The protective effects of apocynin is attributed to a preserved
proteostatic signaling
The main function of muscle-derived IGF-1 is to promote protein
synthesis and muscle growth via the action of an intracellular signal
transducer, mTOR (Nicklin et al., 2009). Given the expression of
muscle-derived IGF-1 was found to be suppressed by CS (Figure 2E) and
CSE (Figure 4C-E and H-J) exposure, we reasoned whether the key signal
transduction pathways responsible for maintaining balance between
protein synthesis and breakdown (i.e. proteostatic signaling) were
impacted. H2O2 exposure
concentration-dependently ablated the phosphorylation level of S6
ribosomal protein and eukaryotic translation initiation factor
4E-binding protein 1 (4E-BP1; Figure 6A, C-D), which are the key
downstream effectors of mTOR (Schiaffino & Mammucari, 2011). The
phosphorylation status of a key repressor of protein synthesis,
eukaryotic translation initiation factor 2A
(eIF2α; Figure 6A and B), was
found to be increased by 5 to 15-fold, suggesting a global inhibition of
protein synthesis. In line with the mRNA expression (Figure 4A),
H2O2 increased the protein abundance of
MAFbx (~50%), a muscle specific E3 ubiquitin ligase
(Figure 6A and E). Furthermore, a significant increase in abundance of
the 19S proteasome (S5a), a regulatory subunit of the 26S proteasomal
complex, was observed following exposure to 100 μM of
H2O2 (Figure 6A and F), suggesting the
activation of the
ubiquitin-proteasome system (UPS). H2O2exposure also resulted in the activation of autophagic pathway evidenced
by the conversion of LC3A/B-I to LC3A/B-II (Figure 6A and G-H) and
decrease in p62 abundance (Figure 6A and I).
Apocynin treatment maintained
phosphorylated S6 ribosomal protein expression against 10 μM of
H2O2 but not 4E-BP1 or eIF2α (Figure
6A-D). Meanwhile, no significant effects were detected for the
activation of UPS and autophagic pathways (Figure 6A, E-I) suggesting
the protective effects of apocynin are unlikely to be modulated through
protein degradative pathways.
Meanwhile, exposure of myotubes to submaximal concentrations of CSE did
not evoke the phosphorylation of eIF2α (Figure 7A-B) or decrease the
phosphorylated S6 ribosomal protein expression (Figure 7A and C),
although a concentration-dependent reduction in the phosphorylation
levels of 4E-BP1 was observed (Figure 7A and D). Like that of
H2O2, CSE exposure increased abundance
of MAFbx (Figure 7A and E), however no detectable changes in 19S
proteasome protein were observed until the maximal concentration (100%)
of CSE was used (Figure 7A and F). Likewise, exposure to the maximal
concentration of CSE resulted in the activation of autophagic pathway
evidenced by the LC3A/B-I to LC3A/B-II conversion (Figure 7A and G-H)
and decrease in p62 abundance (Figure 7A and I), but no significant
effects were observed under submaximal conditions (10-20% of CSE).
Apocynin treatment preserved the phosphorylation of 4E-BP1 without
affecting that of eIF2α or S6 ribosomal protein (Figure 7A and B-D).
Apocynin treatment completely blocked the enrichment of 19S proteasome
elicited by maximal concentration of CSE (Figure 7A and F). To our
surprise, the conversion of LC3A/B-I to LC3A/B-II which was undetectable
at submaximal CSE concentrations under vehicle condition, became
apparent starting at 20% CSE concentration, suggesting apocynin may
selectively enhance cellular autophagic response in the CSE-exposed
myotubes.
Discussion
The present study found that apocynin treatment was effective in
attenuating lung inflammation and prevented the skeletal muscle
dysfunction resulting from CS exposure. Our molecular analysis found
that the CS-induced muscle dysfunction is attributed to oxidative stress
and impaired muscle derived-IGF-1 expression which leads to a disruption
of proteostatic signalling. Apocynin effectively modulated oxidative
stress, thereby preserving muscle derived-IGF1 expression and the
downstream proteostatic signalling in myofibers, protecting them from
the damaging effects of CS/CSE exposure.
In the lungs, CS exposure elicited an abnormal inflammatory response,
which may promote mucous metaplasia and lung destruction leading to the
manifestation of chronic bronchitis and emphysema (O’Donnell, Breen,
Wilson & Djukanovic, 2006). Neutrophils have been suggested to be a key
driver of these deleterious effects in the lungs, by secreting a number
of proteases, such as matrix metalloproteinases and neutrophil elastases
(Vlahos et al., 2006). These proteases degrade components of the
pulmonary extracellular matrix leading to the destruction of the lung
parenchyma (Vlahos et al., 2006). Meanwhile, neutrophilic proteases may
perpetuate lung inflammation by acting on proteinase-activated receptors
(PARs)(Jenkins et al., 2006; Scotton et al., 2009). Destruction of the
lung parenchyma and persistent inflammation not only drives the
development of airflow limitation and emphysema, but also compromises
the integrity of epithelial lining of the airway (Vlahos et al., 2006).
