Key words
calpain - disuse atrophy - proteolysis - muscle remodeling - membrane repair - exercise
Introduction
Skeletal muscle is a highly plastic tissue that undergoes continuous remodeling
(i. e., plasticity), in response to changing levels of contractile activity
(e. g., resistance exercise training-induced fiber hypertrophy or fiber
atrophy resulting from prolonged periods of muscle inactivity). This skeletal muscle
plasticity occurs due to the interaction between protein synthesis and protein
degradation. In regard to the regulation of proteolysis, four major proteolytic
systems exist in skeletal muscle (i. e. autophagy, ubiquitin-proteasome,
caspase-3, and calpain systems) and accumulating evidence suggests that the calpain
protease system plays a unique role in mediating skeletal muscle plasticity in
response to both exercise and prolonged periods of muscle inactivity.
Calpains have been extensively studied since their discovery in skeletal muscle in
1964 [1]. The calpain family consists of a group of
calcium (Ca2+) activated proteases whose proteolytic function
varies according to the calpain isoform and cell type. Calpains function through
cleaving target substrates at specific sites, forming fragmented protein structures
that can be further degraded by other proteolytic systems. However, truncation of
calpain substrates by calpains can result in fragmented proteins with specific
biological functions. Indeed, the first reported function of calpains in skeletal
muscle was calpain-induced activation of phosphorylase kinase which resulted in this
family of proteases being first termed as “kinase activating
factors” [1]
[2].
Despite this early label as a signaling regulator, calpains are currently viewed by
muscle physiologists as proteases that assist in myofibrillar protein turnover.
Although calpains serve an important role in skeletal muscle protein turnover,
accruing evidence reveals that calpains serve diverging roles in skeletal muscle
fibers.
This review will discuss the biological roles that calpains play in skeletal muscle.
In particular, we will highlight how calpains facilitate the adaptive response of
skeletal muscle fibers to chronic contractile activity (i. e. exercise) and
muscle fiber atrophy associated with prolonged muscle inactivity. The first section
of this review will introduce the calpain isoforms that are relevant to skeletal
muscle and briefly discuss their general function and activation. We will then
discuss the role that calpains play in skeletal muscle remodeling in response to
both exercise training and in response to prolonged periods of skeletal muscle
inactivity. Finally, we will also highlight the emerging evidence that calpains play
an important signaling role in skeletal muscle.
Calpains in Skeletal Muscle
Calpains in Skeletal Muscle
The calpain (CAPN) family of proteases in humans consists of 15 calpain isoforms,
CAPN1-CAPN3 and CAPN5-CAPN16 with all isoforms containing a cysteine protease core
[3]. Notably, CAPN4 is a 28 kd subunit of
calpain that lacks the cysteine proteolytic core. Due to this, CAPN4 is not
recognized as an independent calpain isoform and has been reclassified as calpain
subunit 1 (CAPNS1).
Three predominant calpain isoforms exist in skeletal muscle: 1) CAPN1; 2) CAPN2; and
3) CAPN3 [4]. Historically, CAPN1 and CAPN2 were
previously referred to as μ-calpain and m-calpain, respectively. The
nomenclature of μ-calpain and m-calpain evolved from the levels of
Ca2+ (the primary allosteric regulator of traditional
calpains) required to activate calpains in vitro. Specifically, it was
previously believed that activation of CAPN1 occurred when the cellular levels of
free Ca2+ reached the μM range whereas CAPN2 was not
activated until the intracellular Ca2+ concentrations reached the
mM range. However, it has since been recognized that early calpain studies measuring
the in vitro Ca2+ activation requirements did not reflect
what occurred in vivo and thus, more appropriate nomenclature has been
adopted [5]
[6]. Current
understanding of the Ca2+ activation requirements for calpains
will be discussed later in this review. Finally, CAPN3 is sometimes referred to as
p94 in the literature. The use of p94 in reference to CAPN3 is due to its larger
molecular weight of 94 kDa compared to the 80 kDa mass of CAPN1 and
CAPN2.
Overview of calpain structure and function
CAPN1 and CAPN2 form heterodimers consisting of a similar, but distinct,
80 kDa catalytic subunit (i. e. CAPN1 and CAPN2) and a
28 kDa CAPNS1 regulatory subunit. In contrast, CAPN3 exists as a
homodimer with two CAPN3 subunits. Note that while CAPN3 does have proteolytic
function, several unique structural features of CAPN3 differentiate it from
CAPN1 and CAPN2 (e. g. formation as a homodimer vs heterodimer) and
allows CAPN3 to serve roles outside of proteolytic function; this topic will be
discussed in more detail later in this review.
Ca2+ binding to calpain serves as an essential allosteric
regulator for activating the proteolytic function of traditional calpains. The
structural site on calpains responsible for proteolytic cleavage is labeled the
catalytic triad; this catalytic triad is composed of an active site that is
modulated by Ca2+. When Ca2+ is not bound
to calpains the catalytic triad retains a structure that is not catalytically
active [7]. Conversely, Ca2+
attachment to two Ca2+ binding sites on calpains results in a
conformational change in the cysteine protease region of calpain and subsequent
activation. A detailed discussion of the structure and mechanisms responsible
for activation of calpains is beyond the goals of this review and readers are
referred to other reports for more details [5]
[7].
Importantly, the structural composition of calpains provide these proteases with
the ability to selectively cleave specific sites on targeted proteins, as well
as to cleave proteins that are unable to be accessed by other proteolytic
systems (e. g. proteasome) [8]. In this
manner, calpains play important roles for skeletal muscle protein turnover and
can act independent and upstream of other proteolytic systems. Indeed, calpains
were first noted as initiators of proteolytic degradation under post-mortem
conditions whereby skeletal muscle ATP is depleted (thus preventing proteolysis
occurring due to the ubiquitin-proteasome and autophagy systems which require
ATP), yet protein degradation continued [8].
Curiously, it was observed that the protein degradation that occurred after the
autophagy and ubiquitin proteasome systems were inactivated was due to
Ca2+ leak from the sarcoplasmic reticulum (SR) and
consequential activation of calpains [8]
[9]. This observation set the framework for our
current understanding of calpains function in skeletal muscle.
Historically, calpains’ primary role in skeletal muscle has been viewed
as a facilitator of protein turnover through the proteolytic cleavage of
sarcomeric and cytoskeletal proteins such as titin, dystrophin, nebulin, and
desmin [7]
[10]
[11]. However, our understanding of calpains has
evolved to suggest that calpains participate in roles beyond protein turnover
[12]
[13]. In
this regard, the cleavage of select calpain substrates can result in
biologically active fragments; these fragmented proteins can possess enzymatic
activity or perform other biological functions. In this manner, calpains can
function outside of their role in protein turnover and instead, influence cell
signaling events leading to a diverse array of biological events (e. g.,
apoptosis). At present, direct evidence that calpains function outside the role
of cytoskeletal proteolysis in skeletal muscle is limited. Despite this,
abundant evidence exists in other cell types indicating that calpain acts in
numerous roles including processes of cell motility, insulin signaling, and
apoptosis. [14]
[15]
[16]
[17]
[18]. Further discussion of the
expansive biological roles that calpain plays in skeletal muscle will be
highlighted in future sections.
Factors regulating proteolytic activity of calpains
While Ca2+ is the primary regulator of the proteolytic
activity of calpains, several other factors are also involved in regulating
calpain activity. Key positive allosteric modifiers of calpain activity include
autoproteolysis, binding of phospholipids, and phosphorylation. In contrast, the
inhibitory protein calpastatin serves as the primary negative allosteric
modifier of calpain activity. A brief overview of these allosteric regulators of
calpain follow.
