Cigarette smoke exposure and muscle function analysis
Mice were placed in 18L perspex chambers and exposed to CS from three cigarettes (Winfield Red, 16 mg or less of tar, 15 mg or less of carbon monoxide, 1.2 mg or less of nicotine; Philip Morris, Australia) spaced evenly over 1 hour and carried out three times per day (09:00, 12:00, and 15:00 h), five days a week (Monday to Friday) for 8 weeks. The sham mice were handled identically and exposed to room air. We have previously shown that this CS exposure protocol in male Balb/C mice replicates key clinical traits of human COPD, including lung inflammation and pathology (emphysema , mucous hypersecretion, impaired lung function), increased lung and systemic oxidative stress and comorbidities including skeletal muscle dysfunction (Austin, Crack, Bozinovski, Miller & Vlahos, 2016; Chan et al., 2020; Vlahos & Bozinovski, 2014). At the end of protocol, in situ muscle function analysis was performed as previously described (Chan et al., 2020). In brief, mice were anaesthetised with ketamine (80 mg/kg BW) /xylazine (16mg/kg BW) and small incisions were then made on the skin to expose the tibialis anterior (TA) muscle taking care not to damage the fascia. The mouse was secured on the heated platform (37ºC) of anin situ contractile apparatus (809B in situ Mouse Apparatus, Aurora Scientific, Canada) with a pin behind the patellar tendon and a foot clamp. The distal end of the TA was tied firmly to a lever arm attached to an isometric force transducer. Two fine electrodes (3-5mm apart) were inserted into the belly of the TA muscle. The muscle was stimulated by two field stimulating platinum electrodes coupled to an amplifier. The TA muscle was contracted via square wave (0.2 ms) pulses at 10 V from the stimulator (701C stimulator, Aurora Scientific, Canada). Forces were converted to a digital signal and recorded by DYNAMIC MUSCLE ANALYSIS 611ATM (Aurora Scientific, Canada). Optimum muscle length (Lo) was first determined by eliciting twitch contractions by incrementally adjusting muscle length with a micromanipulator until a repeatable maximum peak twitch force was obtained. Optimal muscle length (Lo) was measured with precision digital calipers from the beginning of the distal tendon to the insertion of the TA at the base of the knee. Subsequently, the TA was stimulated at 100 Hz tetanic contraction, followed by a 2 min rest interval, and then twitch contraction. Comparable twitch forces pre and post 100 Hz stimulation indicated that the knots were both secure and unlikely to slip during the remaining protocol. If a decrease in twitch force was observed, the muscle was incrementally tensioned and stimulated between 2 min rest intervals until peak twitch force (Pt) was re-established. To establish the force frequency relationship, the TA was stimulated supramaximally (10 V) for 500 ms at 10, 20, 30, 40, 50, 80, 100, 150, 200, 250, 300 Hz, with a 2 min rest interval in-between.
As contractile force is closely related to muscle mass which is directly proportional to the cross-sectional area (CSA) of myofibers and the intrinsic properties of the contractile machinery within the muscle (Hakim, Wasala & Duan, 2013). To differentiate whether the observed muscle weakness induced by CS was attributed to reduced muscle mass and/or an impaired excitation–contraction coupling, the maximal contractile force at 120 Hertz is normalized to the whole-muscle CSA to produce the specific muscle force. CSA can be approximated from the gross mass and L0 of the muscle, together with the muscle density (~1.06 g·cm-3) (Close, 1972). Hence, the following equation is used: