The signaling pathways involved in the regulation of protein turnover in skeletal
muscle
Regulation of protein synthesis
Protein synthesis rates are determined by de novo production of ribosomes
(increasing translational capacity) and by ribosome composition (determining
translation selectivity). In fact, ribosome biogenesis is an early event in
several conditions with an impact on SkM growth and maintenance [20]
[32]. The
association between the accumulation of rRNA levels and muscle hypertrophy was
reported in an incremental overload model [33] and
after 4 weeks or 8 weeks of resistance training [34]
[35]. Ribosomes are highly conserved
supramolecular complexes involved in the translation of mRNA into protein. These
structures consist of two subunits in eukaryotic cells: the small 40 S
subunit (contains 33 ribosomal proteins and 18 S rRNA) and the large
60 S subunit (comprises 47 ribosomal proteins and 3 rRNAs) [32]. Mitochondria also harbor a small proportion of
ribosomes (mitoribosomes) involved in the translation of mtDNA-encoded oxidative
phosphorylation (OXPHOS) polypeptides. In response to defects in the assembly of
mitoribosomes that leads to mitochondrial dysfunction, the upregulation of the
transcription of cell-survival mechanisms takes place as a compensatory measure
[36]. Still, the direct association between
mitoribosomes and SkM mass is poorly understood despite the recognized role of
mitochondria on the regulation of protein turnover, as analyzed below.
mTOR is a key regulator of protein synthesis
Translation initiation is the most studied process of protein synthesis with a
focus on the regulatory role of mTOR, which is also a key regulator of ribosome
biogenesis [37]. From the two multi-protein
complexes of mTOR, mTORC1 has been viewed as the main regulator of protein
synthesis, linking energetic and hormonal signs to protein synthesis [38]. Once activated by increased loading or amino
acid consumption, mTORC1 phosphorylates the downstream kinase ribosomal protein
of 70 kDa S6 kinase 1 (p70S6K1) and the 4E-binding protein-1 (4EBP1)
that promotes the initiation of protein synthesis through ribosomal binding to
mRNA [30]. mTOR is targeted by Akt [or protein
kinase B (PKB)], which is activated by the phosphatidylinositol 3-kinase (PI3K),
a downstream player of insulin signaling [39]
([Fig. 1]). Akt also phosphorylates and
inhibits glycogen synthase kinase (GSK) 3β, thus preventing the
inhibition of translation initiation via eukaryotic initiation factor
2Bε (eIF2Bε) [40]. The role of
mTORC1 in regulating SkM mass has been extensively analyzed, being widely
accepted that mTORC1 activation results in increased protein synthesis, whereas
suppression of mTORC1 signaling and decreased protein synthesis contributes to
SkM loss (reviewed by [40]). Indeed, Guridi et al.
[41] showed that the inhibition of mTORC1 (in
raptor muscle knock-out (RAmKO) mice) causes a significant reduction of muscle
mass, paralleled by a decreased expression of proteins involved in glucose and
fatty acid oxidation. In fact, the SkM phenotype of RAmKO was characterized by a
reduced oxidative capacity and decreased mitochondrial density. The same authors
also demonstrated that the chronic activation of mTORC1 (in tuberous sclerosis
protein 1 (TSC1) muscle knock-out (TSCmKO) mice) promotes severe metabolic
changes characterized by glucose intolerance (due to decreased translocation of
glucose transporters (GLUT) 4 to the sarcolemma) though without an impact on SkM
structure. In either case, mice were unable to gain lean mass with age,
suggesting that a proper balance in mTORC1 signaling is essential for SkM
homeostasis [41].
Intriguingly, the activation of mTORC1/p70S6K signaling was reported in
rat soleus muscle at the initial stage of hindlimb unloading (24 hours
after mechanical unloading) and was associated with the upregulation of
E3-ligases from the UPP [42]. The suppression of
contractile activity at the onset of hindlimb unloading may cause a fast
accumulation of high-energy phosphates (ATP, ADP, and creatine phosphate), which
was reported to be elevated in rat soleus muscle after 24 hours [42] and 10 days of unloading [43]. Increased levels of high-energy phosphates
inhibit AMP-activated protein kinase (AMPK) by decreasing its phosphorylation at
Thr172 [44]. AMPK is a heterotrimeric holoenzyme
comprising three subunits: a catalytic α (α1 or α2), a
scaffolding β (β1 or β2), and a regulatory γ
(γ1, γ2, or γ3) subunit [43]. AMP (or ADP) can bind to the γ subunit, changing its
conformation and exposing the Thr172 site of the catalytic α subunit.
The phosphorylation of this amino acid residue activates AMPK [45]. Thus, AMPK phosphorylation at Thr172 affects
protein synthesis in SkM by interfering with mTOR signaling through
phosphorylation of TSC2 (mTORC1 inhibitor) and of Raptor [46]
[47]
[48]. Regulated in development and DNA damage 1
(REDD1) is also a negative regulator of mTORC1 and was proposed to inhibit
protein synthesis in SkM during exercise [49]. In
addition, AMPK impairs translation by detaching polysomes into free ribosomal
subunits [37]. In cancer cachexia, the increase of
AMPK activity and REDD1 content was associated with decreased mTOR activity in
SkM [50]
[51].