Autoproteolysis refers to the proteolytic removal of the N-terminal domain of
calpains and is considered a key regulator of calpain activity [19]. In this regard, autoproteolysis allows for
calpains to become activated at lower Ca2+ concentrations in
the cell. For instance, autoprotoleysis of CAPN2 results in a 25-fold reduction
in the amount of Ca2+ required to elicit half-maximal
activation [20]. Therefore, autoproteolysis is a
key modulator of calpain activity.
Autoproteolysis reduces the mass of the respective calpains, causing CAPN1 to
appear as 76 kDa and CAPN2 as 78 kDa (compared to 80 kDa
for both CAPN1 and CAPN2 prior to autoproteolysis). Additionally,
autoproteolysis of the 94 kDa CAPN3 results in the autoproteolyzed
fragment appearing at ~55 kDa. Notably, the occurrence of
autoproteoloysis is postulated to correspond with the initial activation of
calpains. Due to this, the autolyzed forms of CAPN1 and CAPN2 are often used as
markers of calpain activation in skeletal muscle [21].
Note that, although abundant evidence exists that autoproteolysis occurs within
the calpain molecule, debate exists about the physiological importance of
autoproteolysis in regulating calpain activity [5]. Autoproteolysis is posited to occur as an intermolecular reaction,
thus requiring at least one calpain protein to become activated in order to
cleave the N-terminal of nearby calpains [5].
However, many sites near the N-terminal cleavage point are required for calpain
function and would render calpains inactive if also cleaved. Evidence from in
vitro studies suggest that these adjacent sites are also cleaved in a
similar timed-fashion as the regulatory N-terminal site during calpain
activation [22]. In this regard, it has been
argued that autoproteolysis can be an in vitro artifact in purified
calpains due to their close proximity to one another [5]. Additionally, it has been argued that autoproteolysis would limit
the ability to further regulate calpain activity due to the autoproteolyzed
calpain being unable to return to a non-proteolyzed state. Nonetheless,
autoproteolysis of calpain has been observed to occur in skeletal muscle and the
relevance of autoproteolysis as a physiological regulator in vivo remains
to be determined.
Calpain activity is also regulated by binding of phospholipids to calpains such
as phosphatidylinositol [23]. For instance,
exposure of calpains to phosphatidylinositol 4,5-bisphosphate (PIP2)
reduces the Ca2+ requirement for CAPN1 and CAPN2
autoproteolysis by three to five fold [24].
However, the concentrations of PIP2 required to induce this effect
in vitro appear to be higher than what likely occurs in vivo
[7]. Nonetheless, phospholipid binding in
combination with other factors serves as additional mechanisms to regulate
calpain activity.
Calpain activity is also regulated by its phosphorylation status. At least 6
phosphorylation sites have been identified on both CAPN1 and CAPN2 [7]. Phosphorylation of CAPN1 by protein kinase A
(PKA) can occur at several serine residues on CAPN1, resulting in increased
calpain activity [25]. Conversely,
dephosphorylation of CAPN1 by alkaline phosphatase decreases calpain activity
[25]. However, PKA phosphorylation of CAPN2
results in decreased CAPN2 activity that is posited to occur due to the
prevention of PIP2 binding [26]. To
conclude, although phosphorylation of calpain can be a positive allosteric
modifier of calpain activity, additional research is required to fully elucidate
the complex role that phosphorylation plays in the regulation of calpain
activity.
Finally, the key negative allosteric regulator of calpain activity is the
endogenous protein, calpastatin. Calpastatin contains four inhibitory domains;
only one domain is required to inhibit calpains. Thus, each calpastatin protein
can inhibit up to four calpains [27]. More
specifically, calpastatin inhibits calpain by binding to calpains and
“looping” around the catalytic triad which blocks proteolytic
cleavage from being able to occur [28].
Intriguingly, calpastatin is a calpain substrate itself and can be cleaved by
calpain [29]. However, the looping maneuver that
occurs during calpastatin-binding allows it to avoid the catalytic triad and
prevents calpastatin from being cleaved in its calpain-bound state. The
importance of calpastatin’s regulation of calpains is likely
demonstrated by the fact that the only known function of calpastatin is the
inhibition of calpains [7]. The inhibitory actions
of calpastatin on calpains are also regulated by several cellular events.
Interestingly, calpastatin-calpain binding only occurs when calpains become
activated by Ca2+ binding. Considering that calpastatin only
binds calpain in its activated state, it would appear likely that calpastatin
functions as a mechanism to prevent prolonged calpain activation where more time
has passed and allowed for calpastatin to associate and bind activated calpains.
Calpastatin can also be phosphorylated by PKA and protein kinase C (PKC),
although the effects of calpastatin phosphorylation are not well known [7].
Modification of calpain substrates can increase their susceptibility for
calpain-mediated degradation
Robust evidence exists that post-transcriptional modifications of calpain
substrates can modulate their susceptibility to degradation by calpains. For
example, select kinases and phosphatases can regulate the susceptibility of
specific calpain substrates to proteolytic degradation via phosphorylation or
dephosphorylation [30]. For instance, PKC can
phosphorylate troponin-I and increase its susceptibility for degradation by
calpain [30]. The interconnection between calpains
and phosphorylation as a regulating event is further illustrated by the fact
that PKC is also a calpain substrate; however, calpain cleavage of PKC results
in a catalytically active PKC fragment. Due to calpain cleavage increasing the
kinase activity of PKC, the calpain-cleaved PKC fragments have an increased
phosphorylation rate of calpain substrates and subsequently increases the
susceptibility of calpain substrates to proteolytic degradation by calpains
[31].
Further, oxidation of skeletal muscle proteins also increases their
susceptibility to be cleaved by calpain due to conformational changes such as
protein unfolding [32]. Oxidation of proteins
affects their secondary and tertiary structure [33]. In this manner, protein unfolding increases the accessibility of
calpains to access calpain cleavage target sites. Importantly, oxidative stress
is a causative force in inactivity-induced muscle atrophy and the activation of
calpains in inactivity-induced muscle atrophy will be discussed later in this
review [34].
Measurements of calpain activity
Although several experimental approaches exist for measuring calpain activity,
precise measurements of in vivo calpain activity remains challenging
because of the allosteric regulation of calpains. For example, homogenizing
skeletal muscle fibers releases calcium from the sarcoplasmic reticulum,
resulting in calpain activation. Hence, this activation of calpain during the
homogenization process masks the in vivo calpain activity that existed in
the fibers prior to assay. Historically, calpain activity is measured in three
ways: 1) zymography; 2) cleavage of exogenous fluorescent substrates; and 3)
measurement of calpain-specific αII-spectrin breakdown products.
Zymography assays of calpain activity involve incubating homogenized tissue or
cells with known calpain substrates and measuring the resulting cleavage
products with gel electrophoresis. This technique can be useful for determining
the factors that regulate calpain activity by adjusting variables within the
assay (i. e. Ca2+ concentration) and observing the
effects on calpain activity. However, these assays do not reflect in vivo
calpain activity because of disruption of the cellular environment that calpains
are exposed to during assay preparation. As mentioned previously, homogenizing
muscle fibers disrupts the sarcoplasmic reticulum, releasing
Ca2+ and activating calpains. Although this problem can
be addressed by co-incubation with Ca2+ chelating agents
(e. g., EGTA), it remains unclear how much of the released
Ca2+ activates calpains during this process.
