Cigarette smoke exposure impairs early-stage recovery from lengthening contraction-induced muscle injury in male mice

Abstract

The use of tobacco cigarettes produces locomotor muscle weakness and fatigue intolerance. Also, smokers and chronic obstructive pulmonary disease patients have a greater incidence of muscle injury and a deficient myogenic response. However, the effects of smoke exposure on the recovery from eccentric exercise-induced muscle injuries are unknown. Mice were exposed daily to cigarette smoke (CS) or room air (Air) for 4 months; the anterior crural muscles from one limb were injured by a lengthening contractions protocol (LCP) and recovered for 7 days. Lung compliance was greater, and body weights were lower, in CS-exposed than in the Air group. In LCP-subjected limbs, CS exposure lowered tibialis anterior myofiber cross-sectional area, decreased the size of centrally nucleated myofibers, and decreased extensor digitorum longus (EDL) mass, but did not affect EDL force from both limbs. CS exposure upregulated the mRNA levels of several myogenic (Pax7, Myf5, nNOS) genes in the EDL. The combination of CS exposure and LCP decreased Myf5 and nNOS mRNA levels and exacerbated pro-inflammatory mRNA levels. These data suggest that smoke exposure leads to an excessive pro-inflammatory response in regenerating muscle that is associated with a lower muscle mass recovery from a type of injury that often occurs during strenuous exercise.

Keywords: eccentric contractions; myofiber growth; sustained inflammation; tobacco smoke.

FIGURE 1

Effects of 4 months of daily CS or Air exposures on body weight and torque responses during lengthening contractions. (a) Weekly body weight gain in Air‐ and CS‐exposed groups during the 16 weeks of exposures. N = 12 mice per group (Air or CS), two‐way ANOVA, p‐values obtained by the Bonferroni post‐test are shown above time points; (b) Peak torque normalized by mouse body weight (N.mm.kg−1) during LCP performed at Week 15 of exposures (indicated by the arrow in [a]). N = 6 mice per group, two‐way ANOVA.

FIGURE 2

Myofiber cross‐sectional area from tibialis anterior (TA) muscles 7 days after lengthening contractions in Air‐ and CS‐exposed mice. (a) Representative immunofluorescence image of TA muscle sections from control and LCP legs from Air and CS exposed groups. Scale bar = 100 μm; (b) Average of myofiber CSA of muscles from control and LCP legs from both exposure groups. N = 4 mice per group, two‐way ANOVA repeated measures (control vs. LCP); (c, d) Myofiber CSA distribution in muscles from control legs (c) and LCP legs (d) between Air‐exposed and CS‐exposed groups. N = 4 mice per group, two‐way ANOVA. p‐values obtained by the Bonferroni post‐test are shown above bars.

FIGURE 3

Detection of centrally nucleated myofibers and Pax7+ cells from TA muscle sections 7 days after lengthening contractions in Air‐ and CS‐exposed mice. (a) Comparison of centrally nucleated myofiber CSA distribution between Air and CS exposure groups in muscle sections from LCP legs; (b) Representative immunofluorescence image of TA muscle sections immunostained with laminin (red), nuclei (DAPI) and Pax7 (green) from control and LCP legs from Air and CS exposed groups. Scale bar = 100 μm; (c) Number of Pax7+ cells relative to the area (in mm²) of each muscle section. N = 4 mice per group, two‐way ANOVA, Air versus CS groups. p‐values obtained by the Bonferroni post‐test are shown above bars.

FIGURE 4

Changes in mRNA expression of myofiber repair and maturation (a), muscle wasting (b), and inflammatory (c) genes in EDL muscles from control and LCP legs from both exposure groups. Data was normalized by the mRNA expression measured in control muscles from Air‐exposed mice. N = 3–6 mice per sample, two‐way ANOVA. p‐values obtained by the Bonferroni post‐test are shown above bars.

FIGURE 5

Extensor digitorum longus (EDL) muscle morphometrics and force production 7 days after lengthening contractions in Air and CS exposed mice. (a) EDL mass from legs subjected to LCP (LCP leg) and from contralateral non‐injured legs (control leg). N = 11 mice for Air exposed and 12 mice for CS exposed groups, two‐way ANOVA repeated measures. p‐values obtained by the Bonferroni post‐test are shown above bars. (b) Force production evoked by different frequencies of pulse‐stimulation in muscles from control and LCP legs. N = 11 mice for Air exposed and 12 mice for CS exposed groups, three‐way ANOVA repeated measures.

FIGURE 6

Lung mechanical properties of mice exposed to CS or Air for 4 months. (a) In vivo lung pressure‐volume responses. Two‐way ANOVA; (b) total lung capacity (TLC); (c) residual volume (RV); (d) lung compliance (C). Student's t‐test p‐values are shown above bars in b–d. N = 11 mice for Air exposed and 8 mice for CS exposed groups.