Treadmill exercise attenuated this trend by increasing the expression of IGF-1
and, therefore, mTORC1 signaling [50]. Indeed,
exercise training has been recommended to preserve or increase the SkM mass in
subjects at risk of muscle wasting. Resistance training is a well-established
exercise mode that stimulates myofibrillar protein synthesis; however, endurance
training has been more prescribed for preventing muscle wasting [23]. According to Jee et al. [52], only high intensity endurance training
preserved mice SkM mass (compared to moderate intensity). Still, in a rat model
of cancer that develops cachexia, treadmill exercise was shown to prevent and
counteract muscle loss, an effect that was associated with exercise-induced
lower tumor malignancy and the decrease of circulating pro-inflammatory
cytokines [53]. Eccentric resistance exercise has
a higher anabolic effect in type II fibers (the type of muscle fibers more
susceptible to atrophy in cachexia) compared to concentric and to eccentric
combined to concentric exercise (reviewed by [23]). Moreover, the type of protein that is synthetized depends on the
type of exercise. In general, endurance training leads to an increase in the
expression of mitochondrial genes, and ultimately to enhanced mitochondrial
density, leading to a shift toward an oxidative phenotype and fatigue
resistance. Resistance exercise mostly induces the transcription of gene
encoding for myofibrillar proteins [30]. The
ability of a muscle fiber to alter the type and amount of protein is dependent
on the protein’s half-life; the ones with a shorter half-life and higher
synthesis rates are capable of attaining a new steady state faster in response
to a stimulus [54]. These attributes of protein
synthesis contribute to the SkM phenotypes seen after regular exercise.
Myostatin signaling inhibits protein synthesis in skeletal muscle
Myostatin, also known as growth and differentiation factor 8 (GDF-8), belongs to
the transforming growth factor (TGF)-β family and is a key regulator of
SkM mass. This ligand of type IIb activin receptors (particularly ActRIIB) is
expressed in SkM, and its upregulation has been reported in several catabolic
conditions such as cancer cachexia [55]. Following
ligand binding, ActRIIB recruits and activates the activin type I receptors
(ALK4 and ALK5), which phosphorylate and activate Smad2 and Smad3. In the
nucleus, Smad proteins regulate the expression of target genes [56]. Moreover, myostatin was shown to block
Akt/TORC1 signaling, through Smad2 inhibition of Akt activity, thus
reducing protein synthesis [57]
[58]. In cultured human myotubes, the reduction of
phosphorylated Akt resulted in the accumulation of active Forkhead box-O (FoxO)
transcription factors that regulate the expression of E3 ligases from the UPP
[59], as detailed in the section entitled
“The ubiquitin-proteasome pathway oversees skeletal muscle
proteolysis”. Overexpression of myostatin was also reported to elicit
the downregulation of muscle structural proteins (e. g., MHCIIb,
troponin I, and desmin) and myogenic transcription factors (MyoD and myogenin),
resulting in a significant loss of SkM mass [60].
Thus, blocking myostatin increases protein synthesis and SkM size, which
involves not only the activation of Akt/mTOR signaling but of other
pathways such as Hippo signaling [55]. Exercise
training can be seen as a therapeutic strategy to downregulate myostatin
expression in SkM, as recently reported in ovariectomized rats submitted to
weight-bearing exercise for 8 weeks [61].
Wnt and Hippo signaling interplay in the regulation of protein
turnover
Other important regulators of SkM mass in response to exercise include
Wnt/β-catenin signaling. This pathway also mediates the gain of
SkM mass induced by electric stimulation following spinal cord injury [62] and is critical to SkM development, formation
of the neuromuscular junction, and activation of stem cells. During
overload-induced SkM hypertrophy, Wnt activates its receptor, mFrizzled
(mFzd)-1, and the disheveled (Dvl)-dependent inhibition of β-catenin
complex degradation. Consequently, β-catenin accumulates in the nucleus
and acts as a transcription factor by interacting with T-cell
factor/lymphoid enhancer factor (TCF/Lef) [21]
[63] ([Fig. 1]). Wnt/β-catenin interferes
with other pathways such as Hippo signaling, which can inhibit Wnt pathway by
blocking Dvl or avoiding β-catenin translocation to the nucleus [63]. Yes-associated protein (Yap) and its ortholog
transcriptional co-activator with PDZ binding motif (Taz) are the main players
of the Hippo signaling. Yap/Taz may bind to TEA domain (TEAD)
transcription factors and regulate the expression of specific genes such as
L-type amino acid transporter 1 (LAT1). However, when Yap/Taz are
phosphorylated by the large tumor suppressor kinases (Lats) 1 and 2, they are
maintained in the cytoplasm and targeted by the UPP. Yap can also be
phosphorylated by AMPK and sequestered in the cytoplasm [63]
[64]. Yap was reported to accumulate
in the nucleus and to interact with TEAD after disruption of the neuromuscular
junction, regulating gene expression that maintains SkM mass [65]. Yap may be activated by integrin-mediated
stress fiber formation in response to a mechanical stimulus [25]. In fact, increased mechanical loading (using
the synergist ablation mice model) was shown to promote Yap overexpression in
the plantaris muscle, which was associated with increased content of
phosphorylated Akt (Thr308) and total Akt but not of mTORC1. Mechanical
loading-induced overexpression of Yap was also associated with the increase of
c-Myc expression, a potent activator of ribosomal biogenesis, and of MyoD, a
myogenic regulator factor, and to decreased levels of Smad2/3 and MuRF1.
