Skeletal muscle fatigue – regulation of excitation–contraction coupling to avoid metabolic catastrophe

Brian R. MacIntosh1,*, Robert J. Holash1 and Jean-Marc Renaud2

1Human Performance Laboratory, Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada2Department of Cellular and Molecular Medicine, Neuromuscular Research Centre, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5,Canada

*Author for correspondence (brian.macintosh@ucalgary.ca)

Journal of Cell Science 125, 2105–2114� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.093674

SummaryATP provides the energy in our muscles to generate force, through its use by myosin ATPases, and helps to terminate contraction bypumping Ca2+ back into the sarcoplasmic reticulum, achieved by Ca2+ ATPase. The capacity to use ATP through these mechanisms issufficiently high enough so that muscles could quickly deplete ATP. However, this potentially catastrophic depletion is avoided. It has been

proposed that ATP is preserved not only by the control of metabolic pathways providing ATP but also by the regulation of the processes thatuse ATP. Considering that contraction (i.e. myosin ATPase activity) is triggered by release of Ca2+, the use of ATP can be attenuated bydecreasing Ca2+ release within each cell. A lower level of Ca2+ release can be accomplished by control of membrane potential and by direct

regulation of the ryanodine receptor (RyR, the Ca2+ release channel in the terminal cisternae). These highly redundant control mechanismsprovide an effective means by which ATP can be preserved at the cellular level, avoiding metabolic catastrophe. This Commentary willreview some of the known mechanisms by which this regulation of Ca2+ release and contractile response is achieved, demonstrating thatskeletal muscle fatigue is a consequence of attenuation of contractile activation; a process that allows avoidance of metabolic catastrophe.

Key words: Endurance, Membrane excitability, Skeletal muscle contraction

IntroductionTypically, when exercise scientists think of control of skeletal

muscle contraction, they consider motor unit recruitment and ratecoding, the primary means by which the brain regulates the

magnitude of muscle contraction. However, the magnitude ofskeletal muscle contraction can also be regulated at the cellular

level. This level of control is necessary to avoid cellular

metabolic catastrophe, i.e. the situation in which ATP depletionreaches a level that is damaging to the fiber.

ATP is the common final source for energy in the cell, and itsconcentration [along with the concentrations of ADP and

inorganic phosphate (Pi)] affects the magnitude of energy thatis made available from its hydrolysis. At rest, skeletal muscle has

a low metabolic rate, not unlike many other passive tissues, and

the intracellular concentration of ATP is in the 5–7 mM range(Hochachka and Matheson, 1992). However, during muscle

activity, there are three major ATPases that require ATP for their

function (Box 1); during exercise, skeletal muscle can increase itsrate of ATP use by more than 100 times (Hochachka and

McClelland, 1997), a rate that is much faster than can bereplenished by aerobic metabolism. This latter point is clear from

the fact that the muscle power output can greatly exceed the level

that can be supported by aerobic metabolism (MacIntosh et al.,2000). To accommodate this additional energy requirement, ATP

is provided by non-aerobic means (through phosphocreatine

hydrolysis and glycolysis resulting in lactate formation), but thisalternative source of ATP has limited capacity (Monod and

Scherrer, 1965). For this reason, a high rate of ATP use cannot besustained in muscle and ATP use must thus be regulated

(Hochachka, 1994; Hochachka and Matheson, 1992; Myburgh,

2004). We know that muscle fibers can preserve ATP because theATP level usually decreases by only 20–25% during exercise(Argov et al., 2000; Dawson et al., 1978) and rarely falls below

50% of its initial value (Karatzaferi et al., 2001; Spriet et al.,1987).

We have proposed that the cellular regulation of ATP use is the

principal mechanism for the decreased contractile response that isassociated with acute repetitive muscle use, typically calledmuscle fatigue (MacIntosh and Shahi, 2011). We also have

recognized that regulating Ca2+ release from the sarcoplasmaticreticulum (SR), and thus the intracellular Ca2+ concentration

([Ca2+]i), dictates the rate of ATP use in the muscle cell becauseit affects Ca2+ ATPase and myosin ATPase activity, whichtogether account for 90% of ATP use (Box 1). It should be kept in

mind that a multitude of highly redundant factors contribute tothis regulation of ATP usage. We will not present all of thesehere. Instead, this Commentary concentrates on the following

three major areas: (1) regulation of the contractile proteins actinand myosin by Ca2+ and myosin light chain phosphorylation, (2)

regulation of membrane excitability through K+ and Cl2

channels, and (3) the regulation of Ca2+ availability and the SRCa2+ release channel. A summary of the process of excitation–

contraction coupling is presented in Fig. 1 and described in detailin Box 1. This figure gives the context for the remainingdiscussion in this Commentary.

Regulation of the interaction between actinand myosinMuscle contraction is the consequence of the interaction betweenactin and myosin. When the myosin head binds to actin, forming

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a cross-bridge, it undergoes a sequence of reactions referred to as

the cross-bridge cycle (Box 2). The force of isometric contraction

is primarily a function of the number of cross-bridges that are

engaged simultaneously in the strong-binding state. This number

is governed, at a given sarcomere length, by the proportional

occupation of troponin with Ca2+ and the rate constants for the

transition to the strong-binding state, as well as the dissociation

of the cross-bridge. Troponin is a regulatory protein associated

with the thin filament. As explained in Box 1, binding of Ca2+ to

troponin allows cross-bridge cycling to occur.