More recently, calpain activity has also been measured in situ by
administering cell-permeable substrates that become fluorescent when cleaved by
calpains. For instance, the synthetic construct
7-amino-4-chloromethylcoumarin-t-butoxycarbonyl-L-leucyl-L–methionine
amide (Boc-Leu-Met-CMAC) can cross skeletal muscle membranes and is subsequently
transformed by glutathione S-transferase which makes it impermeable. This
compound can then be cleaved by calpains resulting in a fluorescent chromophore
that can be measured to reflect calpain activity [35]. However, this assay has several limitations that are independent
of calpain activity such as the rate of thiol conjugation by glutathione
S-transferase, the rate of substrate entry into the cell, and the intracellular
concentration of the substrate.
Finally, specific biomarkers exist that can be measured as an index of calpain
activity. Specifically, the universally expressed, membrane-associated
cytoskeletal protein αII-spectrin is cleaved by calpain resulting in a
calpain-specific 150 kd fragment that can be detected via western blot.
The 150 kd spectrin cleavage fragment has a relatively long half-life of
~4.2 h and is widely-used for assessing the calpain activity
that occurs in vivo
[36]
[37].
Summary of structure and activation of calpains
The unique proteolytic actions of calpains provides them the ability to perform
several important roles in skeletal muscle tissue. Calpains are regulated by
several allosteric regulators and cellular events that increase
calpain’s sensitivity to Ca2+ or increase the
susceptibility of protein substrates to calpain-mediated degradation. While,
historically, research on calpains’ role in skeletal muscle has focused
on degradation of sarcomeric proteins in muscle wasting conditions
(e. g., inactivity-induced muscle atrophy), evidence also exists that
calpains are activated following exercise. The next section focuses upon the
role of calpain activation in skeletal muscle adaptations to exercise.
Calpains and Exercise
It is well-established that chronic exercise results in numerous adaptations to
skeletal muscle fibers. In particular, skeletal muscle fibers undergo systematic
remodeling in response to regular bouts of exercise; this occurs via a coordinated
interaction between catabolic and anabolic reactions (i. e., increased
protein degradation and increased protein synthesis) [38]. While four major proteolytic systems exist in skeletal muscle
(i. e. the ubiquitin-proteasome, autophagy, caspase, and calpain systems),
calpain is particularly interesting given that Ca2+, the primary
allosteric regulator of calpain activity, is released from the SR in order to
facilitate actin-myosin contractions during exercise. Thus, it is feasible that
calpains become active during exercise training sessions that result in a prolonged
increase in free Ca2+ in the cytosol; hence, if calpains are
activated during exercise it is predicted that active calpain participates in
skeletal muscle adaptation to exercise. The following sections highlight the
evidence indicating that calpains are activated during exercise followed by a
discussion of the physiological role that active calpains play in skeletal muscle
remodeling in response to exercise training.
Activation of calpains during exercise
A long-debated question related to calpain activation during exercise is
“do cytosolic levels of Ca2+ reach the level required
to activate calpains?”. In this regard, it is believed that the in
vivo Ca2+ concentrations required to activate
calpains occurs at ~0.5–2 μM for CAPN1 and
~50–150 μM for CAPN2 [7]. Resting free Ca2+ concentrations exist at
~100 nM in the cytosol of skeletal muscle and
~390 μM in the sarcoplasmic reticulum [39]
[40]. While
measuring Ca2+ levels in contracting myofibers remains a
difficult task, SR release of Ca2+ in response to an action
potential in isolated muscle fibers has been measured at a peak increase of
~120 μM in slow-twitch muscle fibers and
~358 μM in fast-twitch muscle fibers [41]. Therefore, it is plausible that calpains are
activated within these ranges of cytosolic Ca2+
concentrations. Further, it is possible that cytosolic Ca2+
concentrations in skeletal muscle may reach even higher levels during exercise
because sustained Ca2+ levels within muscle fibers are
determined by both the intensity and duration of exercise. Moreover, high
intensity and/or eccentric exercise (i. e. muscle contractions
during muscle lengthening) is capable of inducing damage to myofiber membranes
allowing extracellular Ca2+ to enter the myofiber which often
results in elevated levels of cytosolic Ca2+ for
12–36 h post exercise [42].
Therefore, when comparing experimental results regarding exercise-induced
activation of calpain in skeletal muscles, it is important to consider the
exercise protocol used in the experiments.
Calpain activation following exercise has predominantly been observed following
two types of exercise modalities: prolonged endurance exercise and eccentric
exercise. In this regard, several rodent experiments have demonstrated that
calpain activity is increased following prolonged endurance exercise or
continuous low frequency contractions [43]
[44]
[45]
[46]
[47]. In addition,
evidence from rodent models also reveal that calpains are activated following
eccentric exercise [48]
[49]
[50]
[51]. However, the evidence of calpain activation in humans following
exercise is limited to a few studies that employ widely varying experimental
protocols. A brief summary of these experiments follows.
Three independent human studies utilized autoproteolyzed calpain as a biomarker
for calpain activation following exercise [52]
[53]
[54]. In regard to the exercise modality, only one of these studies
measured calpain activation following prolonged exercise; they reported no
autoproteolysis of CAPN1 or CAPN3 immediately following a prolonged cycling
exercise bout in a small number of trained cyclists [53]. A follow-up study demonstrated that eccentric exercise in humans
resulted in autolyzed CAPN3 at 24 h following the completion of exercise
with no evidence of calpain activation within the first three hours following
exercise [52]. Importantly, these studies suffer
from experimental shortcomings because both experiments used the cytosolic
(i. e. soluble) fraction of muscle proteins to measure autoproteolysis
of calpains. This is problematic because autoproteolyzed CAPN1 is concentrated
within the myofibrillar fraction (i. e. insoluble) of rodent skeletal
muscle proteins following both prolonged immobilization and endurance exercise
[45]
[55]. Thus,
use of the cytosolic muscle protein fraction to investigate calpain activation
is likely a fatal experimental flaw. Indeed, a subsequent study illustrates this
point with evidence that the observations of autolyzed CAPN3 in total human
muscle homogenate is predominantly due to autoproteolysis of CAPN3 in the
myofibrillar fraction following eccentric exercise [54].
Another study utilizing in vitro measures of calpain activity demonstrated
that calpain activation occurred immediately following eccentric exercise in
humans [56]. Calpain activity increased 3-fold
immediately after a rigorous bout of eccentric exercise and increased calpain
activity persisted for 95 h following completion of the exercise bout
[56]. Notably, the majority of elevated
calpain activity occurred in the myofibrillar (i. e. insoluble) fraction
[56]. The fact that calpain activity is
differentially regulated in various cell compartments within the myofiber
highlights the need for stringent methodological approaches when measuring
calpain activation in future studies.
Although few human studies have investigated the impact of prolonged endurance
exercise on calpain activation, combining both the human and animal studies, it
appears clear that calpains are activated in skeletal muscle in response to both
prolonged endurance exercise and eccentric exercise [43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[54]
[56]
[57]. The following sections discuss the proteolytic roles that
activated calpains serve in the skeletal muscle response to exercise, as well as
a recently discovered nonproteolytic role of calpain with exercise.
Proteolytic function of activated calpains with exercise
Exercise-induced activation of calpains serve multiple roles in skeletal muscle.
Calpains disassemble myofibrils in order to facilitate protein turnover of
myofibrillar proteins and aid in remodeling with exercise training. Emerging
evidence also suggests a role for calpains in the skeletal muscle response to
exercise-induced damage. In this regard, calpains have recently been
demonstrated to participate in membrane repair and altered
excitation-contraction (EC) coupling. The following sections will discuss the
proteolytic roles that calpains serve with exercise.