Thus, Yap seems to induce SkM hypertrophy by increasing c-Myc and MyoD
expression ([Fig. 1]) and by suppressing protein
breakdown rates through the inhibition of Smad-mediated E3 ligase expression
[66]. However, in the SkM fiber-specific
knock-in (MCK-tTA-hYAP1 S127A) mouse model, the overexpression of constitutively
active Yap (hYAP1 S127A) resulted in SkM atrophy rather than hypertrophy. This
atrophic phenotype was reverted after removing hYAP1 S127A expression, with mice
recovering body and muscle weight [67]. These
apparent contradictory findings on the impact of Hippo signaling in SkM mass
regulation may be explained by the experimental models used. Still, this topic
deserves further investigation.
Notch signaling regulates skeletal muscle mass
Muscle fibers express Notch2 receptors that may be activated by the ligand DII4,
which is upregulated and released by the microvascular endothelium in atrophic
conditions. The endothelial Dll4–SkM Notch2 axis was recently shown to
be a central mechanism that regulates catabolic signals from mechanical and
metabolic stimulation [22] ([Fig. 2]). In addition, the Notch target gene Hey1
was reported to be upregulated in the rat soleus muscle after 4 months of spinal
cord transection (by approximately 1.5-fold), and further increased by 7 days of
electric stimulation, suggesting a role for Notch signaling in the hypertrophy
of atrophied muscle in response to exercise [62].
Data from the microarrays used to profile the transcriptome of the biceps
brachii from young women and men after an acute unilateral resistance session
followed by 12 weeks of progressive training highlighted the activation of
atrophic factors from the Notch pathway (e. g., the transcriptional
repressors of Hey and Hes family members) in women, concomitantly with the
hypertrophic effect of resistance exercise seen in both sexes [68]. Thus, Notch signaling seems to be differently
modulated by sex, being upregulated in women following resistance exercise.
Overall, there are multiple signaling pathways controlling protein synthesis
during loading and wasting conditions. The stimulation of protein synthesis and
SkM mass gain by exercise is dose- and threshold-dependent. Different loading
patterns result in similar anabolic responses in untrained muscle; however, in
trained subjects, muscle protein synthesis depends on the mode of exercise [69]. Resistance exercise training has been
consistently reported to increase protein synthesis; however, no changes in
protein synthesis were detected in rats’ gastrocnemius after 6 weeks of
progressive resistance-loaded voluntary wheel running, despite mass gain [17]. Both resistance and endurance exercise blunt
muscle protein synthesis during contraction [3]
[69], perhaps to channel energy
supplies for contraction. Contracting muscle itself and/or humoral
factors seem to interplay to activate/suppress the signaling pathways
involved in the regulation of protein synthesis in SkM, with AMPK and mTOR
taking a key role in such regulation. The contribution of these pathways to the
rate of protein turnover depends on their interaction with the pathways that
control proteolysis and should be assessed in an integrated perspective.
Regulation of muscle protein breakdown
Mammalian SkM contains four major proteolytic systems: lysosomes, UPP, calpains,
and caspases [70]. The role of lysosomal proteases
(e. g., cathepsins) in myofibrillar protein turnover is expected to be
small, since low pH is required for their optimal activity, and engulfment of
myofibrils would be necessary. Nevertheless, small autophagosomes were described
in the perinuclear region and between myofibrils, and several mechanisms of
autophagy are involved in bringing bulk cytoplasmic, long-lived proteins inside
lysosomes [71]. Oxidative muscles exhibit higher
levels of cathepsins, which are up-regulated (particularly cathepsin L) in
wasting conditions such as cachexia [71]
[72]. The caspase system is usually associated with
apoptosis. Still, caspase-3 may initiate SkM proteolysis by cleaving
myofibrillar proteins into smaller fragments, providing substrate for UPP [72]
[73]. The
contribution of calpains and the UPP to SkM protein turnover, particularly of
myofibrillar proteins, is expected to be higher [24]
[74]. In fact, the activation of
calpains and UPP has been reported under conditions promoting muscle atrophy.
However, increased contractile activity (e. g., in the early moments of
exercise training) also stimulates these proteolytic systems, possibly to remove
the damaged proteins resulting from the mechanical and metabolic challenges
imposed on muscle fibers [74]. The relevance of
UPP and calpains in SkM adaptation to pathophysiological conditions has been
discussed in numerous reviews [70]
[75]. Still, some issues on the role of calpains and
UPP in SkM protein turnover are highlighted herein.
Proteolysis mediated by calpains
Calpains (calcium-activated cysteine proteases) were shown in 1976 to initiate
myofibrillar protein turnover by selectively releasing filaments from the outer
layer of myofibril [76], making them available for
degradation by other proteolytic systems, mainly UPP. Indeed, myofibrillar
proteins to be degraded without affecting the contractile activity of muscle
must be disassembled from the myofibril and then proteolytically decomposed
[7]. Yet a small number of myofilaments can be
easily released by myofilaments, primarily the ones lacking in or with lower
amounts of α- actinin, an actin crosslinking protein [10]. Calpains are concentrated in the Z disk of
myofibrils [77], and they were suggested to
penetrate the myofibrillar matrix and cleave sites inside the myofibril [7]. At least 15 different calpains were described
in mammals; however, only six are expressed in SkM. Of these, μ- and
m-calpains (activated by μM and mM concentrations of
Ca2+, respectively) have been the isoforms most studied
in SkM remodeling. Both μ- and m-calpains (also known as calpain-1 and
calpain-2, respectively) have nearly identical substrate specificities. Among
their substrates are myofibrillar and signaling proteins such as protein kinase
C (PKC), calcineurin, and calcium/calmodulin-dependent protein kinase IV
(CaMK) [70]
[75].