This increases lung permeability allowing for the overspill of
pro-inflammatory mediators into the systemic circulation, which has been
postulated to be a key mechanism for the onset of skeletal muscle
dysfunction (Bernardo, Bozinovski & Vlahos, 2015; Passey, Hansen,
Bozinovski, McDonald, Holland & Vlahos, 2016).
Indeed, skeletal muscle dysfunction was observed following 8 weeks of CS
exposure, characterized by the loss of mass and contractile function
(Figure 2A-D). In patients with COPD, muscle dysfunction is most
frequently reported in the lower limbs than the upper limbs (Gea, Pasto,
Carmona, Orozco-Levi, Palomeque & Broquetas, 2001; Man et al., 2003),
suggests leg muscles are more susceptible to dysfunction in patients
with COPD. Strikingly, symptoms of muscle weakness, which are hallmarks
of functional impairment, have been reported in smokers without
detectable decline in respiratory function (Maltais et al., 2014). This
not only suggests that CS may directly impair leg muscle function, but
also that the onset of limb muscle dysfunction may well precede that of
respiratory symptoms. On this note, impaired quadricep function was
detected in asymptomatic smokers with matching physical activity levels
to non-smokers, which may be attributed to an acute toxicity of CS
exposure on oxygen delivery and mitochondrial function (Wust, Morse, de
Haan, Rittweger, Jones & Degens, 2008).
In addition to exerting acute toxicity, our study suggests that muscle
loss and dysfunction may also arise from chronic oxidative stress
elicited by repeated CS exposure. It is understood that CS represents an
external source of oxidants (>1016 free
radicals per puff) which exert adverse effects on tissues through
oxidative damage of biological structures (Bartalis, Chan & Wooten,
2007). Moreover, CS also activates inflammatory cells of the airway and
lungs which may enhance oxidant production in pulmonary and
extra-pulmonary tissues. Through these sources, chronic CS exposure
generates transient and repeated bouts of oxidative stress which may
modify key proteins involved in muscle metabolism or function, leading
to the manifestation of muscle dysfunction seen in patients with COPD
(Barreiro et al., 2010). Indeed, our results demonstrated the presence
of oxidative stress and increased protein oxidation following CS
exposure. This took place independent of muscle inflammation but was
linked to an altered myogenic homeostasis characterised by a blunted
expression of IGF-1 and increased expression of myostatin, suggesting a
disrupted proteostasis. In C2C12 myotubes, we found that oxidative
stress suppressed mTOR-driven protein synthesis, while activating the
UPS degradative pathway resulting in myofiber wasting. Myostatin is a
member of the transforming growth factor beta (TGF-β) family and a
potent inducer of muscle atrophy. By inhibiting myogenic signalling,
myostatin activates the UPS pathway through Forkhead box class O 3a
(FoxO3a), thereby promoting the expression of the muscle-specific
ubiquitin ligases: Muscle RING finger 1 (MuRF1) and MAFbx, resulting in
a net loss of muscle protein and atrophy (Zhou et al., 2010). In muscle,
Sriram et al (Sriram et al., 2011) demonstrated that oxidative
stress is a potent stimulator of myostatin expression. Intriguingly, the
same study also showed that myostatin itself also causes oxidative
stress via the action of Nuclear Factor Kappa B (NFκB) and Nox2, meaning
that a self-perpetuated mechanism may exist to sustain protein
degradation in atrophic muscles. Nevertheless, these findings highlight
the instrumental role of oxidative stress in CS-induced myostatin
expression and muscle loss observed in our study.
In accordance with this, attenuation of oxidative stress by apocynin
markedly ameliorated the CS-induced lung inflammation and muscle
dysfunction. In the muscle, apocynin prevented the induction of
myostatin and its inhibitory effects on myogenic signalling, thereby
preserving muscle proteostasis. In human COPD patients, muscle loss has
been postulated to be a result of unintended weight loss due to
malnutrition (Collins, Yang, Chang & Vaughan, 2019). Our in vivodata certainly reflects an association between loss of TA mass with
reduced weight gain and food intake by CS exposure. However, apocynin
treatment was able to preserve muscle mass and function despite both
weight gain and food intake remaining suppressed, suggesting that the
CS-induced muscle loss is unlikely a result of simple weight loss from
malnutrition. Moreover, loss of muscle mass was mainly observed in the
TA and soleus muscles, but not the gastrocnemius and plantaris,
highlighting the selective nature of CS-induced muscle loss. While
malnutrition and weight loss may be a major contributor to muscle loss
in advanced COPD where respiratory function is severely compromised,
they are unlikely to be accountable for the direct effects of CS induced
muscle loss observed in this study.