When force is plotted as a function of [Ca2+], a sigmoid curve

is obtained. At very low [Ca2+], no force is generated (Fig. 2).

Resting [Ca2+] is in this range. Next, there is a range for which

increases in [Ca2+] are associated with increases in force, as

additional binding of Ca2+ to troponin reveals an increased

number of actin myosin interaction sites. This range of [Ca2+] is

referred as submaximal [Ca2+]. Finally, there is a range, for

which increases in [Ca2+] produce no further increase in force

because, in that range, all Ca2+-binding sites of troponin are

occupied; i.e. the contractile apparatus is maximally activated.

The [Ca2+], at which force is half-maximum is known as Ca50

and reflects the Ca2+ sensitivity of the contractile components. A

decrease in Ca50 is observed when the force–[Ca2+] curve is

shifted towards lower [Ca2+] and represents an increased Ca2+

sensitivity.

There are two primary mechanisms that affect Ca2+ sensitivity,

altered binding of Ca2+ to troponin and altered cross-bridge

kinetics. Cross-bridge kinetics is the more dominant of these

mechanisms and its kinetics is affected by several factors,

including: temperature, pH, sarcomere length, myosin regulatory

light chain (RLC) phosphorylation, and concentration of Pi. How

these factors affect cross-bridge kinetics and Ca2+ sensitivity has

been discussed in detail elsewhere (MacIntosh, 2003). Here, we

will discuss the mechanism for increased Ca2+ sensitivity that is

affected by myosin RLC phosphorylation.

Myosin RLC phosphorylation increasesCa2+ sensitivityThere are two myosin light chains (small peptides) associated

with each myosin head. One of these light chains, the RLC, can

be phosphorylated by myosin light chain kinase (MLCK). In

resting muscle, the RLCs of myosin are mostly unphosphorylated

(MacIntosh et al., 1993). When [Ca2+]i increases during

contraction, Ca2+ binds to another small peptide, calmodulin,

and the Ca2+–calmodulin complex subsequently binds to MLCK,

thereby activating it. Therefore, the repetitive activation of

muscle leads to a progressive phosphorylation of RLCs

(MacIntosh et al., 1993). MLCK is eventually deactivated when

[Ca2+]i decreases to resting levels and RLC is dephosphorylated

over the course of a few minutes by the slow activity of myosin

light chain phosphatase (Hartshorne et al., 1998).

When muscles are elicited to contract with a single stimulus,

force rapidly rises then falls in a contraction called a twitch. Peak

twitch force represents about 5–20% of the maximum force a

muscle can generate when stimulated at high frequency

(.150 Hz, yielding a completely fused tetanic contraction).

When a twitch is elicited immediately after a 1-second long

tetanus, the peak force is much higher than that measured before

the tetanic contraction; this phenomenon is known as post-tetanic

twitch potentiation. When muscles are stimulated at frequencies

below 10 Hz, each subsequent contraction develops more force

for the first several seconds, a phenomenon known as staircase.

During staircase and post-tetanic potentiation, the twitch peak

rate of force development increases proportionally to the increase

in active force, whereas the timecourse of the twitch is not altered

(Hainaut and Desimedt, 1968; MacIntosh and Gardiner, 1987).

Both staircase and post-tetanic twitch potentiation are thought to

be due to RLC phosphorylation (MacIntosh et al., 1993;

MacIntosh and Rassier, 2002; Moore and Stull, 1984). Skinned

fiber experiments have confirmed that RLC phosphorylation, as

achieved with addition of MLCK and calmodulin, results in

increased Ca2+ sensitivity (Persechini et al., 1985), which now

has been related to changes in cross-bridge kinetics, as explained

below.

Sweeney and Stull (Sweeney and Stull, 1990) addressed the

question of whether potentiation relies on an increased number of

engaged cross-bridges or an increased force per cross-bridge.

They used the quick longitudinal stretch of the muscle to measure

stiffness, which is assumed to be proportional to the number of

cross-bridges engaged in the strong-binding state. They reported

that longitudinal stiffness increases in proportion to the increased

active force at submaximal [Ca2+]i when myosin light chains are

phosphorylated by MLCK. This was interpreted as indicating that

potentiation is a function of increased number of cross-bridges

engaged, not an increase in force per cross-bridge. They also

reported that myosin ATPase activity increases in proportion to

the increased active force, indicating that the cycle time per

cross-bridge is not altered. This allowed them to conclude that

the increase in the number of cross-bridges is a function of an

Box 1. Excitation–contraction coupling andATP usage

Contractions in skeletal muscle are triggered when action potentials

are generated in the outer cell membrane (Fig. 1). Under normal

conditions, an action potential consists of a depolarization from a

resting membrane potential (Em) of 280 mV to +30 mV, followed by

a repolarization, returning Em to its resting value. The depolarization

depends on Na+ influx through voltage-sensitive Na+ channels (Nav),

and the repolarization involves K+ efflux through voltage-sensitive K+

channels (Kv). Each action potential propagates along the surface

membrane and into the transverse tubules (Fig. 1). Depolarization of

the transverse tubules is detected by voltage-sensitive Ca2+

channels (Cav), also known as dihydropyridine receptors (DHPRs),

and these communicate directly with Ca2+ channels, also known as

ryanodine receptors (RyRs), to release Ca2+ from the sarcoplasmic

reticulum. At low [Ca2+]i, tropomyosin blocks the myosin-binding site

on actin. When [Ca2+]i increases in response to an action potential, it

binds to troponin and a segment of actin, seven monomers long,

becomes available to myosin as one tropomyosin is moved away

from the myosin-binding site on actin. The number of such available

segments is a function of the [Ca2+]i. The subsequent binding of

myosin to actin generates force and/or shortening of sarcomeres.