GLUT4 was also shown to be a substrate of m-calpain, which supports the
participation of calpains in the regulation of the SkM metabolism [78]. There is another calpain isoform, calpain-3
(or p94), with recognized physiological relevance in SkM protein turnover. This
isoform works at sub-μM Ca2+concentrations and it is
not inhibited by calpastatin, the endogenous inhibitor of calpains. Calpain-3
mainly localizes in the sarcomere bound to titin (at two regions, N2A line and M
line), the largest whole-body protein (3 million Dalton), and the third most
abundant in SkM (following actin and myosin). Calpain-3 interaction with titin
protects calpain-3 from auto-degradation and maintains the enzyme in an inactive
state, despite being near its substrates [79].
Calpain-3 also binds at the Z-band to α-actinin through the N-terminus
[80]. Titin damage may be an important signal
for the activation of this calpain isoform [79].
Calpain-mediated cleavage of sarcomere proteins such as titin was reported in
muscle unloading and is associated with the decrease of force-generating
capacity [81]. The activation of calpains in
disuse-induced atrophy was shown to depend on time and muscle type. For example,
increased calpain activation was observed in the soleus within 12 hours
of hindlimb suspension, whereas in gastrocnemius calpain activity was not
detected until the muscles had been unloaded for more than 3 days [82]. Calpain activation also occurs following a
stimuli like eccentric exercise (but not isometric contractions). The increase
of intracellular Ca2+concentration activates calpain-3 that
cleaves titin and other myofibrillar proteins [83]
[84]
[85]. Consequently, the transmission of force will tend to decrease.
The activation of calpains following exercise is also time-dependent. For
example, μ-calpain was reported to be activated 30 minutes after
eccentric exercise in humans [86], whereas
calpain-3 activation was detected 24 hours after the activity [85]. Immediately after a single bout of aerobic
exercise training, calpain activation was detected in mice plantaris muscle;
however, 48 hours after the end of the exercise session, activity was no
longer observed [74].
The ubiquitin-proteasome pathway oversees skeletal muscle proteolysis
The peptides resulting from myofibrillar protein cleavage must be removed to
avoid the toxicity of their accumulation. Thus, the cleavage products are
targeted by ubiquitin for subsequent elimination by 26 S proteasome
([Fig. 2]). The UPP is an ATP-dependent
process, linking proteolysis to energy availability. In SkM, this proteolytic
system includes the E3-ligases MuRF1 (or Trim63) and Muscle Atrophy F-box
(MAFbx), also known as atrogin-1 [38]. These
atrogenes have been seen as the gold-standard markers of SkM proteolysis, and
their expression varies with the atrophic stimuli. For example, the peak of
atrogin-1 and MuRF1 mRNA expression in the gastrocnemius was reported 3 days
after denervation, before the manifestation of atrophy, and returned to basal
levels by 14 days (atrogin-1) and 28 days (MuRF1) [87]. The expression of these E3-ligases in the soleus and plantaris
was also observed to peak 3 days after hindlimb immobilization. Still, the
expression of atrogin-1 and MuRF1 was higher in plantaris than in soleus muscle;
however, the percentage of muscle mass decrease was similar in both muscles,
suggesting that E3-ligases have a higher contribution to the atrophy of
fast-twitch than slow-twitch muscles [88].
The gene encoding for MuRF1 and atrogin-1 seems to be upregulated together under
most atrophy conditions. In fact, the proximal promoter region of each gene
contained several consensus Class O FoxO binding sites (FBE), capable of binding
FoxO1, FoxO3a, and FoxO4 transcription factors, all of which are expressed in
SkM and under Akt regulation [89]. Akt is
determinant in the control of both anabolic and catabolic signals. Its activity
may be inhibited by TRB3 (the mammalian Drosophila tribbles homolog 3), a
pseudokinase that directly binds to Akt. The overexpression of TRB3 was
associated to decreased mTOR/S6K1 phosphorylation and, consequently,
protein synthesis rate, and to increased FoxOs activation and atrogenes
expression, reducing SkM mass [90]. However,
contradictory findings on the effect of TRB3 overexpression in SkM were reported
[91], possibly justified by the mice model
used and diet. Phosphorylation of FoxO transcription factors promotes their
export from the nucleus to the cytosol, inhibiting their activity. However, the
MuRF1 and atrogin-1 promoters do not seem to be equally activated by these
transcription factors. FoxO1 was reported to increase MuRF1 expression, whereas
FoxO3a seems to increase atrogin-1 [89]. Still,
FoxO3 was suggested to be the most critical regulator of atrophy since its
deletion in SkM was not compensated by other factors. FoxO3 activity is
regulated by several molecules such as REDD1, which prevents its
dephosphorylation in SkM from cachectic mice [51].
FoxO transcription factors were shown to regulate the expression of other
E3-ligases besides MuRF1 and atrogin-1, as for example the muscle ubiquitin
ligase of the SCF complex in atrophy-1 (MUSA1), also known as Fbxo30 [92]
[93]. This ligase
was reported to be critical for denervation and fasting-induced SkM atrophy.
FoxO-dependent atrogenes include autophagy-related genes such as LC3 and Bnip3
[93], whose role in protein turnover is
discussed below.