Another interesting finding of the present study is that the impaired
contractile function by CS exposure was only partially improved by
apocynin, despite a fully preserved muscle mass. This apparent mismatch
raises an important notion that muscle mass and function may not always
correlate in a linear fashion in patients with COPD, unlike that in
healthy individuals. In agreement with this, Mantoani et al(Mantoani et al., 2017) reported no correlations between muscle mass and
muscle function assessed by quadriceps maximal voluntary contraction,
although baseline physical activity was found to be related to greater
muscle strength. In addition to its deleterious effects on muscle mass,
CS exposure has been shown to directly impair excitation-contraction
coupling (Nogueira et al., 2018) suggesting muscle contractile apparatus
are sensitive to redox modifications. Barreiro et al . (Barreiro
et al., 2010) reported that a number of muscle proteins involved in
force generation are subjected to post-translational oxidative
modifications, including ATP synthase and actin. Oxidative modifications
of protein, such as carbonylation, may result in loss of protein
function and accelerated degradation by the UPS (Barreiro et al., 2010)
which may offer an explanation for the impaired contractile function
observed in our study. Collectively, these findings suggest that the
relationship between muscle mass and function is unlikely to be linear,
particularly in smokers or patients with COPD. Future studies should be
mindful of factors that may influence this relationship, such as muscle
of interest, the type of assessment chosen, age, sex and disease
severity of the test subject, when designing interventional trials for
COPD patients aiming to examine muscle changes.
Since muscle mass and function may be disconnected in the context of
COPD, the finding that not all leg muscles display susceptibility to
CS-induced muscle loss would prompt a new set of research questions on:
1) whether strength is preserved in muscles that are seemingly
unaffected by mass loss; and 2) what effect does apocynin have on the
contractile function of these muscles? Due to the limitation of the
present study, we are unable to shed further light on these questions.
Regarding apocynin, it seems to act as a prodrug, which must be
initially oxidized into its dimeric form, diapocynin, in order to be
active (Johnson et al., 2002). Supporting this, Ximenes et al.(Ximenes, Kanegae, Rissato & Galhiane, 2007) reported the isolation of
diapocynin in apocynin-treated neutrophils, and that the purified forms
of diapocynin have been suggested to be more effective than apocynin
itself (Kanegae et al., 2010; Mora-Pale, Weiwer, Yu, Linhardt &
Dordick, 2009). Despite the controversies regarding its potency and
selectivity as a Nox inhibitor, apocynin remains one of the most
promising drugs for experimental models of disease involving ROS since
its characterization in 1994.
In summary, we show that Nox-driven oxidative stress may be an
underlying mechanism for the skeletal muscle loss and dysfunction caused
by CS exposure. The induction of oxidative stress disrupts proteostasis
by dampening myogenic signalling and enhancing UPS activation, resulting
in muscle loss. Meanwhile, the oxidative modification of muscle proteins
may also give rise to contractile impairment. By inhibiting Nox-driven
oxidative stress, apocynin treatment attenuated lung inflammation and
preserved myofibrillar proteostasis, thereby preventing muscle loss and
dysfunction. Therefore, targeted inhibition of oxidative stress may be
utilized to improve pulmonary and systemic outcomes associated with
COPD.
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Table 2. Summary of tissue weight expressed as mean ±
SEM
Data are expressed as mean ± SEM.
* p< 0.05 compare to the relevant Sham; analyzed with
two-way ANOVA with multiple comparisons and Tukey post-hoc test.
Figure legends
Figure 1 . Effectof apocynin on body
weight, food intake and lung inflammation induced by CS exposure. Mice
were exposed to CS (smoke) or room air (sham) for 8 weeks with or
without i.p. injection of apocynin (5
mg·kg-1·day-1) or vehicle (saline).
Progressive body weight of CS-exposed (smoke) and room air-exposed (sham
mice with or without apocynin (A ) and average food intake
(B ) across the experimental period. Total number of cells
(C ), macrophage (D ), neutrophils (E ) and
lymphocytes (F ) in BALF. Quantitative PCR was performed to
assess the expression of Gmcsf (G ), Ccl2(H ), Cxcl2 (I ), and Tnfα (J ) in
homogenized lung tissues. Data are expressed as mean + SEM (n= 8-10 mice
per group) and analyzed by two-way
ANOVA with multiple comparisons and Tukey post-hoc test.