Calpains and myofibrillar disassembly
As introduced earlier, regular exercise training is well-known to induce
skeletal muscle remodeling. For instance, chronic endurance exercise
training results in a fast-to-slow shift in skeletal muscle fiber types.
While the processes of muscle remodeling include a balance between protein
synthesis and degradation, protein turnover is necessary to remove proteins
in order for newly synthesized proteins to be incorporated. In this regard,
myofibrils within skeletal muscle cells present a challenge for turnover of
myofibrillar proteins due to its unique structure. The myofibril apparatus
is tightly packed with myofilaments such as actin, myosin, tropomyosin,
troponin, and actinin. Myofibrils are dense in nature and this density
limits diffusion of large proteins (i. e.>200 kDa)
within the myofibril. This is problematic for processes of protein turnover,
as the proteasome complex, which is responsible for degradation of numerous
muscle proteins, is unable to directly interact with intact myofibrils due
to its large size of ~2000 kDa. Instead, calpains facilitate
myofibrillar protein turnover via cleavage of cytoskeletal proteins
responsible for maintaining the structural integrity of myofibrils [58]. Calpain cleavage of these proteins
functions to disassemble myofibrils, release myofibrillar proteins, and
allow myofibrillar protein degradation by the ubiquitin-proteasome system
([Fig. 1a]) [59]. Examples of the cytoskeletal proteins cleaved by calpains
include α-actinin, tropomyosin, desmin, nebulin, troponin, and
titin.
Fig. 1 a Exercise-induced damage activates calpains and
results in degradation of cytsoskeletal proteins encapsulating
contractile proteins in order for contractile apparatus dissassembly
and repair. b Exercise-induced damage causes membrane damage.
Resulting activation of calpains causes cleavage of dysferlin into
mini-dysferlin to create lattice for membrane repair. See text for
more details.
In regard to the calpain-mediated cleavage of cytoskeletal proteins, one bout
of exhaustive endurance excise in rodents increases calpain-mediated
cleavage of tropomyosin and α-actinin by 48% and
103%, respectively [44].
α-actinin anchors actin to the z-line and degradation of
α-actinin contributes to myofibrillar disassembly. The disassembly
of the myofibril is believed to play an important role in removing damaged
proteins and replacing them with newly synthesized proteins [59]
[60].
Moreover, disassembly likely plays an essential role for incorporating new
myosin isoforms into the myofibril apparatus. Myosin heavy chains exist at
~220 kDa and fully assembled myosin molecules at
~520 kDa [59], and because of
the large size of this molecule, myofibrillar disassembly is required to
incorporate this large protein structure into the densely packed
myofibrillar apparatus. In this manner, calpains may be necessary for muscle
growth or fiber-type shifts in response to exercise training by facilitating
myosin heavy chain incorporation into the myofibril apparatus. Notably,
while substantial evidence exists that calpains play an important role in
myofibril remodeling [7]
[58]
[59]
[61], no studies have directly assessed the role
of calpains in response to chronic exercise training. Therefore, while it
appears likely that active calpains contribute to exercise-induced
remodeling of skeletal muscles, a detailed understanding of the role that
calpains play in training adaptations is not currently available. This topic
remains an important area for future research.
Calpains and exercise-induced damage
Participation in eccentric or unaccustomed exercise often results in
exercise-induced damage to skeletal muscle followed by a delayed onset of
muscle soreness. Exercise-induced damage can be characterized by many
factors such as sarcomere disruption (e. g. z-line streaming),
damaged cytoskeletal proteins (e. g. titin, desmin, dystrophin,
etc.), diminished membrane integrity, compromised EC-coupling, and decreased
force output [62]
[63]
[64]. Recovery from
exercise-induced damage typically occurs over the span of a few days and
results in skeletal muscle adaptations that help prevent future damage
against a similar exercise bout: known as the repeated bout effect [65]. Recovery from exercise-induced damage
requires protein turnover of dysfunctional proteins and their replacement by
newly synthesized proteins, hence calpains would likely play an important
role. The impact of exercise on protein turnover following damaging exercise
is demonstrated by the fact that whole body protein breakdown can remain
elevated for 10 days following a single bout of eccentric exercise [66].
In regard to the mechanisms responsible for exercise-induced damage,
mechanical sheer forces resulting in microscopic tears and subsequent
oxidative damage are plausible events that have been proposed to explain the
occurrence of exercise-induced damage (e. g. disrupted z-line
streaming) [67]. In addition to these
postulates, recent evidence also implicates calpain activation as a direct
contributor to exercised-induced muscle injury. For example, a study
utilizing ex vivo eccentric muscle contractions demonstrated that
pharmacological inhibition of calpains diminishes biomarkers of eccentric
exercise-induced muscle damage [51].
Specifically, inhibition of calpain activity prevented eccentric
exercise-induced disruption of muscle sarcolemma integrity and loss of
cytoskeletal proteins. Moreover, inhibition of calpain activation blunted
the eccentric exercise-induced reductions in muscle force generating
capacity. The authors posit that eccentric exercise-induced activation of
calpains results in the cleavage of cytoskeletal proteins and the disruption
of myofiber membrane integrity which are characteristic of exercise-induced
damage. A separate group utilized in situ eccentric contractions and
showed that pharmacological blockade of calpain activation resulted in
faster recovery of force production at 3 days following eccentric
contractions [49]. Together, these studies
suggest that activation of calpains are causal in exercise-induced muscle
damage.
Given that calpain inhibition can protect against reductions in muscle force
production following exercise-induced muscle injury, it appears that calpain
activation is a contributory event to muscle damage. Nonetheless, an
alternative interpretation is that calpain activation occurs in response to
exercise-induced “micro-damage” to skeletal muscle and that
calpains degrade damaged proteins and facilitate the recovery from injury,
instead of causing the actual damage per se. Indeed, calpains have
been postulated to play important roles in responding to micro-damage in
skeletal muscle fibers and facilitating sarcolemma repair [68]
[69]. A brief
discussion of the role that calpains play in sarcolemma repair follows.
Calpains facilitate membrane repair
Emerging evidence indicates that calpains can contribute to muscle fiber
membrane damage resulting from damaging exercise ([Fig. 1b]). In this regard, both CAPN1 and CAPN2 play an essential
role in mediating membrane repair following muscle damage [68]. Specifically, CAPN1 and CAPN2 contribute
to the repair process of skeletal muscle membranes by cleaving the protein
dysferlin into mini-dysferlinC72
[69]. Mini-dysferlinC72, but not full length dysferlin,
acts to localize to the damaged membrane site and recruit mitsugumin 53
(MG53), forming a lattice that stabilizes the injured site and further
recruits repair machinery. The injured membrane site results in
Ca2+-induced activation of CAPN1 and CAPN2 leading to
the required cleavage of dysferlin into mini-dysferlinC72.
Importantly, this evidence highlights the important concept that calpains
role is not limited to protein turnover; indeed, active calpains are capable
of cleaving proteins and altering their biological function to facilitate
processes such as membrane repair.
Calpains participation in membrane repair via cleavage of dysferlin likely
has important indications for exercise-induced membrane damage. This is due
to findings that dysferlin-knockout mice show a significantly blunted repair
rate with eccentric exercise-induced membrane damage [70]. A lack of dysferlin following eccentric
exercise would prevent calpains from cleaving dysferlin and creating
mini-dysferlinC72 which is important in the membrane repair
process. Thus, it is predicted that calpain cleavage of dysferlin into
mini-dysferlinC72 is a key event in the cellular response to
damaging exercise. Collectively, these findings support the idea that
calpains respond to micro-damage following exercise and facilitate muscle
repair instead of causing detrimental cell damage. Given this, it may also
be plausible that calpains protect against further muscle damage by reducing
the force generating capacity of skeletal muscle via alterations in
EC-coupling.
