hypo-
calcemia is frequently observed during rhabdomyolysis owing to massive influx of
Ca2+ into the muscle tissue. Administration of Ca2+ in this situation should be very
carefully considered as clinical investigations have shown that it may worsen the
damage considerably.46
GISSEL: CA2+ AND MUSCLE DAMAGE 173

Exercise

It is well documented in both human and animal studies that prolonged or unac-
customed excercise, and in particular lengthening (eccentric) exercise, may lead to
muscle cell damage.47–49 This damage may be ultrastructural alterations leading to
loss of force or loss of sarcolemmal integrity leading to loss of intracellular enzymes,
such as lactate dehydrogenase (LDH) or CK. Although not necessarily a pathologic
phenomenon, it is a frequently occurring consequence of certain types of contractile
activity, and might serve as an adaptive response to exercise. However, if the exercise
is excessive, rhabdomyolysis may occur.
Much attention has been diverted to the milder forms of muscle damage—in par-
ticular, delayed onset muscle damage (DOMS), since it is of great importance for
muscle performance in athletes. A model for the development of DOMS was pro-
posed by Armstrong in 1990.49 The model describes four stages in exercise-induced
muscle injury: (1) the initial stage; (2) the autogenic stage, or the “Ca2+ overload”
stage; (3) the phagocytic stage; and (4) the regenerative stage. The autogenic stage
is characterized by Ca2+ overload. This is supported by evidence that exercise leads
to an increased activation of calpain26 as well as PLA2,50 both of which are activated
by increased [Ca2+]c. Activation of calpain and PLA2 may then further worsen the
damage as described above in the section on the vicious cycle. The increased pro-
duction of ROS may also have deleterious effects on muscle cell integrity.
There has been much debate on the nature of the initial event triggering DOMS.
Some believe that mechanical damage to the fiber initiates exercise-induced muscle
damage.48 This may very well be the case during lengthening contractions, where
the strain on the muscle fiber is high. Our lab has shown that excitation of rat skeletal
muscle both in situ and ex vivo (isometric and shortening contractions) is associated
with an increased influx of Ca2+ (up to 34-fold increase within the first 30 sec of
stimulation) and that prolonged stimulation may lead to accumulation of Ca2+ in the
muscle cells and loss of membrane integrity.5,51,52 Similar, but smaller accumula-
tions of Ca2+ in muscles from athletes after prolonged running has also been report-
ed.47,53 We suggest that the excitation-induced influx of Ca2+ may act as or
contribute to the initiating factor leading to muscle damage during prolonged or oth-
erwise excessive exercise.

Trauma

Trauma affects muscle membrane integrity directly. In this section I will discuss
mechanical and electrical trauma both leading to an increased influx of Ca2+ al-
though by two very different mechanisms.
Mechanical trauma causes direct injury to the cellular membrane causing Ca2+
and Na+ to flood the injured tissue. Often circulation is compromised, causing is-
chemia of the tissue, and the damage process may accelerate in the following reper-
fusion period. The effect is a massive Ca2+ overload of the muscle cells, resulting in
necrosis of many fibers.45
Electrical trauma due to high-voltage electrical injury or lightening may lead to
electroporation of cellular membranes, rendering them permeable to ions and mole-
cules. This will result in an increased influx of Ca2+.54 If resealing does not occur
rapidly enough, cellular Ca2+ homeostasis will be lost, leading to destruction of the
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muscle cell. (Electric force–mediated membrane disruption will be reviewed else-

where in this issue by Raphael Lee.)

Ischemia
Prolonged ischemia and reperfusion results in tissue damage and necrosis in
many organs.
Prolonged ischemia is associated with hypoxia. Results from this laboratory as
well as others have shown that anoxia increases the permeability of the membrane
for Ca2+ and may lead to a loss of cellular integrity.55–57 The mechanism behind the
increased influx of Ca2+ has not been identified, but if anoxia was combined with
electrical stimulation, possibly depleting cellular ATP stores, the stimulation-
induced accumulation of Ca2+ was markedly increased and so was the release of
LDH.55 During prolonged ischemia/anoxia, ATP levels would be expected to be low,
possibly compromising the Ca2+ handling ability of the muscle cell. In line with this
increasing baseline [Ca2+]c was observed during fatiguing stimulation under
hypoxic conditions.58
In vivo reperfusion may be associated with an increased generation of ROS from
circulating neutrophils, causing further damage to the cellular membrane.59

