Clinical significance:
Targeting oxidative stress may improve pulmonary and systemic outcomes associated with COPD.

Abstract

Background and Purpose : Cigarette smoking (CS) is the major risk factor for developing COPD and related skeletal muscle dysfunction. It has been postulated that CS exposure may directly causes muscle dysfunction via the induction of oxidative stress. The present study examined the effect of a potent Nox inhibitor and ROS scavenger, apocynin on CS-induced muscle dysfunction.
Experimental Approach: Male BALB/c mice were exposed to either room air (sham) or CS generated from 9 cigarettes per day, 5 days a week for 8 weeks with or without apocynin treatment (5 mg·kg-1 w/v, intraperitoneal injection). C2C12 myotubes exposed to either hydrogen peroxide (H2O2) or water-soluble cigarette smoke extract (CSE) with or without apocynin (500 nM), was set up as an experimental model in vitro .
Key Results: Eight weeks of CS exposure caused significant lung inflammation and muscle dysfunction in mice; evidenced by a 10% loss in muscle mass and 54% loss in contractile function of tibialis anterior, attributable to altered myogenic homeostasis and protein oxidation. These effects were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2O2 or CSE caused myofiber wasting, which was associated with altered myogenic homeostasis marked by ~50% loss in muscle-derived insulin-like growth factor (IGF)-1 and 1.5-fold increase in myostatin expression. Apocynin treatment completely attenuated CSE-inducedNox2 expression, preserving muscle-derived IGF-1 expression and downstream mammalian target of rapamycin (mTOR) signaling pathway, thereby preventing myofiber wasting.
Conclusion and Implications: Targeted pharmacological inhibition of Nox-derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.
Keywords: Chronic Obstructive Pulmonary Disease, NADPH oxidase, IGF-1, Antioxidants, Protein carbonylation.

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation that is not fully reversible (Vogelmeier et al., 2017). Cigarette smoking (CS) is the major cause of COPD accounting for 80-90% of cases in industrialized countries (Vogelmeier et al., 2017). In addition to the pulmonary pathologies, COPD may also give rise to debilitating conditions (i.e. comorbidities) in extra-pulmonary tissues which may lead to a deterioration of function, quality of life and mortality (Fabbri & Rabe, 2007). Skeletal muscle dysfunction is considered to be one of the most common comorbidities that affects up to 40% of COPD patients (Passey, Hansen, Bozinovski, McDonald, Holland & Vlahos, 2016). Skeletal muscle dysfunction limits exercise performance and capacity, thereby is detrimental to the overall health of those suffering from COPD irrespective of the lung function decline (Swallow et al., 2007). In line with this, muscle dysfunction has also been demonstrated to be a major risk factor for future acute exacerbations and hospital readmission of COPD patients (Vilaro et al., 2010), suggesting skeletal muscle function may be a determinant of health outcomes in these patients.
Muscle dysfunction can be defined as the inability of a muscle to perform its task, as a result of reduced strength and/or endurance, leading to the manifestation of muscle weakness and fatigue (Yamano, Kawai, Minami, Hiraga & Miyata, 2010). By definition, muscle weakness (i.e. loss of strength) and fatigue (i.e. loss of endurance) are distinct conditions, however, the observation that a weak muscle becomes more easily fatigued have highlighted the inseparable nature of the two (Yamano, Kawai, Minami, Hiraga & Miyata, 2010). Indeed, both reduced force-generating capacity and fatigue resistance have been observed in limb muscles of COPD patients leading to exercise intolerance (Vogelmeier et al., 2017).
The observations that even a single session of smoking was sufficient to result in decreased exercise capacity (Hirsch, Sue, Wasserman, Robinson & Hansen, 1985), and that non-symptomatic smokers more often complain of fatigue than non-smokers (Corwin, Klein & Rickelman, 2002), have led to the concept that CS may directly impact on muscle function. In non-symptomatic smokers and patients with COPD, Barreiro et al . (Barreiro et al., 2010) demonstrated that CS exposure directly elicits oxidative stress in the vastus lateralis muscle which may contribute to atrophy and dysfunction. Importantly, the same study also reported no significant rise in muscle inflammation amongst smokers and COPD patients, thus confirming the direct effects of CS exposure on muscle dysfunction which may be exerted through an oxidative stress-driven mechanism that is independent of inflammation.
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is a multimeric enzyme that catalyzes the formation of reactive oxygen species (ROS) - superoxide anion (O2-) which is the parent species amongst a family of molecules that ultimately contribute to oxidative stress (Griffith, Pendyala, Hecker, Lee, Natarajan & Thannickal, 2009). Not only O2- in itself is a potent oxidant, but it can be converted into hydrogen peroxide (H2O2), which is a more influential form of ROS in in terms of redox signaling with a longer half-life (Griffith, Pendyala, Hecker, Lee, Natarajan & Thannickal, 2009). The role of Nox-derived ROS has long been recognized in the pathogenesis of COPD. However, deletion of Nox2 or its catalytic subunit, p47phox, was found to result in greater lung inflammation and alveoli destruction in mice exposed to CS, despite showing decreased ROS production (Yao et al., 2008). This suggests normal expression of Nox2 is essential for maintaining redox and immune homeostasis.
In skeletal muscle, a functional Nox enzyme complex has been detected at the plasma membrane during muscular contraction, suggesting its active involvement in muscle function (Sakellariou et al., 2013). However, the exact role of Nox-derived ROS in CS-induced muscle dysfunction remains unclear. Given the detrimental effects of genetic disruption of Nox, the present study opted a pharmacological inhibitor approach, using apocynin. Apocynin inhibits Nox activation by blocking the cytosolic to membrane translocation of p47phox and p67phox, thereby disrupting the assembly of the active enzyme complex (Johnson et al., 2002). Furthermore, apocynin has also been shown to act as a scavenger for O2- and other ROS (Heumuller et al., 2008). For these reasons, the present study aimed to examine the role of Nox-derived ROS in CS-induced muscle dysfunction using apocynin. We hypothesize that inhibition of Nox-derived ROS would attenuate lung inflammation and muscle dysfunction induced by CS exposure.

Materials and methods