Calpains affect EC-coupling by degrading triad junction proteins
As mentioned previously, calpains are purported to play a role in the
decreased force output that occurs following exercise-induced muscle damage.
While it is established that diminished EC-coupling contributes to exercise
damage-induced reductions in muscle force production, the mechanisms
responsible for this phenomenon remain poorly understood. Previous studies
suggest that eccentric exercise impairs EC-coupling potentially through
decreases in the abundance of triad proteins [71]
[72]. In this regard, recent
evidence has implicated that calpains play a significant role in diminished
EC-coupling following exercise-induced damage through remodeling of the
triad junction. Triad junctions are sites where T tubules and the SR connect
to facilitate the release of Ca2+ in response to an
action potential. Thus, the triad junction is an essential element to
EC-coupling [73].
Pharmacological inhibition of calpains prevents eccentric exercise-induced
decreases in the protein abundance of the triad junction proteins
dihydropyridine receptor (DHPR), junctophilin 1 (JP1), and junctophilin 2
(JP2) following in situ eccentric exercise in rats [49]. Importantly, preventing the degradation of
triad junction proteins by inhibiting calpains also coincided with conserved
force production three days after the eccentric exercise bout. Junctophilins
span across the t-tubule membrane and tether the SR to form the triad
junction and are essential for triad junction formation. Indeed,
junctophilin-knockout mice display greatly reduced triad junction formation
and die soon after birth [74]. Thus, it
appears likely that calpain-mediated reductions in the tethering components
of the triad junction (i. e. junctophillins) and voltage sensing
proteins responsible for facilitating Ca2+ release from
the SR (i. e. DHPR) would result in the diminished EC-coupling that
occurs with exercise-induced damage ([Fig.
2a]).
Fig. 2
a Exercise-induced damage activates calpains and cleaves
triad junction proteins resulting in decreased
excitation-contraction (EC) coupling. b CAPN3 plays a
structural role in triad junction formation in order to facilitate
Ca2+ release required for CaMK activation and
downstream adaptations. See text for more details. (Abbreviations:
CAPN- Calpain,RyR- ryanodine receptor, JP1/2- junctophilin proteins,
DHPR- dihydropyrodine receptor, CaMK- calmodulin dependent
kinase).
The cleavage of triad proteins by calpains as a means to facilitate further
proteolytic degradation of these proteins follows the canonical view of
calpains’ primary function. In addition, calpain initiation of
decreased EC-coupling would also prevent the force that muscle fibers would
experience during recovery (i. e. due to lower contractile capacity)
and would reduce the potential for further damage occurring. Thus, calpain
cleavage of triad junction proteins may serve a protective function that
prevents exercise-damaged muscles from producing high levels of contractile
forces until muscle recovery has occurred. While proteolytic remodeling of
the triad junction by calpains serves an important role following
exercise-induced damage, recent discoveries have indicated that calpains
also play a unique role within the triad junction that is completely
independent of their proteolytic function.
Non-proteolytic function of calpain in exercise
Recent evidence reveals that CAPN3 is an essential structural component for
conformation of the triad junction ([Fig. 1b])
[75]. Moreover, the structural involvement of
CAPN3 at the triad junction plays an important role in the adaptive response of
skeletal muscle to endurance exercise [76].
Notably, the concept that CAPN3 can function in a role other than proteolysis
was first noted in patients with limb girdle muscle dystrophy type 2A (LGMD2A).
LGMD2A develops due to mutations in the CAPN3 gene. Intriguingly, assessment of
the localization of mutations within the CAPN3 gene revealed that several
patients with LGMD2A had mutations in regions of the gene that did not affect
the proteolytic activity of CAPN3 [77]. This
discovery led to the development of knock-in mice containing a mutant CAPN3 gene
that renders CAPN3 proteolytically inactive but structurally intact [78]. It was observed that CAPN3 KO mice lacked
appropriate triad junction formation, however mutant mice with proteolytically
inactive CAPN3 formed triad junctions with functioning Ca2+
release channels. Thus, it was apparent that the proteolytic activity of CAPN3
was not responsible for triad junction formation, but that CAPN3 was acting in a
separate role. This finding led to the discovery that CAPN3 functions as a
structural component that tethers the triad junction to connectin/titin
within skeletal muscle [78].
In regard to exercise, the structural role of CAPN3 appears essential in the
adaptive response to endurance exercise [76]. For
instance, CAPN3 KO mice have reduced presence of ryanodine receptor (RyR) and,
consequentially, impaired Ca2+ release from the SR [75]. This is problematic, as
Ca2+ is a key signal for adaptations with exercise
training. While endurance exercise training normally promotes transcription of
genes involved in oxidative metabolism and a slow-twitch muscle fiber phenotype
[79], CAPN3 KO mice have an impaired response
to endurance training and exhibit blunted expression of genes associated with
the slow-fiber phenotype [76]. Due to the
structural role of CAPN3 in formation of the triad junction, its absence reduces
RyR presence and blunts Ca2+ signaling. In turn, CAPN3 KO
mice lack appropriate signaling through the
Ca2+-calmodulin-dependent protein kinase (CaMK) pathway which
is responsible for upregulated transcription of genes involved in oxidative
metabolism [76]. Therefore, CAPN3 appears to be
essential in the promotion of a slow-twitch phenotype in skeletal muscle in
response to endurance exercise training. The novel finding that CAPN3 serves as
a structural component required for exercise-induced adaptations highlights the
important role that calpains play outside of their canonical function in
proteolysis.
Summary of calpains in exercise and future directions
While research on the role that calpains play in exercise-induced adaptations
remains in its infancy, growing evidence suggests that calpains have an
important role in exercise-induced adaptation in skeletal muscle. Indeed,
calpains have a large impact in the skeletal muscle response to eccentric
exercise. Nonetheless, future studies are required to determine if calpain
activation is a detrimental consequence of eccentric exercise, or if calpains
play an essential role to the muscle adaptations that coincide with exercise
training.
From a resistance training perspective, compared to concentric exercise,
eccentric exercise is postulated to elicit a greater hypertrophy response
purportedly due to the greater load placed on skeletal muscle fibers [80]. Given the role of calpains in skeletal muscle
remodeling, it is plausible that calpain activation by eccentric exercise may
also play a critical role in these higher “returns” for a given
exercise bout. In addition, accruing evidence suggests that the role of calpains
in muscle adaptation extends beyond its involvement in proteolysis. Indeed,
calpains contribution to membrane repair and triad junction formation highlight
that calpains are not limited to their function in proteolysis and may have
untold roles in skeletal muscle function that have yet to be discovered. Future
research is needed in order to fully elucidate the role of calpains in skeletal
muscle and its adaptation to exercise. In the next section, we discuss the role
that calpains play in promoting skeletal muscle atrophy during prolonged periods
of muscle inactivity.
