Smoke-induced neuromuscular junction degeneration precedes the fibre type shift and atrophy in chronic obstructive pulmonary disease.

KEY POINTS: Chronic obstructive pulmonary disease (COPD) is largely caused by smoking, and patient limb muscle exhibits a fast fibre shift and atrophy. We show that this fast fibre shift is associated with type grouping, suggesting recurring cycles of denervation-reinnervation underlie the type shift. Compared to patients with normal fat-free mass index (FFMI), patients with low FFMI exhibited an exacerbated fibre type shift, marked accumulation of very small persistently denervated muscle fibres, and a blunted denervation-responsive transcript profile, suggesting failed denervation precipitates muscle atrophy in patients with low FFMI. Sixteen weeks of passive tobacco smoke exposure in mice caused neuromuscular junction degeneration, consistent with a key role for smoke exposure in initiating denervation in COPD. ABSTRACT: A neurological basis for the fast fibre shift and atrophy seen in limb muscle of patients with chronic obstructive pulmonary disease (COPD) has not been considered previously. The objective of our study was: (1) to determine if denervation contributes to fast fibre shift and muscle atrophy in COPD; and (2) to assess using a preclinical smoking mouse model whether chronic tobacco smoke (TS) exposure could initiate denervation by causing neuromuscular junction (NMJ) degeneration. Vastus lateralis muscle biopsies were obtained from severe COPD patients [n = 10 with low fat-free mass index (FFMI), 65 years; n = 15 normal FFMI, 65 years) and healthy age- and activity-matched non-smoker control subjects (CON; n = 11, 67 years), to evaluate morphological and transcriptional markers of denervation. To evaluate the potential for chronic TS exposure to initiate these changes, we examined NMJ morphology in male adult mice following 16 weeks of passive TS exposure. We observed a high proportion of grouped fast fibres and a denervation transcript profile in COPD patients, suggesting that motor unit remodelling drives the fast fibre type shift in COPD patient limb muscle. A further exacerbation of fast fibre grouping in patients with low FFMI, coupled with blunted reinnervation signals, accumulation of very small non-specific esterase hyperactive fibres and neural cell adhesion molecule-positive type I and type II fibres, suggests denervation-induced exhaustion of reinnervation contributes to muscle atrophy in COPD. Evidence from a smoking mouse model showed significant NMJ degeneration, suggesting that recurring denervation in COPD is probably caused by decades of chronic TS exposure.

Failed upregulation of TFAM protein and mitochondrial DNA in oxidatively deficient fibers of chronic obstructive pulmonary disease locomotor muscle.

BACKGROUND: Low mitochondrial content and oxidative capacity are well-established features of locomotor muscle dysfunction, a prevalent and debilitating systemic occurrence in patients with chronic obstructive pulmonary disease (COPD). Although the exact cause is not firmly established, physical inactivity and oxidative stress are among the proposed underlying mechanisms. Here, we assess the impact of COPD pathophysiology on mitochondrial DNA (mtDNA) integrity, biogenesis, and cellular oxidative capacity in locomotor muscle of COPD patients and healthy controls. We hypothesized that the high oxidative stress environment of COPD muscle would yield a higher presence of deletion-containing mtDNA and oxidative-deficient fibers and impaired capacity for mitochondrial biogenesis. METHODS: Vastus lateralis biopsies were analyzed from 29 COPD patients and 19 healthy age-matched controls for the presence of mtDNA deletions, levels of oxidatively damaged DNA, mtDNA copy number, and regulators of mitochondrial biogenesis as well the proportion of oxidative-deficient fibers (detected histologically as cytochrome c oxidase-deficient, succinate dehydrogenase positive (COX(-)/SDH(+) )). Additionally, mtDNA copy number and mitochondrial transcription factor A (TFAM) content were measured in laser captured COX(-)SDH(+) and normal single fibers of both COPD and controls. RESULTS: Compared to controls, COPD muscle exhibited significantly higher levels of oxidatively damaged DNA (8-hydroxy-2-deoxyguanosine (8-OHdG) levels = 387 ± 41 vs. 258 ± 21 pg/mL) and higher prevalence of mtDNA deletions (74 vs. 15 % of subjects in each group), which was accompanied by a higher abundance of oxidative-deficient fibers (8.0 ± 2.1 vs. 1.5 ± 0.4 %). Interestingly, COPD patients with mtDNA deletions had higher levels of 8-OHdG (457 ± 46 pg/mL) and longer smoking history (66.3 ± 7.5 years) than patients without deletions (197 ± 29 pg/mL; 38.0 ± 7.3 years). Transcript levels of regulators of mitochondrial biogenesis and oxidative metabolism were upregulated in COPD compared to controls. However, single fiber analyses of COX(-)/SDH(+) and normal fibers exposed an impairment in mitochondrial biogenesis in COPD; in healthy controls, we detected a marked upregulation of mtDNA copy number and TFAM protein in COX(-)/SDH(+) compared to normal fibers, reflecting the expected compensatory attempt by the oxidative-deficient cells to increase energy levels; in contrast, they were similar between COX(-)/SDH(+) and normal fibers in COPD patients. Taken together, these findings suggest that although the signaling factors regulating mitochondrial biogenesis are increased in COPD muscle, impairment in the translation of these signals prevents the restoration of normal oxidative capacity. CONCLUSIONS: Single fiber analyses provide the first substantive evidence that low muscle oxidative capacity in COPD cannot be explained by physical inactivity alone and is likely driven by the disease pathophysiology.