There are three major proteins that contribute to the increased

ATP use during contraction: Na+/K+-ATPase, myosin ATPase and

Ca2+ ATPase (Fig. 1). The Na+/K+-ATPase pumps Na+ back out

and K+ back into the fiber after an action potential. The myosin

ATPase uses ATP to generate force and do work, whereas the

Ca2+ ATPase is responsible for pumping Ca2+ back into the SR to

allow muscle relaxation. Each of these ATPases, contributes ,10,

,60 and ,30% of total ATP use, respectively (Homsher, 1987).

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increased rate of engagement, not a decrease in the rate of

dissociation of cross-bridges (Sweeney and Stull, 1990).

Levine and colleagues have shown that RLC phosphorylation is

associated with a change in the way in which the myosin heads are

ordered along the thick filament backbone (Levine et al., 1996). In

the unphosphorylated state, the myosin heads are packed tightly

along the thick filament. However, phosphorylation of the RLC

results in disorder of the myosin heads. This disorder was

interpreted as an increased mobility, such that the myosin head

can swing away from the thick filament, bringing the myosin head

into close proximity to actin and, hence, increasing the probability

of its interaction with actin.

So, the regulation of [Ca2+]i during contraction and

phosphorylation of myosin light chain can have significant

impact on ATP usage. Although RLC phosphorylation is

typically associated with potentiation, this phosphorylation

persists when fatigue begins (MacIntosh et al., 1993). The

same Ca2+–calmodulin complex that activates MLCK also

inhibits Ca2+ release (Boschek et al., 2007; Xiong et al., 2006).

The muscle is capable of decreasing the release of Ca2+, while

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

+ + + + + + + + + + + +

Myosin and actin filaments

+

+ +

+ + +

+ ++ + + +

+ + + + + +

+ +

+ + +

+ + + + + + + + + + + + + + +

++

Sarcolemma

Sarcoplasm

Sarcolemma

3Na+

2K+

K+

K+

Na+

Na+

Na+

K+

ClC–

Ca2+

Ca2+

+ + + +

2Ca2+ 2Ca2+

K+ATP

3Na+

2K+

+ + + + + + + + +

Calmodulin(apoCaM andCaCaM)

DHPR II–III loop

Cytosolic side

Luminal side

S100A1

Calsequestrin

RyR

(DHPR)

(RyR)

Na+ channel

K+ channel

CIC channel

Ca2+-ATPase

Na+/K+-ATPase

K+ATP channel

Sarcoplasmic reticulum

MyosinATPase

2Ca2+

Mg�ATP ADP +Pi +Mg2+

Mg�ATP ADP +Pi +Mg2+

Mg�ATP

ADP +Pi +Mg2+

Key

T-tubule30 A

Myosin

Fig. 1. Excitation–contraction coupling and ATP usage in the cell. Here, the processes that use ATP are illustrated together with the proteins involved in

excitation–contraction coupling. ATP is used by the Na+/K+-ATPase at the surface and transverse tubule membranes, by myosin ATPase throughout the cell and

by Ca2+ ATPase in the membranes of the sarcoplasmic reticulum. The sequence of events in excitation–contraction coupling involve the following: action

potential is generated at the neuromuscular junction and propagates along the surface membrane and into the transverse tubules (gray arrows). The voltage sensor

DHPR detects the associated depolarization and opens the RyR to release Ca2+ into the sarcoplasm. Ca2+ diffuses throughout the cell and binds to several ligands,

including troponin and calmodulin. Binding of Ca2+ to troponin moves the tropomyosin away from the myosin-binding site on actin, permitting cross-bridge

cycling (Box 2). Removal of Ca2+ from the cytoplasm by Ca2+ ATPase results in the recovery of tropomyosin to its blocked position and relaxation occurs. The

detailed image of the RyR is reproduced from Song et al. (Song et al., 2011) with permission.

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preserving its contractile function through increasing Ca2+

sensitivity. Such a mechanism helps in preserving ATP and

prolonging the duration of any muscle activity. The importance

of this process is further illustrated by the fact that muscle

activation by motor neurons is at frequencies (Hennig and Lømo,

1985) that do not give rise to maximal increases in [Ca2+]i and

full force development (Westerblad and Allen, 1991); i.e. most of

the time [Ca2+]i is submaximal.

Analysis of the force–Ca2+ relationship also suggests that the

attenuation of Ca2+ release and thus lower [Ca2+]i can also be an

effective way of decreasing the activity of myosin ATPases as

well as Ca2+ ATPases. Myosin ATPase activity decreases

because less Ca2+ will bind to troponin, and therefore there

will be a reduced level of myosin–actin interaction. A reduction

in Ca2+ ATPase activity occurs because there would be less Ca2+

to pump back into the SR. Decreased Ca2+ release can therefore

be an effective way to preserve ATP during a metabolic stress.

There is considerable evidence that a reduction in Ca2+ release

does occur during repeated muscle activation, either through

reduced Ca2+ availability (Allen et al., 2011; MacIntosh and

Kupsh, 1987) or by regulating the process of its release (Li et al.,

2002). In the next sections, we will look at how changes in

membrane excitability affect Ca2+ release. This is followed by a

discussion of the regulation of the ryanodine receptor (RyR),

which is the Ca2+ release channel of the SR.

