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