Effect of eccentric versus concentric exercise training on mitochondrial function.

INTRODUCTION: The effect of eccentric (ECC) versus concentric (CON) training on metabolic properties in skeletal muscle is understood poorly. We determined the responses in oxidative capacity and mitochondrial H2 O2 production after eccentric (ECC) versus concentric (CON) training performed at similar mechanical power. METHODS: Forty-eight rats performed 5- or 20-day eccentric (ECC) or concentric (CON) training programs. Mitochondrial respiration, H2 O2 production, citrate synthase activity (CS), and skeletal muscle damage were assessed in gastrocnemius (GAS), soleus (SOL) and vastus intermedius (VI) muscles. RESULTS: Maximal mitochondrial respiration improved only after 20 days of concentric (CON) training in GAS and SOL. H2 O2 production increased specifically after 20 days of eccentric ECC training in VI. Skeletal muscle damage occurred transiently in VI after 5 days of ECC training. CONCLUSIONS: Twenty days of ECC versus CON training performed at similar mechanical power output do not increase skeletal muscle oxidative capacities, but it elevates mitochondrial H2 O2 production in VI, presumably linked to transient muscle damage.

Increased sensitivity to mitochondrial permeability transition and myonuclear translocation of endonuclease G in atrophied muscle of physically active older humans.

Mitochondrial dysfunction is implicated in skeletal muscle atrophy and dysfunction with aging, with strong support for an increased mitochondrial-mediated apoptosis in sedentary rodent models. Whether this applies to aged human muscle is unknown, nor is it clear whether these changes are caused by sedentary behavior. Thus, we examined mitochondrial function [respiration, reactive oxygen species (ROS) emission, and calcium retention capacity (CRC)] in permeabilized myofibers obtained from vastus lateralis muscle biopsies of healthy physically active young (23.7±2.7 yr; mean±SD) and older (71.2±4.9 yr) men. Although mitochondrial ROS and maximal respiratory capacity were unaffected, the acceptor control ratio was reduced by 18% with aging, suggesting mild uncoupling of oxidative phosphorylation. CRC was reduced by 50% with aging, indicating sensitization of the mitochondrial permeability transition pore (mPTP) to apoptosis. Consistent with the mPTP sensitization, older muscles showed a 3-fold greater fraction of endonuclease G (a mitochondrial proapoptotic factor)-positive myonuclei. Aged muscles also had lower mitophagic potential, based on a 43% reduction in Parkin to the voltage-dependent anion channel (VDAC) protein ratio. Collectively, these results show that mitochondrial-mediated apoptotic signaling is increased in older human muscle and suggest that accumulation of dysfunctional mitochondria with exaggerated apoptotic sensitivity is due to impaired mitophagy.

Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease.

RATIONALE: Locomotor muscle atrophy develops in patients with chronic obstructive pulmonary disease (COPD) partly because of increased protein degradation by the ubiquitin-proteasome system. It is not known if autophagy also contributes to protein degradation. OBJECTIVES: To investigate whether autophagy is enhanced in locomotor muscles of stable patients with COPD, to quantify autophagy-related gene expression in these muscles, and to identify mechanisms of autophagy induction. METHODS: Muscle biopsies were obtained from two cohorts of control subjects and patients with COPD and the numbers of autophagosomes in the vastus lateralis and tibialis anterior muscles, the levels of LC3B protein lipidation, and the expression of autophagy-related genes were measured in the vastus lateralis muscle. To investigate potential pathways that might induce the activation of autophagy, measures were taken of protein kinase B (AKT), mTORC1, and AMPK pathway activation, transcription factor regulation, proteasome activation, and oxidative stress. MEASUREMENTS AND MAIN RESULTS: Autophagy is enhanced in the locomotor muscles of patients with COPD as shown by significantly higher numbers of autophagosomes in affected muscles as compared with control subjects. Autophagosome number inversely correlates with FEV1. In the vastus lateralis, LC3B protein lipidation is increased by COPD and the expression of autophagy-related gene expressions is up-regulated. LC3B lipidation inversely correlates with thigh cross-sectional area, FEV1, and FEV1/FVC ratio. Enhanced autophagy is associated with activation of the AMPK pathway and FOXO transcription factors, inhibition of the mTORC1 and AKT pathways, and the development of oxidative stress. CONCLUSIONS: Autophagy is significantly enhanced in locomotor muscles of stable patients with COPD. The degree of autophagy correlates with severity of muscle atrophy and lung function impairment.