Control of membrane excitationAs mentioned above, the release of Ca2+ is triggered by an action

potential propagating through transverse tubules (Fig. 1). The

magnitude of Ca2+ release can be modulated by the amplitude

and shape of the action potential. Shape and amplitude are

affected by both ion concentration gradients and by modulation

0

0.2

0.4

0.6

0.8

1

8 7 6 5 4

Rel

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pCa2+

Fig. 2. Ca2+–force relationship. The relationship between [Ca2+] (expressed

as negative log) and active force is presented here for three conditions, control

(black), increased Ca2+ sensitivity (shifted to the left, red) and decreased Ca2+

sensitivity (shifted to the right, blue). Force is primarily a function of [Ca2+],

but clearly the force at a given [Ca2+] can be modified. Ca2+ sensitivity is

altered by several factors including sarcomere length, regulatory light chain

phosphorylation, pH, Pi concentration and temperature.

Box 2. Cross-bridge cycle

In the resting state, most of the myosin heads exist in a primed state [step (ii) in the figure]; that is, with the ATP partially hydrolyzed and the myosin

head cocked, ready for the power stroke. Although it is thought that a weak binding of the myosin head to actin can occur, even when the

tropomyosin is in the ‘blocked’ position [step (i) in the figure], the binding of Ca2+ to troponin moves tropomyosin to the ‘closed’ position, thereby

increasing the probability of this weak binding and permitting the rapid transition from weak to strong binding. The existence of one myosin head in

the strong binding state causes the tropomyosin to move further from the blocked position to a position referred to as ‘open’. This is a form of

cooperativity. The rate constant for the transition to strong binding is low without Ca2+ bound to troponin, but increases with binding of Ca2+ to

troponin. On going from a weak-binding state to a strong-binding state [step (iii) in the figure], inorganic phosphate (Pi) is released from the myosin

head and a swinging lever action generates force and/or permits relative motion of the thick and thin filaments. This strong-binding state will persist

until ATP replaces the ADP that remains after the Pi is released. ATP binding permits dissociation of the myosin head from actin [step (iv) in the

figure]. It is generally accepted that each cycle as described here results in the hydrolysis of one ATP molecule (Barclay, 2003; Gordon et al., 2000).

(ii) Closed (iii) Open

Pi

PartialATP hydrolysis

Ca2+

Ca2+(ii) Blocked

(i) Closed (i) Blocked (iv) Open

Ca2+

Tropomyosin

Myosin

Actin

ADP ADP*Pi ATPKey

Myosin ATPase

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of ion channel activity. During metabolic stress, it is desirable to

decrease Ca2+ release. Here, we will present the mechanisms by

which the cell modifies the amplitude and shape of the action

potential to decrease Ca2+ release. Through the discussion below,

we will see that some of the changes are meant to preserve

contractile function, but when ATP levels are threatened, the

changes result in decreased excitability, decreased Ca2+ release

and preservation of ATP.

To facilitate an understanding of the impact of ion

concentration gradients and permeabilities on the action

potential, it is worthwhile to begin with the resting condition.

The resting state of the cell and the changes in ion permeability

that occur during the action potential are presented in Box 3.

The role of K+ in membrane excitability

At rest, the normal extracellular [K+] ([K+]e) varies between 3.5

and 4.0 mM. However, this increases to 10–14 mM during

muscle activity (Juel et al., 2000; Mohr et al., 2004; Nielsen et al.,

2004; Street et al., 2005). In vitro at 37 C, maximal tetanic force

is not affected until the [K+]e reaches 10 mM; at or above that

concentration, tetanic force decreases (Cairns et al., 2011).

Twitch and submaximal tetanic forces, are potentiated when

[K+]e is increased up to 12 mM (Holmberg and Waldeck, 1980),

whereas above that concentration twitch force is depressed

(Yensen et al., 2002). The mechanism of the K+-induced

potentiation is unknown, but is no doubt beneficial for

maximizing muscle performance at the onset of exercise. The

K+-induced force depression, on the other hand, is due to a K+-

induced depolarization of the cell membrane resulting in

decreased action potential overshoot as Na+ channels are

inactivated (Yensen et al., 2002) (Fig. 3; Box 3). There is

indirect evidence that the amount of Ca2+ that is released by SR is

reduced once the action potential overshoot decreases below 5

mV; twitch force is unaffected when overshoot varies between 5

and 30 mV (Cairns et al., 2003; Yensen et al., 2002). It is also

important to note that the force–[K+]e relationship is dynamic;

i.e. the concentration at which K+ potentiates or depresses force is

modulated by several factors. Two of these factors are the

activities of the voltage-dependent chloride channel protein (ClC-

1, also known as CLC1 and CLCN1) and the ATP-sensitive K+

(KATP) channel, which are discussed in the following two

sections. Ion channel activity can influence action potential

amplitude because the amplitude of the action potential is

dependent not only on the resting membrane potential but, more

importantly, also on the exchange of ions during the action

potential through channels in addition to voltage-gated Na+ and

K+ channels (Nav and Kv, respectively), such as the Cl2 and the

KATP channels (Box 3).

