Neuromuscular mechanisms of weakness in a mouse model of chronic alcoholic myopathy

Abstract

Background: Weakness is a common clinical symptom reported in individuals with chronic alcohol use disorder. However, it remains unclear whether low strength in these individuals is directly related to excessive ethanol intake, other deleterious factors (lifestyle, environment, genetics, etc.), or a combination of both. Therefore, we examined whether (and how) ethanol reduces the muscle's force-producing capacity using a controlled in vivo preclinical mouse model of excessive ethanol intake.

Methods: To establish whether chronic ethanol consumption causes weakness, C57BL/6 female mice consumed 20% ethanol for 40 weeks (following a 2-week ethanol ramping period), and various measures of muscular force were quantified. Functional measures included all-limb grip strength and in vivo contractility of the left ankle dorsiflexors and plantarflexors. Once confirmed that mice consuming ethanol were weaker than age-matched controls, we sought to determine the potential neuromuscular mechanisms of muscle dysfunction by assessing neuromuscular excitation, muscle quantity, and muscle quality.

Results: Mice consuming chronic ethanol were 13 to 16% weaker (p ≤ 0.016) than controls (i.e., mice consuming 100% water) with the negative impact of ethanol on voluntary grip strength (ƞ² = 0.603) being slightly larger than that of electrically stimulated muscle contractility (ƞ² = 0.482). Relative to controls, lean mass and muscle wet masses were 9 to 16% lower in ethanol-consuming mice (p ≤ 0.048, ƞ² ≥ 0.268). No significant changes were observed between groups for indices of neuromuscular excitation at the level of the motor unit, neuromuscular junction, or plasmalemma (p ≥ 0.259, ƞ² ≤ 0.097), nor was muscle quality altered after 40 weeks of 20% ethanol consumption (p ≥ 0.695, ƞ² ≤ 0.012).

Conclusions: Together, these findings establish that chronic ethanol consumption in mice induces a substantial weakness in vivo that we interpret to be primarily due to muscle atrophy (i.e., reduced muscle quantity) and possibly, to a lesser degree, loss of central neural drive.

Keywords: EtOH; atrophy; force; skeletal muscle; strength.

FIGURE 1

Blood alcohol concentrations (BACs) were elevated after chronic EtOH consumption. All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. Blood was collected at study completion via a cardiac puncture within 1 h after the dark cycle ended (5:00 to 7:00 a.m.). bars are mean ± SD. N = 10 for EtOH and 5 for control. EtOH, Ethanol.

FIGURE 2

Chronic EtOH intake resulted in skeletal muscle weakness. Grip strength was evaluated by assessing voluntary all‐limb grip strength (A) while contractility of left plantarflexors (gastrocnemius, soleus, and plantaris muscles) (B) and dorsiflexors (tibialis anterior, extensor digitorum longus, and extensor hallucis muscles) (C) were measured by obtaining tetanic isometric torques from the left hindlimb. All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. *Significantly different than the control group (p < 0.05). Bars are mean ± SD. N = 10 for EtOH and 5 for control. EtOH, Ethanol.

FIGURE 3

No overt electromyographic changes were detected after chronic EtOH consumption. Various in vivo parameters of NMJ transmission and plasmalemma excitability (A to D) were assessed by stimulating the sciatic nerve and measuring the electromyographic signal of the left posterior crural muscles, primarily the gastrocnemius. All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. Bars are mean ± SD. N = 10 for EtOH and 5 for control. CMAP, compound motor action potential; EtOH, Ethanol; MUNE, motor unit number estimate; RNS, repetitive nerve stimulation; SMUP, single motor unit potential. EtOH, Ethanol.

FIGURE 4

Body mass and fat mass were lower in the EtOH group. Body mass (A) was obtained used an electric balance while fat mass (B) was assessed using nuclear magnetic resonance (NMR). Lean mass is presented in Figure 5A. All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. *Significantly different than the control group (p < 0.05). Bars are mean ± SD. N = 10 for EtOH and 5 for control. EtOH, Ethanol.

FIGURE 5

Lean mass and muscle wet mass were reduced by chronic EtOH intake. Atrophy or loss of muscle quantity was assessed using nuclear magnetic resonance (NMR) to estimate lean body mass (A) and by obtaining muscle wet mass of the left plantarflexors (gastrocnemius, soleus, and plantaris muscles) (B) and dorsiflexors (tibialis anterior, extensor digitorum longus, and extensor hallucis muscles) (C). All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. *Significantly different than the control group (p < 0.05). Bars are mean ± SD. N = 10 for EtOH and 5 for control. EtOH, Ethanol.

FIGURE 6

Skeletal muscle quality was maintained after chronic EtOH consumption. Muscle quality was analyzed by normalizing muscle function to lean/muscle mass (i.e., specific force). Grip strength was normalized to lean mass to measure grip strength quality (A) while tetanic isometric torque was normalized to wet muscle mass to measure quality of contractility. Specifically, plantarflexor torque was normalized to the wet mass of the posterior crural muscle (gastrocnemius, soleus, and plantaris muscles) (B) and dorsiflexor torque was normalized to the wet mass of the anterior crural muscle (tibialis anterior, extensor digitorum longus, and extensor hallucis muscles) (C). See Figure 2 for force measurements and Figure 5 for lean/muscle masses. All mice were female and the EtOH group consumed 20% EtOH in drinking water for approximately 40 weeks. Bars are mean ± SD. N = 10 for EtOH and 5 for control. EtOH, Ethanol.