Calpains in Inactivity-induced Muscle Atrophy
Calpains in Inactivity-induced Muscle Atrophy
Skeletal muscle atrophy is a conserved biological process that is essential for
systemic proteostasis and energy balance in times of need (e. g. prolonged
fasting). Skeletal muscle comprises nearly 40% of human body weight,
~75% of total body protein content, and skeletal muscle maintenance
alone can contribute to 20% of resting energy expenditure [81]. Skeletal muscle mass is regulated in response to
the activity load it experiences in order to meet the “needs” of the
body but not exceed those means in order to maintain efficient homeostasis. In this
regard, skeletal muscle responds to periods of muscle inactivity by initiating
processes that facilitate muscle atrophy. Inactivity-induced muscle atrophy occurs
as a result of conditions that significantly decrease the work performed by skeletal
muscle such as limb-immobilization, prolonged bed-rest, and mechanical
ventilation-induced inactivity of inspiratory muscles [12]
[82]
[83]
[84]
[85]
[86]
[87].
Unfortunately, prolonged inactivity results in skeletal muscle atrophy that is
detrimental due to a reduced force-generating capacity of skeletal muscle. Severe
muscle atrophy can be a devastating consequence that can impair the livelihood of
those affected by reducing the ability to perform tasks of daily living and in some
patient populations, muscle atrophy can increase the risk of morbidity and mortality
[88]. In the subsequent sections, we will discuss
both the tools utilized to study inactivity-induced muscle atrophy and the role that
calpains play in mediating atrophy during prolonged muscle inactivity.
Experimental models and measures of inactivity-induced muscle atrophy
Because of the clinical significance of skeletal muscle atrophy, scientific
interest in investigating the mechanism has increased markedly; however,
investigating the mechanisms responsible for inactivity-induced muscle atrophy
in humans remains a challenge for several reasons. For example, human studies
seeking to delineate the mechanisms of inactivity-induced atrophy require muscle
biopsies that are invasive and difficult to obtain in some forms of muscle
inactivity-induced muscle atrophy (e. g. biopsies from diaphragm muscle
in patients undergoing mechanical ventilation). Further, the amount of muscle
tissue obtained in most muscle biopsies is small (e. g.,
50–100 mg) and limits the number of analyses that can be
performed on as single sample. Additionally, human experimental models to study
inactivity-induced muscle atrophy such as prolonged bed-rest and limb-casting
are expensive to perform. Moreover, many of the experimental interventions
utilized to delineate the roles of signaling pathways (e. g.
pharmacological inhibitors, transgenic gene modulation, etc.) are not approved
for use in humans. Therefore, because of these complications in the study of
inactivity-induced atrophy in humans, animal models to study inactivity-induced
muscle atrophy have been developed. For instance, hindlimb-unloading via
suspension of a rodent’s tail can be utilized to unweight the hindlimbs
and simulate the muscle atrophy that occurs during bed-rest or space flight.
Limb casting in rodents can be utilized to immobilize limbs and promote the
skeletal muscle atrophy that occurs due to casting in humans. Denervation of
skeletal muscle motor neurons in animals is also utilized to emulate muscle
inactivity that occurs with spinal cord injury. Additionally, rodent models of
mechanical ventilation exist that decrease the contractile activity of
inspiratory muscles (i. e. diaphragm) and result in inactivity-induced
muscle atrophy similar to what occurs in humans undergoing mechanical
ventilation. All of these animal models have been successfully used to
investigate the mechanisms responsible for inactivity-induced muscle
atrophy.
Inactivity-induced muscle atrophy is measured by reductions in muscle mass or the
cross-sectional area of muscle and individual muscle fibers. Other hallmarks of
inactivity-induced muscle atrophy include alterations in the ultrastructure of
muscle (e. g. disrupted z-line streaming, altered mitochondrial
morphology, etc.) and reductions in the specific force production of muscle
(i. e., force produced per cross-sectional area of muscle) [12]
[89]
[90]. Importantly, calpains have been implicated to
play a mechanistic role in inactivity-induced muscle atrophy.
Evidence of calpain involvement in inactivity-induced muscle atrophy
Several lines of evidence indicate that calpains play an important role in the
occurrence of inactivity-induced muscle atrophy. For instance, pharmacological
inhibition of calpains attenuates muscle atrophy that occurs in rodents during
both limb casting and prolonged mechanical ventilation [12]
[90]. However, it may be important
to note that while calpain inhibitors are capable of inhibiting a broad array of
calpain isoforms, current and previous generations of calpain inhibitors often
exhibit off-target effects such as inhibition of cathepsins (i. e.
lysosomal proteolytic enzymes). Although current generations of calpain
inhibitors have markedly improved selectivity of calpains over cathepsins [91], the potential for off-target effects of most
calpain inhibitors remains a concern in interpretation of data from studies
using these inhibitors. Instead, transgenic overexpression of calpastatin, the
endogenous inhibitor of calpains, is a highly selective approach for inhibiting
calpains in skeletal muscle. Indeed, the only known function of calpastatin is
the inhibition of calpains which makes calpastatin overexpression a powerful
experimental tool to selectively target calpain activation. In this regard, two
separate studies have demonstrated that transgenic mouse models overexpressing
calpastatin are resistant to inactivity-induced muscle atrophy provoked by
hindlimb unloading [89]
[92].
Collectively, strong evidence exists that calpains play a key role in the muscle
atrophy that occurs in response to prolonged muscle inactivity. The following
paragraphs will further elaborate the currently available evidence on how
calpains become activated during muscle inactivity and the specific functions
that activated calpains may serve in inactivity-induced muscle atrophy.
Regulators of increased calpain activity during inactivity-induced muscle
atrophy
Prolonged inactivity in skeletal muscle results in several events that lead to
calpain activation. For example, prolonged muscle inactivity (i. e.,
hindlimb unloading and denervation) result in significant increases of cytosolic
levels of Ca2+ leading to calpain activation [93]
[94].
Additionally, muscle inactivity results in increased production of reactive
oxygen species (ROS) and the oxidation of calpain substrates which increases
their susceptibility to degradation [32].
Moreover, phosphorylation of calpain target substrates also contributes to
increased degradation by calpains [94]. The
following paragraphs will discuss these points in more detail.
Inactivity induces calpain activation due to increased cytosolic free
Ca2+
Cytosolic Ca2+ levels increase during prolonged muscle
inactivity and result in the activation of calpains [93]
[94]. For instance, one study
utilizing denervation as a model of muscle inactivity showed that cytosolic
Ca2+ levels were increased 7 days following
denervation and this rise in Ca2+ levels coincided with
the time-point of calpain activation [94]. The
mechanism(s) to explain why prolonged muscle inactivity results in increases
in cytosolic levels of calcium has been a challenging issue to investigate.
However, emerging evidence points to two potential mechanisms.
One mechanism of increased muscle Ca2+ levels with
inactivity is through an inward flux of extracellular
Ca2+ due to opening of Ca2+ ion
channels. Transient receptor potential, subfamily V, member 2 (TRPV2) and
purinergic ionotropic P2X7 receptor (P2X7R) channels
are upregulated with denervation in rodents and become activated during
prolonged muscle inactivity [95]. Therefore,
activation of extracellular Ca2+ channels would
contribute to increased free Ca2+ levels with inactivity
and lead to activation of calpains.