Regulation and impact of the ClC-1

ClC-1 is the main channel in skeletal muscle by which Cl2

crosses the cell membrane; this channel therefore controls the

muscle membrane Cl2 permeability. In the resting state, Cl2

permeability largely exceeds that of K+ (Palade and Barchi, 1977;

Pedersen et al., 2009a; Pedersen et al., 2005; Pedersen et al.,

2009b) and plays a major role in the maintenance of resting

membrane potential (Em). For example, when [K+]e increases, the

resulting membrane depolarization is slower and smaller when

Cl2 is present in the muscle bathing solution than in its complete

absence (Dulhunty, 1978). This observation indicates that inward

movement of Cl2 counteracts the depolarizing effects of the

accumulation of K+ outside the cell.

ClC-1 can, under some conditions, directly influence the

amplitude of the action potential (Box 3). Under normal

conditions (i.e. 4 mM K+), removal of extracellular Cl2 does

not affect the kinetics of an action potential (Cairns et al., 2004),

because Na+ and K+ fluxes largely exceed that of Cl2. However,

the same is not true at high [K+]e. At 11 mM K+, ,50% of soleus

fibers are completely unexcitable and the remaining fibers

generate action potentials with peaks that only reach 210 mV

(Pedersen et al., 2005). Reducing Cl2 permeability by 50%

allows for a recovery of membrane excitability; the number of

fibers generating action potentials increases to 95% and action

potential peak reaches +10 mV. This recovery occurs because at

11 mM K+, a large number of Na+ channels are inactivated

resulting in a considerably reduced Na+ influx during the action

potential compared with that at 4 mM K+. This inactivation of

Na+ channels allows the Cl2 influx to strongly counteract the

smaller Na+ depolarizing effect, decreasing the action potential

amplitude. Lowering Cl2 permeability by closing Cl2 channels

lowers Cl2 influx, thus diminishing its ability to counteract the

Na+ depolarization and allowing for greater action potential

amplitude. Associated with the increase in membrane excitability

is an increase in force production (Pedersen et al., 2005).

Therefore, Cl2 has two opposing effects on membrane

excitability and force when the [K+]e is elevated; a complete

removal of Cl2 from the extracellular fluid exaggerates the K+-

induced membrane depolarization, loss of membrane excitability

and, subsequently, force, whereas the small decreases in Cl2

permeability that are achieved when up to 50% of the Cl2

Box 3. Action potential and its modulation by Cl2

and K+ permeability

Transmembrane movement of ions is affected by three factors, ion

permeability, concentration gradient and membrane potential (Em).

At rest, Em is about 280 mV and the cell membrane is impermeable

to Na+, somewhat permeable to K+ and most permeable to Cl2. Na+

and Cl2 are at a high concentration outside the membrane and a

low concentration inside. [K+] is high inside and low outside. The

action potential results from increased permeability to Na+ and K+,

while Cl2 permeability does not change. The depolarization phase

involves the activation of Na+ channels (Nav) and the resulting influx

brings Em to +30 mV. In normal conditions, this occurs despite a Cl2

influx due to the predominance of Nav activity. Inactivation of Nav

channels shortly after they open, and increasing activation of K+

channels (Kv) resulting in K+ efflux, and the continuing Cl2 influx

allow the repolarization of the membrane back to 280 mV.

Repolarization of the membrane reprimes Nav channels, so they

can be activated for another action potential. However, small

membrane depolarizations (i.e. less than 15 mV) at rest (e.g. an

increased [K+]e during muscle activity) inactivates a portion of the

Nav channels, decreasing their availability. As a consequence of

lower Nav channel availability there is less predominance of the Na+

inward movement during the depolarization phase. Now, any

changes in Cl2 and K+ channel activity will substantially impact

the rate of depolarization and the amplitude of action potentials. For

example, closure of Cl2 channels will decrease the Cl2 influx during

depolarization allowing for faster and greater depolarization. By

contrast, activation of Cl2 and K+ channels (to increase Cl2 influx

and K+ efflux) will slow down the depolarization and decrease the

action potential amplitude.

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channels are closed improves membrane excitability and forcegeneration.

The modulation of the effect of K+ by regulation of ClC-1discussed above is based on studies that use resting unfatigued

muscle fibers. If the observed modulation has any physiologicalsignificance, one would expect that there would be someregulation of the Cl2 permeability by controlling the ClC-1

activity. In fact, during repeated contractions in rat extensordigitorum longus (EDL) fibers, Cl2 permeability initiallydecreases by 50% within 1 minute (Pedersen et al., 2009a). As

the stimulation is prolonged, Cl2 permeability remains constantfor 3 minutes and then suddenly increases by threefold above thepre-stimulation level, corresponding with opening of a very largenumber of ClC-1 channels. Associated with the increase in Cl2

permeability is a decrease in membrane excitability (Pedersenet al., 2009a). Thus, ClC-1 is controlled during muscle activity.

Here, we consider an exercise example to illustrate the impact

of the regulation of Cl2 channels. Interstitial [K+] reaches 10–11 mM within 5 minutes during 30-watt single leg extensionexercise (Nielsen et al., 2004). Such [K+] largely depresses

tetanic force in vitro (Cairns et al., 2011; Pedersen et al., 2003)and yet the exercise continues without difficulty for 30 minutes.Perhaps, the decreased Cl2 permeability at the onset of muscle

activity is essential in preventing any K+-induced forcedepression, while at the same time favoring a K+-induced forcepotentiation (Fig. 3). In other words, the combined increase in

[K+]e and decrease in Cl2 permeability might help to maximize

muscle performance at the onset of muscle activity. Conversely,any increase in Cl2 permeability might be linked to a fatigueprocess triggered by a metabolic stress to increase muscle

sensitivity to the K+-induced force depression (Pedersen et al.,2009b). In the latter situation, membrane excitability decreases,thereby lowering Ca2+ release and force generation (i.e. fatigue)in order to lower the activity of the Ca2+ and myosin ATPases.

