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: