Tobacco‐Smoke Induced Muscle Atrophy Depends Upon Aryl Hydrocarbon Receptor Activity‐Mediated Activation of Mitochondrial Permeability Transition

Abstract

INTRODUCTION Chronic smokers exhibit skeletal muscle atrophy and mitochondrial impairments, which exacerbate the impact of normal aging and can increase risk of death. Tobacco smoke (TS) exposure drives these adverse effects by unknown mechanisms. TS toxins are degraded by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor which upregulates xenobiotic enzymes. Notably, chronic AHR activation phenocopies changes seen with mitochondrial permeability transition (MPT), a well-established cytotoxic mechanism in ischemia-reperfusion injury. Despite this, the role of chronic AHR activation in TS-induced skeletal muscle impact, and the potential involvement of MPT, has not been considered. Therefore, we designed experiments to establish: (1) the impact of TS exposure on MPT and its reliance on AHR activation, and (2) the necessity of AHR activation and MPT in TS-induced muscle atrophy. METHODS MPT is associated with the release of apoptosis inducing factor (AIF), which can translocate to the nucleus thereby providing a read-out of MPT occurrence. Thus, to evaluate the effect of TS on MPT occurrence, we exposed C2C12 myotubes to TS or vehicle control (DMSO) for 24 h, with and without the MPT inhibitor cyclosporin A (CsA; 1µM) and quantified nuclear:AIF co-localization. To evaluate if AHR antagonism could prevent MPT in TS-exposed cells, myotubes were co-treated with CH223191 (an AHR antagonist; 1µM) and nuclear:AIF co-localization was measured. Next, to establish the dependence of TS-induced muscle atrophy on AHR activation and AHR activity-mediated MPT, we exposed isolated single mouse FDB myofibers to TS, TS + CH223191 (1µM), or TS + isoxazole 63 (an MPT inhibitor; 0.5µM). Muscle atrophy was determined as the change in myofiber diameter from 0 to 24 h by measuring diameter at 10 points along the length of each fiber. RESULTS Nuclear:AIF co-localization was greatest in TS-exposed C2C12’s (Mander's Coefficient in TS: 0.49±0.03) compared with all other treatments (DMSO: 0.39±0.02; CsA: 0.40±0.03; CH223191: 0.40±0.02, p=0.001 for all), consistent with TS causing MPT in skeletal muscle in a manner that depends upon AHR activation. TS reduced single FDB fiber diameter over 24 hrs (0 h: 26.3±6.6 vs. 24 h: 22.1±4.8 µm, p=0.002). In contrast, fibers treated with TS + isoxazole 63 (0 h: 27.5±5.5 vs. 24 h: 25.4±5.4 µm, p=0.085) or TS + CH223191 (0 h: 28.0±5.6 vs. 24 h: 26.7±5.8 µm, p=0.302) did not atrophy significantly over 24 h, suggesting AHR-activity induced MPT is necessary for TS-induced muscle atrophy. DISCUSSION Our results show that TS increases nuclear:AIF co-localization in myotubes, consistent with TS causing MPT in skeletal muscle. Further, this effect was dependent upon AHR activation, linking chronic AHR activity with induction of MPT by TS exposure. Our results also show that AHR antagonism or MPT inhibition can prevent TS-induced atrophy in single myofibers, implicating AHR-mediated MPT in the mechanism by which TS causes muscle atrophy.