The protein substrates of E3-ligases are mostly unknown. In fact, the
identification of the substrates targeted for ubiquitination by an individual E3
ligase has been challenging. Moreover, ubiquitination of a protein not only can
mark it for degradation by the proteasome but may also regulate its activity,
change its cellular localization, and interfere in protein-protein interactions
[89]. Still, MuRF1, and its paralog MuRF2,
were reported to be located in the sarcomere, the sarcomere proteins being an
expected substrate of these ligases [79]. In fact,
MuRF1 (more abundant than its paralog) interacts with the giant muscle proteins
titin and nebulin, cooperating with calpain-3 in sarcomere proteins turnover
[2]. MuRF1 was also shown to ubiquitinate
other myofibrillar proteins such as troponin I and myotilin [94], and metabolic proteins such as M-creatine
kinase and aldolase A. By degrading and, consequently, downregulating metabolic
proteins, MuRF1 regulates energetic pathways [2].
Atrogin-1 seems to regulate the activity of transcription factors such as c-Myc
[95] and eukaryotic initiation factor
3 f (eIF3f) [96] by directing them for
proteasome breakdown. MuRF1 also controls protein synthesis through the
ubiquitination of transcription factors, such as the glucocorticoid modulatory
element binding protein-1 (GMEB1) [97].
Concomitantly, amino acids are provided from the degradation of these proteins
and can be used as energetic fuel. Curiously, 3-hydroxyisobutyrate dehydrogenase
(HIBADH), an enzyme involved in the valine catabolic pathway, is also a target
of MuRF1, suggesting that valine, and eventually other branch-chain amino acids
(BCAA), are not oxidized in SkM and may be released to the bloodstream and used
by other tissues [2]
[48]. Therefore, the E3 ligases MuRF1 and atrogin-1 are key regulators
of muscle protein turnover by controlling both protein synthesis and
proteolysis. Even so, MuRF1 and atrogin-1 are under the control of mTORC1
signaling, which seems to involve the nuclear-cytoplasmic traffic of histone
deacetylases (HDACs), particularly HDCA5. In unloaded soleus muscle, increased
p70S6K activity resulted in the upregulation of E3 ligases via phosphorylation
and nuclear export of HDAC5 [42]. Overall, the
signaling pathways involved in the regulation of protein synthesis and breakdown
interplay in the SkM remodeling induced by wasting and loading conditions,
reflecting and contributing to the energetic status of this organ.
The role of mitochondria in the control of protein turnover
The balance between protein synthesis and breakdown is tightly controlled by
mitochondria, the powerhouses of SkM fibers. Mitochondria harbor the oxidative
phosphorylation (OXPHOS) system, the major ATP supplier of SkM fibers. ATP is
needed for several cellular processes, with protein synthesis being the major
ATP-consuming process of myofibers that face high energy demands during
contraction [31]. Thus, perturbations in
mitochondrial homeostasis and, consequently, on ATP generation can have
deleterious impact on the maintenance of SkM mass and function, as reported in
several SkM wasting conditions [98]
[99]. On the other hand, regulators of protein
synthesis, such as mTORC1 and AMPK, control mitochondrial biogenesis and
function [100]
[101]
[102]. mTORC1 selectively promotes
the translation of nucleus-encoded mitochondria-related genes through the
inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding
proteins (4EBPs) [101]. In SkM, rapamycin (mTOR
inhibitor) decreased the expression of peroxisome proliferator-activated
receptor-γ coactivator (PGC)-1α, estrogen-related receptor (ERR)
α and nuclear respiratory factors (NRFs), and, consequently, reduced
mitochondrial gene expression and organelle functionality. The transcription
factor yin-yang 1 (YY1) seems to be required for rapamycin-dependent repression
of the gene encoding for these mitochondrial transcriptional regulators [103]. By controlling mTORC1, REDD1 affects
mitochondrial functionality in SkM. REDD1 was detected in the mitochondrial
fraction of mice SkM after endurance exercise and appears to interact with
mitochondria-associated endoplasmic reticulum (ER) membranes (MAM, a functional
interaction between mitochondria and ER) proteins, including mitochondrial heat
shock protein (mtHSP) 70 ([Fig. 2]). Thus, MAM
contact sites are disrupted, decreasing ATP availability for ER-dependent
protein synthesis [31]
[49]. In this way, energy is saved to ensure cell survival during
metabolic challenges [49].
A great amount of ATP is consumed by sarcoendoplasmic reticulum calcium ATPase
(SERCA) pumps and actomyosin contraction. Transient changes in cytosolic
Ca2+generated by physiological stimuli prompt large
increases in the Ca2+concentration of the mitochondrial
matrix, stimulating the Ca2+-sensitive dehydrogenases of the
tricarboxylic acid (TCA) cycle (e. g., NAD-isocitrate dehydrogenase and
oxoglutarate dehydrogenase). Ca2+entry into mitochondria is
mediated by the mitochondrial calcium uniporter (MCU). Mitochondrial
Ca2+accumulation via MCU was positively associated with
the size of myofibers. Moreover, MCU expression was correlated with
PGC1-α4, an isoform of the transcriptional regulator of mitochondria
biogenesis PGC-1α [104]
[105]. This isoform is highly expressed in trained
SkM but does not regulate most of the known targets of PGC-1α
(e. g., OXPHOS genes). PGC1-α4 specifically stimulates the
anabolic pathway IGF1/Akt and represses the catabolic route triggered by
myostatin [104]
[106]. PGC1-α4 is particularly responsive to resistance exercise
and drives muscle hypertrophy, whereas PGC-1α induces many of the
adaptations promoted by endurance training including mitochondrial biogenesis,
fiber-type switching, upregulation of fatty acid oxidation and angiogenesis
[106]
[107].
PGC-1α may be phosphorylated (at Thr177 and Ser538) by AMPK when ATP
levels are depleted and the AMP/ATP ratio increases, which in turn
induces PGC-1α promoter and the transcription of many AMPK target genes
(e. g., GLUT4, mitochondrial genes). Thus, anabolic pathways are
inhibited, and catabolic ones are stimulated to restore the ATP content [108].
