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 (BI ). 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 (BI ). 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.