Tobacco Smoke Extract Causes Mitochondrial Dysfunction Associated with Myotube Atrophy

Abstract

INTRODUCTIONWhereas the link between chronic tobacco smoke (TS) exposure and development of disease (e.g., chronic obstructive pulmonary disease; COPD) is well established, how chronic TS exposure relates to the mitochondrial dysfunction and muscle atrophy seen in COPD remains unclear.METHODSWe exposed C2C12 myotubes to TS extract (TSE) on day 5 post‐differentiation to determine the impact of TS on mitochondrial function (respiration, reactive oxygen species [ROS] emission and sensitivity to mitochondrial permeability transition [MPT]), and myotube atrophy. Briefly, for respiration experiments myotubes were treated with 0.02% TSE or vehicle (DMSO) for 24 h, and subsequently washed and assessed for maximal uncoupled respiration in an oxygraph using carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; 250 nM – 1.5 μM). For ROS experiments, following 24 h TSE or DMSO treatment myotubes were incubated with the superoxide indicator MitoSox (500 nM) for 15 min and imaged for MitoSox positive area. For MPT experiments, after 24 h TSE or DMSO treatment myotubes were fixed in 4% PFA and incubated with anti‐Apoptosis Inducing Factor (AIF; a protein released from mitochondria during MPT) and NucBlue (to stain nuclei). Images (n=5 per well, 8 wells per treatment) were analysed using Image J to quantify the fraction of AIF co‐localizing with nuclei using the Manders coefficient. For myotube atrophy experiments, 24 h following TSE or DMSO treatment myotubes were fixed, labelled with anti‐sarcomeric myosin, and subsequently imaged to assess myotube area using automated batch processing to avoid human bias. Two‐tailed Students t‐tests were used to evaluate differences between DMSO vs. CSE with an alpha level of 0.05. Data are presented as mean±SD with exact p‐values.RESULTSTreatment of C2C12’s with TSE reduced maximal uncoupled respiration (TSE: 312.0±24.0 vs. DMSO: 415.1±45.5 pmoles/sec/μg, p=0.002) and increased MitoSox positive area (TSE: 8,607.0±2,963.1 vs. DMSO: 2,254.7±1,034.7 μm2, p=0.0001); the latter suggesting TSE increases ROS. AIF: nuclear co‐localization was greater in C2C12’s treated with TSE compared with vehicle (Manders coefficient in TSE: 0.66±0.08 vs. DMSO: 0.46±0.05, p=0.0001), suggesting TSE causes MPT. We also observed significantly smaller myotube area in TSE‐treated cells compared with vehicle (TSE: 62,972.9±43,527.5 vs. DMSO: 85,284.9±28,136.1 μm2, p=0.048), consistent with TS inducing myotube atrophy.DISCUSSIONOur experiments demonstrate that TSE exposure causes significant mitochondrial impairment, as evidenced by reduced respiratory capacity, increased ROS generation, and nuclear co‐localization with a mitochondrial protein released during MPT. Furthermore, TSE treatment caused significant myotube atrophy. These observations are consistent with chronic TS exposure playing an important role in the mitochondrial dysfunction and muscle atrophy seen with the TS‐related disease COPD.