The translation of mtDNA-encoded genes into the corresponding proteins is also
essential for mitochondrial homeostasis and is impacted by several
pathophysiological conditions. Mitochondrial translation is divided into four
phases: initiation, elongation, termination, and mitoribosome recycling. The
mitochondrial translation factors involved are two mitochondrial initiation
factors (mtIF2 and mtIF3), three mitochondrial elongation factors (mtEFTu,
mtEFTs, and mtEFG1), one mitochondrial release factor (mtRF1L), and two
mitochondrial recycling factors (mtRRF1 and mtRRF2) [73]
[109]. Changes in the levels of
these mitochondrial translation factors and, consequently, on mtDNA-encoded
proteins were reported in wasting and loading conditions. Indeed, 3 days after
denervation, a decrease was observed in the expression of mitochondrial
initiation factor mtIF2, elongation factor mtEFTu, recycling factor mtRRF1, and
translational activator TACO1 in soleus muscle. After 7 days, other
mitochondrial translation factors were downregulated, such as mtIF3, mtEFTs,
mtEFG1, mtRF1L, mtRRF1, and mtRRF2. This decrease in the expression of
mitochondrial translational factors resulted in the diminished content of
mtDNA-encoded proteins and reduced mitochondrial biogenesis [110], supporting the involvement of mitochondrial
translation in the regulation of mitochondrial biogenesis and functionality. In
opposition, endurance exercise has been consistently reported to increase
mitochondrial biogenesis (meaning an increase in muscle mitochondrial number and
volume, and changes in organelle molecular composition) by enhancing the
translation of both nDNA and mtDNA-encoded mitochondrial proteins [30]
[111]. Still, the
impact of mitochondrial translation on SkM remodeling depends on muscle type,
being higher in type I fibers that present higher mitochondrial density [109]. Overall, mitochondrial adaptations to regular
exercise not only enhance exercise performance but also impact a broader range
of health issues [111].
Quality control systems balance biogenesis mechanisms to maintain
mitochondrial health
Being a postmitotic tissue, SkM mass not only depends on mitochondrial biogenesis
but also on mitochondria quality control (MQC) systems to keep mitochondrial
proteostasis and function [99]. MQC systems
require the activity of molecular chaperones and mitochondrial proteases
(e. g., Lon and AAA proteases). Besides, UPP degrades misfolded proteins
located in the outer membrane of mitochondria. Mitochondrial proteases and UPP
work together to remove oxidized and misfolded mitochondrial proteins ([Fig. 2]), avoiding the toxicity of their
accumulation [112]. In fact, the accumulation of
oxidized proteins was associated with decreased mitochondrial content of Lon and
m-AAA paraplegin in the gastrocnemius muscle from diabetic [113] and cancer cachectic rats [114]. Moreover, mitochondria are highly dynamic,
being able to fuse, mix, and replenish their content. Mitochondrial fusion may
be seen as an attempt to avoid autophagy and maximize ATP generation. However,
when mitochondrial membrane potential is lost, fission occurs. This process
provokes organelle removal through mitophagy, which is an important mechanism of
MQC [112].
Mitophagy may be triggered by AMPK that phosphorylates and activates Unc-51-like
autophagy activating kinase (ULK1), the most upstream known mitophagy protein.
This kinase recruits other autophagy-related proteins (Atg) that form complexes
[115]
[116] and
may promote the fusion of lysosome with mitochondria-containing autophagosome
[44]. The activation of AMPK and ULK1 was
reported in SkM 6 hours after acute treadmill exercise [117]. More recently, Drake et al. [45] demonstrated that tissue-specific isoforms of
AMPK are localized on the outer mitochondrial membrane (known as mitoAMPK) and
are required for mitophagy. This kinase becomes phosphorylated at Thr172 in SkM
in response to treadmill exercise. When mitoAMPK activity is inhibited,
exercise-induced mitophagy is attenuated. Data suggest that mitoAMPK acts as an
energetic sensor to fine-tune mitochondrial functionality, thus maintaining the
energetic homeostasis in SkM. Mitophagy may also be initiated by phosphatase and
tensin homolog-induced putative kinase protein (PINK)1. When mitochondrial
membrane potential is lost, PINK1 accumulates in the outer mitochondrial
membrane and recruits its substrate, the E3 ligase parkin, which in turn is
phosphorylated and activated by PINK1. Parkin mediates the ubiquitination of
proteins from the mitochondrial surface [98]
[118] ([Fig. 2]).
Ablation of PINK1 and parkin was reported to reduce mitophagy in SkM and to
increase mitochondria susceptibility to oxidative stress [99]. In denervated gastrocnemius,
PINK1/Parkin mediated mitophagy was observed and resulted in significant
reductions in mitochondrial number [119]. An
opposite effect was reported following exercise training and was characterized
by increased mitophagy though balanced by increased mitochondria biogenesis
[99]. Autophagy receptors are recruited and
interact with autophagosome membrane protein light chain 3-I (LC3-I) from the
autophagosome, which is converted in the phosphatidylethanolamine conjugated
form LC3-II [98]
[118]. Increased LC3-II and parkin levels were reported immediately
following acute exhaustive exercise in mice SkM, suggesting enhanced mitophagy
and mitochondrial proteins breakdown. These processes were compromised in mice
lacking PGC-1α, supporting the master role of PGC-1α in the
regulation of exercise-induced mitophagy [117].