Another possibility that could explain elevated Ca2+
levels during prolonged muscle inactivity is the occurrence of
Ca2+ leak from the SR. For example, evidence reveals
that the SR in diaphragm muscle becomes “leaky” during
prolonged mechanical ventilation resulting in unregulated
Ca2+ release [96]. The
occurrence of Ca2+ leak from the SR is linked with
oxidative stress that occurs with prolonged muscle inactivity. Prolonged
inactivity in skeletal muscles is associated with increased ROS production
leading to oxidative stress [34]
[97]
[98]. In this
manner, increased ROS production results in oxidation of the RYR1 and
dissociation of calstabin1 from the RYR1 complex [96]. This is important because Calstabin1 stabilizes the RYR1
complex and its dissociation results in Ca2+ leak during
muscle inactivity; however, antioxidant treatment prevents the
disassociation of calstabin1 and mitigates SR Ca2+ leak
with inactivity [96]. Therefore, oxidization
of the RYR1 complex and resultant Ca2+ leak from the SR
during muscle inactivity would serve as one mechanism to activate calpains
and induce their proteolytic functions. Notably, ROS also oxidize calpain
target substrates and increase their susceptibility for cleavage by
calpains. Indeed, post-transcriptional modifications such as oxidation and
phosphorylation of calpain substrates can increase the susceptibility of
degradation by calpains [32]
[94].
Oxidation and phosphorylation of target substrates increases calpain
degradation
ROS-mediated oxidation of calpain substrate proteins results in a higher
susceptibility to calpain cleavage [32]. Oxidation
of proteins results in alterations in the secondary or tertiary structures of
proteins such as protein unfolding; the unfolding of oxidized calpain substrates
increases the accessibility of the calpain proteolytic core to cleavage sites
within substrate proteins. As mentioned, skeletal muscle inactivity results in
increased oxidant production [34]
[97]
[98]. While many
sources of ROS exist during muscle inactivity, mitochondria are noted to become
damaged in skeletal muscle during prolonged inactivity and are a key source of
elevated ROS emissions [99]. Increased emission of
ROS from mitochondria results in protein oxidation and higher cleavage rates of
these oxidized proteins by calpains. Further, increased production of ROS in
skeletal muscles also increases calpain activation. Specifically, provision of a
mitochondrial targeted antioxidant prevents an increase in calpain activity in
skeletal muscles during both mechanical ventilation and limb-immobilization
[97]
[98].
Importantly, preventing increased mitochondrial ROS emissions and subsequent
calpain activation also protects against muscle atrophy due to prolonged
inactivity [97]
[98].
Hence, increased ROS emissions in skeletal muscles during long periods of
inactivity plays an important role in calpain activation.
Phosphorylation of calpain substrates during muscle inactivity is another factor
that increases calpain degradation of proteins. For instance, recent evidence
reveals that phosphorylation of desmin by glycogen synthase kinase 3-β
(GSK3-β) substantially increases its degradation by CAPN1 in skeletal
muscle in response to denervation [94].
Conversely, inhibition of GSK3-β by expression of a dominant negative
GSK3-β prevented the degradation of desmin. Thus, phosphorylation of
calpain substrates also likely play a key role in determining a
proteins’ susceptibility to calpain cleavage.
Summary of calpain activation with muscle inactivity
Collectively, increased calpain activity during muscle inactivity can occur
by increases in intracellular Ca2+ levels. Increased
intracellular Ca2+ levels serve to activate calpains and
can happen due to Ca2+ leak from the SR and/or
influx of extracellular Ca2+ owing to activation of
membrane Ca2+ ion channels. Additionally, allosteric
modification of calpain substrates via oxidation or phosphorylation can also
increase the susceptibility of calpain substrates to cleavage and result in
an increased proteolytic cleavage of target substrates.
Calpains role in inactivity-induced muscle atrophy
Skeletal muscle atrophy occurs when muscle protein degradation rates exceed the
rate of protein synthesis. Protein degradation is facilitated by four major
proteolytic systems in skeletal muscle: 1) autophagy; 2) ubiquitin-proteasome;
3) caspase-3; and 4) calpain systems. While all four proteolytic systems are
activated in skeletal muscle during prolonged periods of inactivity [99], each proteolytic system likely plays a
different role in promoting muscle atrophy. Both the caspase-3 and calpain
systems function to cleave their targeted proteins to form fragmented sections
while the autophagy and ubiquitin-proteasome systems further breakdown proteins
and protein fragments to amino acids. While calpains are unable to fully degrade
their target proteins, the cleavage of these proteins has marked effects on
skeletal muscle function and proteostasis.
Inactivity-induced activation of calpains disrupt skeletal muscle
ultrastructure
As mentioned earlier in this review, disruptions in the ultrastructure of
skeletal muscle, such as disrupted z-line streaming, are often observed with
inactivity-induced muscle atrophy [100]
[101]. The streaming of z-lines is an important
factor for effectively transmitting force throughout the length of the
muscle fiber. Intriguingly, exposure of skeletal muscle to high levels of
Ca2+ results in near disappearance of z-lines [102]. Indeed, many of calpains’
substrates (e. g. desmin, nebulin, titin, etc.) are responsible for
structural integrity and force transmission of muscle fibers. For instance,
desmin is responsible for attaching the z-disk to the sarcolemma, nebulin
anchors actin to the z-line, and titin acts as a molecular spring that
facilitates passive elasticity. Thus, calpain cleavage of its substrates
disrupt muscle ultrastructure during inactivity-induced muscle atrophy. In
turn, calpain-induced alterations in muscle ultrastructure would also
contribute to decrements in force production observed with inactivity. In
this regard, calpastatin overexpressing mice that underwent 14 days of
hindlimb unloading had complete protection against reductions in soleus
muscle specific force [89]. Importantly, this
protection in muscle specific force coincided with preserved sarcomere
structure (i. e. z-line streaming) [89]. Furthermore, pharmacological inhibition of calpains prevents
the decrease in muscle-specific force that occur due to mechanical
ventilation [90]. These studies suggest that
calpains facilitate the disruptions in muscle fiber ultrastructure and
diminished force production, which have long been regarded as hallmarks in
inactivity-induced muscle atrophy. In addition, calpain-mediated turnover of
myofibrillar proteins may also contribute to reductions in the force
generating capacity of inactive muscles.
Calpains initiate protein degradation that contributes to muscle
atrophy
As mentioned earlier in this review, calpains play important roles in
myofibril disassembly. Myofibril disassembly is necessary in order for
myofibrillar proteins to be degraded during the atrophic process. Recall
that the proteasome complex is unable to degrade proteins within intact
myofibrils due to spatial limitations imposed by the large size of the
proteasome complex and the densely packed myofibrillar apparatus [59]. In contrast, calpains’ size and
selective cleavage of specific calpain substrates provide them the ability
to cleave proteins responsible for maintaining the dense structure of the
myofibril apparatus.
Activated calpains cleave sarcomeric proteins (e. g. desmin, nebulin,
titin) that encapsulate the myofibril and result in freeing of myofibrillar
proteins [10]
[11]
[103]. Indeed, actin and myosin
are released from the myofibrillar apparatus in a Ca2+
dependent manner and this observation was first noted over four decades ago
[104]. Today, calpain-mediated disassembly
of myofibrils is considered the rate limiting factor for
ubiquitin-proteasome degradation of contractile proteins [105]. The important role that calpains serve in
initiating protein degradation is demonstrated by pharmacological and
transgenic studies in rodents showing that inhibition of calpains attenuates
inactivity-provoked muscle atrophy due to limb immobilization, hindlimb
unloading, and mechanical ventilation [12]
[90]
[92].
In addition to myofibril disassembly, recent evidence has also indicated that
calpains can also cleave contractile proteins. While calpains do not readily
degrade undenatured forms of actin and myosin [7]
[11], recent evidence indicates
that oxidation of actin and myosin increases their susceptibility to
degradation by calpains [32]. Given that
prolonged-inactivity induces oxidative stress in skeletal muscle, calpains
may also function to participate in breakdown of actin and myosin during
inactivity-induced muscle atrophy.
Collectively, the activation of calpains leads to the cleavage of sarcomeric
and cytoskeletal proteins that results in the disassembly of myofibrils.