*p< 0.05 denotes differences from the relevant sham
group; †p< 0.05 denotes difference between the compared
groups.
Figure 2 . Effect of CS exposure on tibialis anterior
(TA) muscle weight, contractile performance and homeostatic changes . TA
muscle weight (A ), maximum contractile force (B ),
specific force at 120 Hertz (C ), and maximum contraction rate
measurements (D ) were analyzed at the end of the experimental
period. Quantitative PCR was performed to assess the expression ofIgf1-eb (E ), Myostatin (F ) andTnfα (G ) in homogenized TA muscle. Total oxidized
proteins (carbonylation; H ) in the TA muscle was detected using
the Oxyblot method and analyzed for densitometry (I ). Data are
expressed as mean + SEM (n= 8-10 mice per group, Oxyblot analysis was
conducted on n= 5 mice per group) and analyzed by two-way ANOVA with
multiple comparisons and Tukey post-hoc test. *p< 0.05
denotes differences from the relevant sham group;
†p< 0.05 denotes difference between the compared groups.
Figure 3 . Effect of
H2O2- and CSE-exposure on C2C12 myotube
size, viability and cellular stress response. C2C12 myotubes were
exposed to increasing concentrations of either
H2O2 (A ) or CSE (B )
for 24 hours. Cell viability was assessed using the MTS assay following
H2O2- (C ) or CSE- (H )
exposure. Quantitative PCR was performed to assess the expression ofNox2 (D & I ), Gpx1 (E &J ) and Il-6 (F & K ). IL-6 released
into the medium in response to H2O2(G ) or CSE (L ) was quantified using ELISA. For myotube
size assessments, data are represented as mean + SEM of 3 independent
experiments (n = 270 myotubes counted per condition), other data are
represented as mean + SEM of 3 independent experiments (n = 7-9 per
condition). *p< 0.05 denotes differences from the
relevant sham group; †p< 0.05 denotes difference between
the compared groups. Scale bars = 100 µm (A & B ).
Figure 4 . Effect of
H2O2- and CSE-exposure on C2C12 myotubes
proteostassis. C2C12 myotubes were exposed to increasing concentrations
of either H2O2 or CSE for 24 hours.
Quantitative PCR was performed to assess the expression of MAFbx(A & F ), Mstn (B & G ),Igf-1ea (C & H ) and Igf-1eb (D& I ). Mature IGF-1 released into the medium in response to
H2O2 (E ) or CSE (J )
was quantified using ELISA. Data are represented as mean + SEM of 3
independent experiments (n = 7-9 per condition).
*p< 0.05 denotes differences from control (i.e.
concentration zero).
Figure 5 . Effect of apocynin on C2C12 myotubes size and
cellular stress. C2C12 myotubes were exposed to increasing
concentrations of either H2O2 or CSE
with or without apocynin (500 nM) for 24 hours. Changes in myotube
diameters were quantified (A & F ) from 3 independent
experiments (n = 270 myotubes counted per condition). Quantitative PCR
was performed to assess the expression of Nox2 (B &G ), Il-6 (C & H ), Igf1-ea(D & I ) and Igf1-eb (E &J ). Data are represented as mean + SEM of 3 independent
experiments (n = 7-9 per condition unless otherwise stated).
*p< 0.05 denotes differences from vehicle control (i.e.
concentration zero); †p< 0.05 denotes difference between
the compared.
Figure 6 . Effect of
H2O2 on C2C12 myotubes proteostasis .
C2C12 myotubes were exposed to increasing concentrations of
H2O2 with or without apocynin (500 nM)
for 24 hours. At the end of experiment, samples were harvest for western
blotting analysis. Representative images of the western blots
(A ) and their respective densitometry analyses (B –I ). Data are represented as mean + SEM of 3 independent
experiments (n = 6 per condition), with open bar represents vehicle
conditions and closed bar represents apocynin conditions.
*p< 0.05 denotes differences from vehicle control (i.e.
concentration zero); †p< 0.05 denotes difference between
the compared.
Figure 7. Effect of CSE on C2C12 myotubes proteostasis. C2C12
myotubes were exposed to increasing concentrations of CSE with or
without apocynin (500 nM) for 24 hours. At the end of experiment,
samples were harvested for western blotting analysis. Representative
images of the western blots (A ) and their respective
densitometry analyses (B – I ). Data are represented
as mean + SEM of 3 independent experiments (n = 6 per condition), with
open bar represents vehicle conditions and closed bar represents
apocynin conditions. *p< 0.05 denotes differences from
vehicle control (i.e. concentration zero); †p< 0.05
denotes difference between the compared.