Endogenous mitochondrial membrane-bound receptor proteins, such as
BCL2/adenovirus E1B 19kD interacting protein 3 (Bnip3), may drive
mitophagy by binding to LC3. Increased expression of this protein was observed
in plantaris muscle after 4 weeks of voluntary physical activity and occurred in
parallel with the overexpression of PGC-1α, suggesting that mitophagy
occurs in tandem with mitochondrial biogenesis to improve SkM functionality
[120]. The fusion of lysosomes with
autophagosome generates an autolysosome, allowing lysosomal enzymes to complete
the mitophagy process. These autolysosomes are characterized by the presence of
lysosomal-associated membrane protein 1 or 2 (LAMP1 or LAMP2) [118]. The accomplishment of all steps of mitophagy
is crucial to avoid the accumulation of dysfunctional mitochondria. In fact, in
age-related loss of SkM there is an accumulation of dysfunctional mitochondria
due a decline in mitophagy process [115]
[121].
Taken together, there are multiple molecular mechanisms involved in the
continuous reshaping of the mitochondrial network towards the maintenance of
healthy mitochondria. Collectively, these mechanisms represent a MQC system that
recognizes and counteracts mitochondrial dysfunction, essential for protein
turnover and SkM mass maintenance. Thus, the dysregulation of this system
negatively impacts SkM mass and function, which is at the core of several
pathophysiological conditions.
Regulation of extracellular matrix protein turnover
Intramuscular ECM plays a key role in intercellular communication, force
transmission, and structure maintenance. ECM mediates the transmission of
contractile force, regulating the efficiency of muscular contraction and
protecting muscle fibers from excessive stress and assisting in the healing
following microtrauma [122]. ECM homeostasis is
regulated by protein turnover, which in turn is reflected by the rates of
protein synthesis (mostly of collagen) and breakdown, particularly those
mediated by metalloproteases (MMPs) [123].
Collagen represents the most abundant ECM protein within the muscle and SkM
contains collagen types I, III, IV, V, VI, XII, XIII, XIV, XV, XVIII, and XXII
(from the 28 members of the collagen superfamily) [122]. Of these, collagens I and III account for 75% of total
SkM collagen and are present in the form of fibrils [122]
[124], with slow-twitch muscles
containing more collagen than fast-twitch ones. In SkM, collagen is mostly
produced by fibroblasts and is characterized by several posttranslational
modifications of the polypeptide chains, which are enrolled in the
triple-helical procollagen structures secreted by exocytosis to the ECM. After
N-terminal cleavage, the collagens self-assemble into fibrils [125]. Collagen transcription is rather slow (around
3 days), whereas secretion takes less than 1 hour [122].
Collagens, independently of their type, are substrates of MMPs, whose activity is
regulated by tissue inhibitors of metalloproteinases (TIMPs) ([Fig. 2]). MMP-2 and MMP-9 (also known as
gelatinases A and B, respectively) target type IV collagen, the major
collagenous component of the basement membrane [122]. TIMP-1 forms a complex with MMP-9 and TIMP-2 with MMP-2. MMP-2
is constitutively expressed in fibroblasts and its activity is enhanced in
inflammatory conditions. MMP-9 is secreted by endothelial and inflammatory
cells. As regulators of ECM integrity, MMPs, particularly gelatinases,
participate in the SkM remodeling in response to stimuli such as exercise
training [126]. Despite similar substrate
specificity, the expression pattern and transcriptional regulation of these MMPs
are very different. For instance, increased expression of MMP-9 was reported
after a single bout of exercise, whereas the content of MMP-2, MMP-14, and
TIMP-1 was raised after 10 days of exercise training [127]. MMPs activity counteracts the increased collagen content
reported in trained subjects, which appears to be due to the upregulation of
posttranscriptional and translation events; however, the amount of collagen and
other ECM proteins seems to be dependent on the exercise mode, particularly in
aged individuals [124]. Four days of eccentric
training promoted a greater upregulation of collagens I and III expression, as
well as of TGF-β and lysyl oxidase (that form cross-links in ECM
proteins), in female rat gastrocnemius compared with 4 days of concentric or
isometric training [128]. In human SkM biopsies,
collagen synthesis was reported to increase 4.5 hours after maximal
contractions (shortening or lengthening contractions), suggesting an increase of
protein synthesis activity of SkM fibroblasts promoted by both types of
contractions [129]. After forced lengthening
contractions, the activity of prolyl 4-hydroxylase (which catalyzes the
formation of 4-hydroxyproline in collagens) and type IV collagen concentration
was enhanced in the rat tibialis anterior muscle and were paralleled by the
activation of MMP-2 and MMP-9 and their inhibitors during recovery [130]. In aged SkM, increased collagen concentration
with changed composition (shift towards higher type I to type III collagen) was
reported. Alongside, hydroxylysylpyridinoline cross-linking and advanced
glycation products (AGE) were also raised. Decreased MMP activity may explain
uncontrolled ECM expansion, causing enhanced muscle stiffness and reduced whole
muscle function [122]
[131]. During immobilization, SkM becomes fibrotic with increased
collagen content and disorganization, which contribute to the reduced capacity
of immobilized SkM to elongate in response to stretching without tissue damage
[132].