The ultimate benefit is a reduced ATP demand, avoidingmetabolic catastrophe.

Regulation and impact of the KATP channel

The KATP channel is an ATP-sensitive K+ channel. The channelwas named after it was discovered that ATP binding closes thechannel (Noma, 1983). It is now established that KATP channels

are primarily activated by the changes in metabolite levels thatoccur during metabolic stress, including decreases in intracellularATP and pH, and increases in intracellular ADP and extracellular

adenosine (Barrett-Jolley et al., 1996; Davies, 1990; Noma, 1983;Vivaudou et al., 1991). The KATP channel is thus considered to bean energy sensor that is activated during metabolic stress. Beingan ion channel, it also links the excitability of the sarcolemma to

the metabolic state of the fiber. There is now clear evidence thatactivation of KATP channels during repetitive stimulationcoincides with the activation of Cl2 channels as discussed

above (Pedersen et al., 2009a), and that the channel is crucial inpreventing fiber damage and severe muscle dysfunction duringexercise and fatigue by decreasing muscle cell excitability

(Cifelli et al., 2008; Cifelli et al., 2007; Kane et al., 2004;Stoller et al., 2009; Thabet et al., 2005).

One mechanism by which the KATP channel prevents fiberdamage was revealed by studies in which channel openers were

used, which allows for a large K+ efflux during the actionpotential (Box 3) and results in a reduction in the action potentialamplitude (Gong et al., 2003; Matar et al., 2000). The resulting

decreases in membrane excitability and action potentialamplitude lead to a decrease in Ca2+ release and force (Burtonand Smith, 1997; Duty and Allen, 1995; Gong et al., 2003; Matar

et al., 2001; Weselcouch et al., 1993; Wickenden et al., 1996).The physiological benefit of a reduced Ca2+ release would bereduced activity of Ca2+ ATPases and myosin ATPases, therebypreserving ATP (Fig. 3). Indeed, decreases in ATP levels during

metabolic inhibition are faster in the presence KATP channelblockers (Gramolini and Renaud, 1997; Weselcouch et al., 1993).However, modulation of KATP channel activity during muscle

fatigue has no effect on ATP levels in mouse EDL comparedwith that in control conditions, possibly as other regulatorymechanisms prevail. Nevertheless, in soleus muscle, blocking the

KATP channel accentuates ATP loss compared with that in controlconditions, whereas channel activation prevents a reduction ofATP (Matar et al., 2000). These results therefore support the

hypothesis that KATP channels affect energy metabolism, butmore studies are necessary to fully understand how exactly theseeffects prevent fiber damage and muscle dysfunction.

A second mechanism by which KATP channels prevent fiber

damage and dysfunction is through the maintenance of restingEm. This finding came from studies in which the KATP channelactivity is abolished by either exposing normal muscles to

channel blockers or by using muscles from Kcnj11-null mice[mice in which the Kcnj11 (Kir6.2) gene that encodes the channelpore protein has been deleted]. Normally, resting Em depolarizes

Action potential

Contraction Myosin ATPase

Ca2+ release

ClC1 channel ( Cl– permeability)

ATP

RelaxationCa2+ ATPase

[K+]e

ClC1 and KATP channel ( Cl– and K+ permeability)

Fig. 3. Regulation of membrane excitability during muscle activity. Blue

arrows indicate the normal process by which action potentials activate the

release of Ca2+ from the SR to activate the contractile proteins, resulting in

force generation and/or work. Relaxation is associated with the pumping of

Ca2+ back into the SR by the Ca2+ ATPase. Brown arrows indicate that

repetitive action potentials result in a large K+ efflux and eventually increased

[K+]e, which then reduces action potential amplitude. Small increases in [K+]e

potentiate submaximal force generation (by an unknown mechanism). Green

arrows indicate that the increase in [Ca2+]i during contraction is involved in

the closing of ClC1, possibly through the activation of protein kinase C

(Pedersen et al., 2009a; Pedersen et al., 2009b). The subsequent decrease in

Cl2 permeability improves action potential amplitude, thus counteracting the

K+-depressing effect. The events depicted by the brown and green arrows are

expected to predominate at the onset of exercise and as long as there is no

metabolic stress. Red arrows indicate what happens when ATP use exceeds

the rate of production causing a decrease in ATP concentration; the resulting

metabolic stress then triggers the fatigue mechanism, including activation of

ClC1 and KATP channels. Activation of these channels will cause very large

decreases in action potential amplitude and as a consequence less Ca2+ is

released by SR and less force is generated – all in order to preserve ATP and

prevent a metabolic catastrophe.

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at between 10 and 15 mV during repeated muscle contractions(Cifelli et al., 2008; Comtois et al., 1995; Light et al., 1994),

whereas it does not change during metabolic inhibition(Gramolini and Renaud, 1997). In the absence of KATP channelactivity, the depolarization can be as high as 50 mV under both

conditions. This depolarization has been shown to be sufficientlylarge enough to activate the Ca2+ channels in transverse tubules,leading to an uncontrolled Ca2+ influx, large increases in restingintracellular Ca2+ and in force (Cifelli et al., 2008; Cifelli et al.,

2007; Gong et al., 2000; Light et al., 1994; Matar et al., 2000). Asa consequence of the increases in resting [Ca2+], there is greateruse of ATP by the Ca2+ and myosin ATPases, which worsen the

possibility of a metabolic catastrophe. Furthermore, chronicelevation of [Ca2+]i is a well known factor that causes fiberdamage (Jackson et al., 1984; Jones et al., 1984).