DMD
The human Duchenne muscular dystrophy (DMD) is the best known of the mus-
cular dystrophies, since it is one of the most frequent genetic human diseases.1 It is
characterized by muscle fiber necrosis and progressive muscle wasting and weak-
ness. Ca2+ seems to be critically involved in the pathology of DMD. The primary de-
fect in DMD is the lack of dystrophin, a subsarcolemmal cytoskeletal protein. In
normal muscle dystrophin links the cytoskeleton, via a complex of membrane pro-
teins, to laminin in the extracellular matrix mechanically stabilizing the cellular
membrane.60 Lack of dystrophin renders the sarcolemma more vulnerable to me-
chanical damage. The number of transient disruptions of the sarcolemma was found
to be sixfold higher in muscles from exercised mdx mice compared to controls.61
These transient disruptions may give rise to Ca2+ entry, leading to local activation of
calpain. Studies have shown that [Ca2+] in the subsarcolemmal compartments was
4.5-fold higher in mdx myotubes following depolarization compared to controls.62
Furthermore an increased calpain activity has been observed in muscle from mdx
mice.29 It was suggested that the activation of calpain would lead to proteolysis of
the Ca2+ leak channels (possibly TRP channels63), causing an increased activity.
This in turn would further increase Ca2+ influx and Ca2+-activated proteolysis.64 An
increased Ca2+ leak channel activity has been observed in mdx fibers and this was
thought to be the mechanism behind the greater levels of Ca2+ influx, leading to in-
creased [Ca2+]c and increased proteolysis.65 In addition to the increased calpain ac-
tivity an increase in PLA2 activity was shown in DMD patients.66

Muscle Cachexia
Muscle cachexia is a common metabolic response to several disease states seen
in surgical patients including sepsis, severe injury, renal failure, and cancer.67 Al-
though not identical, the intracellular mechanisms and molecular regulation of mus-
GISSEL: CA2+ AND MUSCLE DAMAGE 175

cle atrophy are similar in many of these conditions. The early phase of muscle
cachexia may be beneficial, providing amino acids for gluconeogenesis and acute-
phase protein synthesis. However, prolonged and severe cachexia has significant del-
eterious consequences.67
It is quite likely that early cachexia is associated with an increased influx of Ca2+.
Sepsis has been shown to increase 45Ca uptake and Ca2+ content in isolated rat skel-
etal muscle, which was associated with an increased protein breakdown.68,69 Fur-
thermore, muscle cachexia induced by severe injury, sepsis, and cancer is associated
with increased gene expression and activity of calpain 1, 2, and 3.67 It has been pro-
posed that early cachexia is associated with a calcium-calpain–dependent degrada-
tion of Z-disks and release of myofilaments from the myofibrils. This is followed by
ubiquitination of the myofilaments and a subsequent degradation by the protea-
some.67 Supported for this was provided by morphologic evidence showing disinte-
gration of the Z-disk and release of myofilaments from myofibrils in cachectic
muscle from septic rats. Treatment with dantrolene (inhibiting the release of calcium
from intracellular stores) prevented the sepsis-induced release of myofilaments.70
In addition, mitochondria from septic animals demonstrate substantially higher
(PLA2-mediated) H2O2 formation compared to controls,35 increasing the oxidative
stress on the muscle fiber.

COUNTERACTING MEASURES

Inhibiting Influx of Ca2+

One obvious way of preventing Ca2+-induced muscle damage is to inhibit or re-
duce the increase in [Ca2+]c. This can be done by resealing of the cellular membrane
or reducing Ca2+ flux through Ca2+ channels in the sarcolemma and SR membrane.

Resealing
A lot of research has gone into developing agents that assist in resealing or stabi-
lization of the membrane. Muscle damage can be considered as a continuous process
of membrane degradation and repair. Assisting membrane repair may stop the vi-
cious cycle, resulting in survival of the muscle fiber. The use of synthetic surfactants
to stabilize membranes and thus restore cellular integrity has yielded promising re-
sults both in vitro and in vivo.71 (Membrane-sealing therapy will be reviewed else-
where in this volume by Lee and Lee.)

