8 and 16 weeks of Chronic Tobacco Smoke Exposure Negatively Impacts Peripheral Motor Axon and Neuromuscular Junction Morphology in the Diaphragm of Mice
Authors: Willms Alexander John , Miguez Kayla , Leduc‐Gaudet Jean‐Phillipe , Baglole Carolyn , Hussain Sabah N.A. , Hepple Russell T. 
Published: June 2021

IntroductionGlobally, chronic tobacco smoke (TS) exposure is the leading cause of preventable disease, and is the primary risk factor for the development of the top three causes of death: chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Importantly, TS‐related diseases are accompanied by similar skeletal muscle abnormalities, which contribute to poor outcomes and prognosis. Recent findings have implicated TS‐induced denervation in TS‐related locomotor muscle abnormalities. Given the important physiological role of the diaphragm, and that muscle abnormalities in tobacco smokers and people with TS‐related diseases manifest differently in the diaphragm than in locomotor muscle, the objective of this study was threefold: (1) to further assess the impact of TS exposure on the diaphragm; (2) to resolve whether the TS‐induced neuromuscular junction (NMJ) impact observed in locomotor muscle also occurs in the diaphragm; and (3) to investigate the progressive impact of TS exposure on morphological features of the motor axon and NMJ.Methods15‐wk‐old male C57Bl/6 wild‐type mice were randomly assigned to an 8‐ or 16‐wk period of either TS or control air exposure. Body mass was weighed at the beginning and end of the exposure periods. Immediately following sacrifice, epididymal fat, spleen, and liver were harvested and weighed in mice undergoing 16‐wk exposures. Diaphragm, gastrocnemius, plantaris, soleus, TA, and EDL muscles were harvested and weighed immediately following sacrifice in all mice. The diaphragm was used to analyze the morphology of motor axons and NMJs using immunofluorescent staining and confocal microscope imaging.Results8 and 16 weeks of TS exposure attenuated body growth (p < 0.0005). Furthermore, 8 weeks (p < 0.0001), but not 16 weeks, of TS exposure reduced muscle mass normalized to their respective controls in all muscles harvested. In the diaphragm, 8 and 16 weeks of TS exposure increased the fraction of NMJs lacking axonal input (p < 0.05) or pre‐synaptic nerve terminal structures (p < 0.005), the latter identified as denervated structures. Pre‐synaptic axon diameter was reduced following 16 (p < 0.05), but not 8, weeks of TS exposure. 8 and 16 weeks of TS exposure led to reduced compactness of AChR structures (p < 0.005), in the absence of AChR fragmentation.ConclusionsChronic TS exposure leads to reduced muscle mass of 8‐week TS‐exposed mice, as well as abnormal morphology of diaphragm motor axons and NMJs in 8‐ and 16‐wk TS‐exposed mice. Increased exposure length resulted in an exacerbation of the impact of TS on reduced pre‐synaptic axon growth and AChR compactness in 16‐wk versus 8‐wk TS exposed mice. These findings illustrate that chronic TS exposure results in specific morphological abnormalities in motor axons and NMJs in the diaphragm muscle.Support or Funding InformationGouvernement du Canada | Canadian Institutes of Health Research (Instituts de recherche en santé du Canada): Russell T. Hepple, 119583This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.