Thus, the regulation of membrane excitability is complex anddepends not only on changes in extra- and intra-cellular ionconcentrations, such as that of K+, but also on changes in ion

channel activity. At the onset of exercise, [K+]e increases rapidlyand is likely to potentiate force generation similar to that upon anincreased RLC phosphorylation. At the same time, the activity of

ClC-1 decreases, possibly to prevent any K+-induced forcedepression. When metabolic stress occurs, the activity of ClC-1increases and this results in a depression of membraneexcitability, which is further enhanced by a concomitant

activation of KATP channels. As membrane excitability isreduced by these two channels, less Ca2+ is released and lessforce is generated. The outcome of these mechanisms is the

preservation of the limited supply of ATP through reducing itsuse by the Ca2+ and myosin ATPases.

Control of Ca2+ releaseAction potentials on the membrane are translated into a release of

Ca2+ in a process referred to as excitation–contraction coupling(Box 1). The dihydropyridine receptors (DHPRs) are specializedchannels that detect the action potential in the transverse tubulemembrane (Fig. 1) and, through a direct connection (in skeletal

muscle), open and close the Ca2+ release channels in the SR, theRyRs. The release of Ca2+ into the myofibril initiates musclecontraction and, as a result, increases ATP demand through the

activity of the myosin and Ca2+ ATPases. Although an actionpotential is the main controlling factor detected by the DHPR, andtherefore the main determinant that results in opening and closing

of the RyR, this ion channel is further regulated by a large numberof ionic and protein ligands that influence the opening probability(Po). We will examine further a small well-known collection of

these ligands and how they prevent ATP depletion. It is also knownthat reactive oxygen species can modulate Ca2+ release through theRyR (Oba et al., 2002), but this will not be discussed further here.

Regulation of RyR by ATP, Ca2+ and Mg2+

The three most studied regulatory ligands of the RyR are ATP,

Ca2+ and Mg 2+ (Lamb and Stephenson, 1992; Laver et al., 1997;Meissner, 1984; Meissner et al., 1986). There are two distinctsites on RyR for these ligands that either enhance or reduce its

Po. These sites are referred to as the high-affinity A-site,regulating activation, and the low affinity I-site, leading toinhibition (Lamb, 2000). Both Ca2+ and ATP bind to the A-site to

increase the Po of RyR (Meissner, 1986), whereas both Ca2+ andMg2+ bind to the lower affinity I-site to reduce its Po (Laver et al.,1997; Meissner et al., 1986). In the absence of Mg2+, Ca2+ opens

RyR by binding to the A-site if it is present at a concentration of1 mM and closes the receptor through binding at the I-site at high

concentrations of 1 mM (Meissner et al., 1986). Under normalresting conditions with [Mg2+] being present at 1 mM, the I-siteis occupied with Mg2+ and the subsequent inhibition of the

channel overrides any activating effect of Ca2+ and ATP. Mg2+

also competes with Ca2+ for binding to the A-site, but this doesnot result in an activation of the channel (Laver et al., 1997). Thestrong activating effect of ATP is most clearly observed when

Mg2+ is removed from the solution (Lamb and Stephenson,1991). ATP, in the absence of external Ca2+, by itself is capableof fully activating RyR and initiating Ca2+ release (Meissner

et al., 1986). This result further emphasizes how important theinhibitory effect of Mg2+ at rest is.

Considering the effects of Mg2+ described above, it would

appear that the RyR would never be activated! However, with eachaction potential, some RyRs are activated and Ca2+ is released intothe cell. The fact that Mg2+ reduces the Po of RyR becomes more

important when the concentration of ATP decreases, because asshown in Fig. 1, a net ATP hydrolysis results in an increase in[Mg2+]. This occurs because Mg2+ has a lower affinity for ADP

than for ATP. This increase in [Mg2+] and decrease in theconcentration of ATP combine to cause a decrease in Po,attenuating Ca2+ release in subsequent activations.

Several studies have examined the effect of higher [Mg2+] onexcitation–contraction coupling (Blazev and Lamb, 1999; Lamband Stephenson, 1991; Laver et al., 1997; Owen et al., 1997). The

general consensus of these studies is that [Mg2+] at rest is a stronginhibitor of RyR, which keeps the channel closed and unresponsiveto the strong activating effect of ATP and Ca2+ until an actionpotential activates the cell. Ca2+-induced activation of RyR, a key

mechanism in cardiac muscle, is apparently not relevant in skeletalmuscle (Endo, 2009). At 3 mM [Mg2+], a concentration that can bereached during fatiguing contractions (Westerblad and Allen,

1992), the magnitude of Ca2+ release achieved by transverse tubuledepolarization is reduced (Blazev and Lamb, 1999; Laver et al.,1997; Owen et al., 1996). Thus, inhibition by Mg2+ is important to

prevent spontaneous Ca2+ release at rest and it provides anothermechanism, by which less Ca2+ is released during metabolic stress.