Altogether, ECM remodeling is characterized by increased collagen synthesis,
which is balanced by MMP activity following exercise but not in aging or other
pathophysiological conditions, resulting in muscle fibrosis. In fact, fibrosis
is a hallmark of maladaptive remodeling of SkM, which is a sign of muscle
weakness and impaired regeneration after injury. Strategies for tackling SkM
fibrosis entails the targeting of TGF-β1, a potent pro-fibrotic
cytokine. This M2 macrophage secreted cytokine not only stimulates fibroblasts
to produce and secrete collagen and other ECM proteins but also promotes the
secretion of TIMPs, inhibiting MMP activity. This cytokine also fosters the
transdifferentiation of several resident cell types into myofibroblasts,
boosting a pro-fibrotic phenotype [133].
Amino acids pool and protein turnover
The balance between protein synthesis and breakdown from and to the intracellular
pool of free amino acids is determinant to protein turnover in SkM. Still,
different SkM types present different protein amounts and turnover as well as
amino acid composition, making it difficult to disclose the impact of changes in
amino acid pools under different stimuli. Indeed, distinct SkM types are
differently affected by wasting conditions and starvation that result in the
release of amino acids. For instance, soleus (slow-twitch muscle) was reported
to have higher concentrations of His and Lys and the most non-essential amino
acids compared with EDL (fast-twitch muscle) in the postprandial state [134]. Greater activity of proteasome was also
reported in the soleus and was associated with the release of 3-methylhistidine
(a marker of proteolysis of myofibrillar proteins) [135]
[136].
Changes in the protein balance occur daily, depending on food intake
(particularly of essential amino acids) and physical activity, which mainly
affect protein synthesis [88]. One day of
starvation blunts protein synthesis and proteolysis is enhanced, increasing the
release of amino acids from SkM to feed liver gluconeogenesis. Still, the
circulating levels of these amino acids decrease except for BCAA (meaning Val,
Leu, and Ile). Why BCAA circulating levels increase during starvation is not
clear, though it was suggested that their synthesis from branched-chain keto
acids rises in the liver and their uptake is reduced due to decreased insulin
production [134]. SkM is the main site of BCAA
metabolism, with the highest activity of BCAA aminotransferase (BCAT2), the
enzyme that catabolizes the first step of BCAA degradation. BCAA are essential
amino acids used for protein synthesis and energy production, and perform
signaling roles (e. g., via mTOR) [137].
BCAA, specifically the proteogenic amino acid Leu, activate Rag GTPase and,
consequently, the translocation of mTORC1 from the cytoplasm to the surface of
lysosomes, where it interacts with the heterodimer formed by GTP-loaded RagB and
GDP-loaded RagD. Thus, the downstream players from mTOR pathway are activated
[138]. However, Leu levels are not sensed
directly by Rag GTPase or mTOR but by Leucyl-tRNA synthetase (LeuRS) ([Fig. 1]) and sestrin2, which were identified as
Leu sensors [139]
[140]. Four weeks of resistance exercise training was reported to
promote gastrocnemius hypertrophy, which was associated with the enhanced
phosphorylation of mTOR, p70S6K and 4E-BP1, and the upregulation of LeuRS but
not of sestrin2 [141]. Some authors reported that
sestrin1, instead of sestrin2, is highly expressed in the SkM and mediates Leu
activation of mTORC1 [142]. Indeed, the
downregulation of sestrin1 expression was described in the mice soleus within
24 hours of hindlimb immobilization and was associated with diminished
mTOR signaling [143]. Increased levels of sestrin1
were detected in human SkM biopsies following 12 weeks of resistance exercise
[144]. Acute treadmill exercise increased
sestrin1 accumulation and induced a discrete enhancement of sestrin2, whereas 4
weeks of aerobic exercise decreased the basal levels of both sestrins in mice
quadriceps muscles. Moreover, the expression of sestrin1 and 2 was associated
with AMPK activation in trained muscles [145]. Arg
can also activate the mTOR pathway by binding to the cytosolic arginine sensor
for mTORC1 subunit 1 (CASTOR1) and disrupts its interaction with
GTPase-activating protein toward rags 2 (GATOR2), thereby activating mTORC1
[146].
The exercise-induced increase of BCAA metabolism in SkM is also mediated by
PGC-1α, which seems to enhance the expression of BCAT2 and
branched-chain α-keto acid dehydrogenase (BCKDH) (the enzyme that
catalyzes the second step of BCAA metabolism) [146]
[147]. PGC-1α also
activates alanine aminotransferase (ALT2) in SkM during fasting [148]. Likewise, PGC-1α overexpression was
associated with increased levels of GABA and Glu in the gastrocnemius muscle
[149]. Thus, PGC-1α seems to play a
role in the regulation of amino acid metabolism in SkM.
Amino acids increase the synthesis of SkM proteins, either myofibrillar,
sarcoplasmic and mitochondrial ones, probably in a dose-dependent manner. In
fact, feeding acts synergistically with exercise to promote protein synthesis in
SkM [69], and the gain of muscle mass after
exercise is dependent on amino acid availability (particularly Leu) during the
postexercise period. The maximum protein synthesis may be achieved with the
intake of approximately 20 g of high-quality protein (such as whey
proteins) [69]. Nevertheless, the ingestion of
40 g of whey protein after resistance exercise resulted in greater
stimulation of protein synthesis in SkM compared to 20 g of whey
protein, though without affecting the p70S6K1 activity. This apparent disconnect
between mTOR signaling and protein synthesis seems to be explained by a temporal
discrepancy between both processes [150]. Still,
BCAA supplementation did not improve muscle soreness and function during
recovery of untrained young adult (average age of 24 years old) from resistance
exercise [151]. Overall, more than BCAA
supplements, the ingestion of high-quality protein boosts protein synthesis
following exercise training, improving muscle health.