Ca2+ Channel Blockers

A certain influx of Ca2+ occurs via the L-type Ca2+ channels in the outer mem-
brane and in the T-tubules when the muscle is excited. If the muscle cell experiences
an increased Ca2+ load, blocking any further influx may allow the muscle cell to
maintain control over Ca2+. Studies using the Ca2+ channel blockers have showed a
reduction in the exercise-induced damage both in human and animal studies.72,73
The ryanodine receptor Ca2+-release channels in the SR likewise release Ca2+
into the cytoplasm following an action potential. Blocking of this (by, for example,
dantrolene) has proven efficient during malignant hyperthermia and sepsis.70
176 ANNALS NEW YORK ACADEMY OF SCIENCES

Inhibiting the Actions of Ca2+

A second approach to prevent or reduce muscle damage is to prevent the conse-

quences of the increased [Ca2+]c. This may be done by blocking the actions of
calpain, PLA2, and ROS.

Calpain Inhibitors
Much discussion regarding the role of calpain in muscle damage has come from
the conflicting evidence that an increase in [Ca2+]c results in cellular damage, but in
some cases this could not be prevented by the use of calpain inhibitors. This discrep-
ancy may be the result of the different types of calpain, all with different character-
istics. The calpain inhibitor calpastatin only inhibits calpains 1 and 2, with little
effect on calpain 3. Leupeptin on the other hand inhibits all three calpains, but calpain
3 needs to undergo autolysis before leupeptin can bind to the active site.22 Ca2+ caus-
es autolytic activation of single calpain 3 molecules, and these can then proteolyti-
cally activate other nearby calpain 3 molecules in a strongly self-reinforcing
activation cascade.74 Since leupeptin cannot bind before calpain 3 is autolyzed it may
not be possible for leupeptin to prevent proteolytic activity of calpain 3 when [Ca2+]
is appreciably above the threshold level for calpain activation.25 However, if the ac-
tivating [Ca2+] is relatively low (e.g., ~2 µM) the actions of calpain may be blocked
by leupeptin.25 This may be the case in DMD/mdx, where it was recently shown that
muscle fiber degeneration in mdx mice was delayed by intramuscular administration
of leupeptin.29

PLA2 Inhibitors
Inhibition of PLA2 has been found to reduce enzyme release (e.g., CK and LDH)
from skeletal muscle,31,32 although PLA2 inhibitors failed to protect against myo-
fibrillar damage.33 Thus blocking PLA2 activity may reduce further damage to the
membrane, but does not provide protection for structural damage within the muscle
fiber.

ROS Scavengers
There is little doubt that ROS are involved in the development of skeletal muscle
damage over a range of situations. Production of ROS is dramatically accelerated
during contractile activity, mainly owing to the increased oxygen consumption.34
Many studies have shown that an increased Ca2+ load also leads to an increased pro-
duction of ROS. In addition, as mentioned above (Section 3.3), the actions of ROS
may result in an increased Ca2+ load on the muscle cell.
The effect of antioxidants in reducing muscle damage after exercise has been
moderate, with some studies showing no effect and others showing small benefits.
Recent studies have suggested that xanthine oxidase–mediated oxidative stress leads
to useful cellular adaptions to exercise and that the practice of taking antioxidants
prior to exercise may have to be re-evaluated.75
Nevertheless antioxidant treatment may prove to be beneficial in other situations
(e.g., sepsis), where a pathologic increase in ROS production leads muscle cell
damage.
GISSEL: CA2+ AND MUSCLE DAMAGE 177

CONCLUDING REMARKS

There is good reason to believe that Ca2+ plays an important role in the develop-
ment of many types of muscle damage. Situations of an increased influx of Ca2+ may
initiate a vicious cycle, leading to muscle damage and death. Therefore, inhibiting
the actions of Ca2+ may be important in preventing or reducing skeletal muscle dam-
age. This may involve the use of a combination of compounds, as in combining
membrane-stabilizing agents with antioxidants and calpain and PLA2 inhibitors.

ACKNOWLEDGMENTS

This study was supported by the Danish Medical Research Council (Grant 22-02-
0523). Thanks are extended to Dr. William McDonald for useful comments on the
manuscript.

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