The reduced activation of RyR, which results from low

concentrations of ATP, has also been explored. When theconcentration of ATP is held artificially low (,0.5 mM ATP),Ca2+ release is significantly reduced when RyR is directly

activated by DHPR (Blazev and Lamb, 1999; Owen et al., 1996).Although this ATP decrease might appear extreme, it has beenreasoned that a localized decrease in the concentration of ATP to

below 1 mM could occur in the vicinity of RyR (Owen et al.,1996). Therefore, both the concomitant decrease in ATPconcentration and increase in [Mg2+] upon an impendingmetabolic catastrophe can contribute to a decrease in Ca2+

release by lowering RyR Po to eventually preserve ATP. Thesensitivity of the RyR to changes in [Mg2+] and ATPconcentration, can also make it an energy sensor, in a similar

manner to the KATP channel.

Regulation of RyR by calmodulin and the S100 Ca2+-binding protein A1 (S100A1)

There are several additional ligands that interact with RyR. Here

we will discuss two that have known regulatory impact on Po ofthe RyR, calmodulin and S100 Ca2+-binding protein A1(S100A1).

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The small ubiquitous peptide calmodulin (CaM) was one of thefirst ligand regulators of RyR to be discovered (Chen and

MacLennan, 1994; Hamilton et al., 2000; Yang et al., 1994). Atrest, when [Ca2+] is low, calmodulin exists in the apocalmodulin(apoCaM) form, and upon increase in [Ca2+]i binds to Ca2+,becoming Ca2+-bound (CaCaM), and resulting in a change of its

shape and its effect as a ligand (Boschek et al., 2007; Boscheket al., 2008; Xiong et al., 2006). At low [Ca2+]i (up to 0.1 mM),the Ca2+-free apoCaM binds to RyR and increases its Po.

Conversely, as [Ca2+]i increases, the amount of the CaCaMcomplex also increases, which binds to RyR but decreases its Po

(Boschek et al., 2007; Xiong et al., 2006).

RyR has many distinct sites for ligand binding (Box 1) (Songet al., 2011), although it will probably be many years before wefully understand the range of ligands for RyR and their functions.Recent work has provided some insight. One of the important

ligands for RyR is S100A1. Prosser and colleagues used aknockout mouse (S100A1) to demonstrate that S100A1 enhancesCa2+ release (Prosser et al., 2008). Twitch and tetanic contractile

amplitudes are attenuated, and the corresponding Ca2+ transientsare smaller in the S100A1 mice compared with muscles of wild-type control mice. Yamaguchi et al. contributed to our

understanding of ligand control of RyR by using a mouse withan altered RyR CaCaM-binding site (RyRD/D) (Yamaguchi et al.,2011). Similar to the S100A1 mice, the RyRD/D mice produce

lower twitch forces, suggesting that they lack the S100A1-binding site. However, Ca2+ release during a maximal tetaniccontraction is enhanced and there is a greater peak [Ca2+]i

compared with that in muscles of wild-type mice (Yamaguchi

et al., 2011). On the basis of these results, the authors suggest thatthere is one site on the RyR that binds both CaCaM and S100A1,and that with S100A1 bound, this site is important for promoting

Ca2+ release at low [Ca2+], such as occurs during a twitch. Thesame site is also important for inhibiting Ca2+ release duringrepeated or sustained activation by binding CaCaM at higher

[Ca2+]i. In this way, Ca2+ is able to slow energy expenditure laterin contraction. It is interesting to note that CaCaM activatesMLCK and inhibits Ca2+ release. These two processes will haveopposing effects on muscle, enhancing contraction by promoting

RLC phosphorylation and attenuating contraction by decreasingCa2+ release.

ConclusionsThis Commentary has aimed to illustrate, with a few examples,how skeletal muscle contraction is regulated at the cellular level.

Although most researchers concentrate on the depression ofcontraction during fatigue, we point out here that there areregulatory processes that can enhance muscle performance. Thefirst example is that of myosin RLC phosphorylation, which

allows for increased force production at a given [Ca2+]i. Thiseffect permits a decrease in Ca2+ release without any loss of forceor power, but with the advantage of a reduced Ca2+ ATPase

activity, as less Ca2+ needs to be pumped back into the SR.Overall, this effect might be important in reducing ATP usageand prolonging the muscle activity. The second example is that of

K+-induced force potentiation that occurs when [K+]e increases.The underlying mechanism of this potentiation is still unknown.Finally, a third mechanism that helps maintain a high muscle

performance at the onset of exercise is the decrease in Cl2

channel activity; this effect is important to counteract the likelydepression of force that would otherwise be the result of

increasing [K+]e. We also discuss some mechanisms thatprevent a metabolic catastrophe when energy demand exceedsthe capability to replenish ATP. This regulation is highlyredundant and appears to concentrate on diminishing Ca2+

release, because this simultaneously reduces the activity ofboth Ca2+ ATPase and myosin ATPase and serves to preserveATP. This regulation occurs at the level of membrane

excitability, where the activation of both Cl2 and KATP

channels reduces excitability, thereby reducing the Ca2+ releaseby RyR. There is also regulation at the level of the RyR, which

responds to a decrease in [ATP] and increase in [Mg2+], whichoccur during repetitive or sustained contractions. In this case,both of these changes reduce the Po of RyR. RyR Po is also

regulated by Ca2+, either directly or through CaM as sustainedelevation of [Ca2+]i results in an inhibition of RyR by the CaCaMcomplex. Collectively, these regulatory processes allow exerciseto continue, as long as additional motor units are available for

recruitment, while preventing depletion of ATP in individualcells.

AcknowledgementsThe authors would like to thank Barbara Holash for creating Fig. 1.Research in the laboratories of the authors is supported by theNatural Sciences and Engineering Research Council of Canada.

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