Myofibril disassembly functions to allow degradation of myofibrillar
proteins via the proteasome complex. Furthermore, during periods of
oxidative stress in skeletal muscle (e. g., prolonged muscle
inactivity), calpains also assist in degradation of contractile proteins
such as actin and myosin. Therefore, calpains contribute to muscle atrophy
during inactivity by accelerating muscle proteolysis ([Fig. 3]).
Fig. 3 Prolonged inactivity induces muscle atrophy via
activation of calpains. Activated calpains cause dissassembly of the
contractile apparatus, disruptions in z-line streaming, activation
of caspase system, mitochondrial dysfunction, and increased
proteolysis. See text for more details.
Activated calpains promote atrophic signaling
The specificity of calpains’ cleavage mechanism provides this unique
protease with the ability to cleave proteins to form biologically active
protein fragments with altered function. In this manner, calpains have the
capacity to serve as upstream signaling regulators. Accruing evidence has
revealed that calpains are signaling effectors for numerous cellular
processes in several types of non-muscle cells [14]
[15]
[16]
[17]. However, our understanding
of calpain cleavage resulting in downstream signaling events in skeletal
muscle is not well understood. Nonetheless, specific calpain cleavage
products may act as mechanisms for mediating muscle atrophy [90]. Examples of calpain signaling in skeletal
muscle fibers follows.
An important pathway of calpain signaling is the calpain-mediated cleavage of
pro-caspase-3 into its active form, caspase-3. Following cleavage, activated
caspase-3 initiates numerous downstream signaling events [106]. For example, activated caspase-3 degrades
proteins involved in DNA repair such as poly(ADP-ribose) polymerase (PARP),
as well as contractile proteins such as actin and myosin [107]
[108]. The
effects of calpain cleaved caspase-3 on DNA repair and proteolysis
highlights an important capability of active calpain.
Calpains also participate in cell signaling events by influencing
mitochondrial function. For example, calpain-mediated cleavage of the BCL-2
family member, Bid, results in its truncated form known as tBid. Evidence in
cardiac myocytes reveals that tBid can translocate to the mitochondria and
recruit Bax; this leads to permeabilization of the mitochondrial membrane
and increased ROS production [109]
[110]. As mentioned previously, increased ROS
production plays an important role in the occurrence of inactivity-induced
atrophy and dysfunctional mitochondria are the major contributor [99]. Similar to cardiac myocytes, calpain
cleavage of Bid into tBid occurs in diaphragm muscle during prolonged
mechanical ventilation and the appearance of tBid is prevented with calpain
inhibition [90]. Therefore, it is plausible
that calpain formation of tBid may play an important role in the causation
of the mitochondrial dysfunction that occurs with muscle inactivity.
Calpain also participates in cell signaling by cleaving apoptosis-inducing
factor (AIF) in mitochondria [111]. AIF is an
important signaling molecule that is capable of inducing cell death. The
proteolytic activity of calpains is required for AIF to be released from
mitochondria where it then translocates to the nucleus and causes chromatin
condensation and DNA degradation. Although AIF plays important roles in
signaling for cell death pathways, intact AIF also serves an important role
in mitochondrial function. Indeed, muscle-specific loss of AIF results in
mitochondrial dysfunction and muscle fiber atrophy [112]. Although it is currently unclear if calpain cleavage of AIF
contributes to inactivity-induced muscle atrophy, cytosolic levels of AIF
are increased with denervation and hindlimb unloading [107]
[113].
Therefore, it is feasible that calpain cleavage of AIF and subsequent
release from mitochondria may play a role in inactivity-induced muscle
atrophy.
The relationship between calpains and mitochondrial function is further
demonstrated by the existence of a mitochondrial targeting motif on CAPN1
[114]. CAPN1 has been shown to translocate
and accumulate in mitochondria of cardiac myoctes resulting in mitochondrial
dysfunction [115]. However, it currently
remains unknown if CAPN1 enters the mitochondria in skeletal muscle. If
CAPN1 is capable of translocating to mitochondria within skeletal muscle, it
is plausible that calpain-mediated cleavage of Bid, AIF, and CAPN1 targeting
of mitochondria may contribute to the mitochondrial dysfunction that occurs
in skeletal muscle during prolonged inactivity.
A final example of the importance of calpain signaling is the discovery that
calpain activation results in decreased Akt activity in skeletal muscle
[116]. Although the mechanism(s)
responsible for this observation remain unclear, Akt abundance is not
decreased in skeletal muscle indicating that calpain-mediated downregulation
of Akt activity is likely due to decreased phosphorylation [117]. Regardless of the mechanisms responsible
for calpain-mediated downregulation of Akt, the finding that active calpains
decrease Akt signaling is another important example of how active calpains
plays a key signaling role in skeletal muscles and highlights the importance
of future research on this topic.
Conclusions and Future Directions
Conclusions and Future Directions
Accumulating evidence indicates that calpains play important roles in skeletal muscle
remodeling in response to both exercise training and inactivity-induced muscle
atrophy. While calpains have been traditionally viewed as a simple proteolytic step
in the processes of protein turnover, exciting new evidence reveals that calpains
serve roles in skeletal muscle fibers that extend beyond their canonical involvement
in cleavage of proteins. For instance, calpains contribute to the cellular
adaptation to stressors by cleaving proteins and altering their biological function
(e. g., calpain cleavage of dysferlin contributing to membrane repair).
Further, calpains also function in roles independent of their proteolytic functions
such as the structural involvement of calpains in the conformation of triad
junctions. Therefore, the accumulating evidence that calpains have biological
functions beyond protein turnover highlights the importance of broadening the scope
of studies into calpain function.
While the study of skeletal muscle calpains may offer untapped potential, it is
important to note that several limitations remain in the study of calpain biology.
In an effort to stimulate future work in this field, the remainder of this section
will focus on these limitations and how addressing them will lead to heightened
understanding of calpain function. First, measuring calpain activity in vivo
remains a difficult task due to the multitude of factors involved in regulating
calpains; improvements in this area will allow investigators to further establish
the physiological paradigms that result in activation of calpains (e. g.
prolonged exercise in humans). In addition, the tools utilized to identify
calpain-truncated proteins is currently complex and time-consuming. While
computational models that predict calpain-cleavage sites are improving, our current
understanding of the specific structural components that dictate calpain cleavage
sites within calpain substrates is still limited. Further development of predictive
computational algorithms would allow for researchers to more easily identify
calpain-truncated proteins. Indeed, the current methodology utilized for
identification of calpain-truncated proteins has occurred largely through
recognition of these fragmented proteins via western blotting followed by
biochemical identification of the cleaved fragment. Due to the laborious efforts
currently required to identify calpain-mediated fragments, future development of
predictive modeling in tandem with tools such as mass spectrometry would lead to the
development of databases capable of identifying biologically important targets at a
larger and more efficient scale. This would then require in vivo studies to
assess the physiological impact of specific calpain-truncated proteins. In this
regard, the significance of calpain-truncated proteins has already begun to emerge
in studies of non-muscle cells. For example, it was recently discovered that
calpain-mediated tau fragmentation is associated with disease progression in
Alzheimer’s [118]. Hence, the study of
calpains and calpain-truncated proteins may yield new-insight in skeletal muscle
biology. While current dogma suggests that calpains are simple
“cleavers” in the proteolytic chain, this perception evolved from
their first proclaimed functions as “kinase activating factors”
[2]. Perhaps it is time to once again revisit the
role of calpains in skeletal muscle physiology.
