Neuromuscular junctions (NMJs): ultrastructural analysis and nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in offspring subjected to protein restriction throughout pregnancy

Abstract

Protein restriction during gestation can alter the skeletal muscle phenotype of offspring; however, little is known with regard to whether this also affects the neuromuscular junction (NMJ), as muscle phenotype maintenance depends upon NMJ functional integrity. This study aimed to evaluate the effects of a low protein (6%) intake by dams throughout gestation on male offspring NMJ morphology and nicotinic acetylcholine receptor (nAChR) α1, γ and ε subunit expression in the soleus (SOL) and extensor digitorum longus (EDL) muscles. Four groups of male Wistar offspring rats were studied. The offspring of dams fed low-protein (6% protein, LP) and normal protein (17% protein, NP) diets were evaluated at 30 and 120 days of age, and the SOL and EDL muscles were collected for analysis. Morphological studies using transmission electron microscopy revealed that only SOL NMJs were affected in 30-day-old offspring in the LP group compared with the NP group. SOL NMJs exhibited fewer synaptic folds, the postsynaptic membranes were smooth and myelin figures were also frequently observed in the terminal axons. With regard to the expression of mRNAs encoding nAChR subunits, only 30-day-old LP offspring EDL muscles exhibited reduced α, γ and ε subunit expression compared with the NP group. In conclusion, our results demonstrate that a low-protein diet (6%) imposed throughout pregnancy impairs the expression of mRNAs encoding the nAChR α, γ and ε subunits in EDL NMJs and promotes morphological changes in SOL NMJs of 30-day-old offspring, indicating specific differences among muscle types following long-term maternal protein restriction.

Keywords: neuromuscular junction; pregnancy; protein restriction; skeletal muscle.

Figure 1

Experimental design.

Figure 2

Electron micrographs of soleus (SOL) motor endplates. Panels a and e depict 30‐day‐old and 16‐week‐old NP offspring respectively; panels b, c and d represent 30‐day‐old LP offspring; panel F represents 16‐week‐old LP offspring. A, muscle fibre‐associated axon terminal; F, junctional folds; gg, glycogen granules; M, mitochondria; MVB, multivesicular body; N, myonuclei; S, sarcomere; SV, synaptic vesicles; asterisks indicate reduced junctional folds and arrows indicate myelin figures. The thick arrow indicates dilated endoplasmic reticulum. Scale bars in a and b are 0.43 μm, and 0.23 μm in c–f.

Figure 3

Electron micrographs of extensor digitorum longus (EDL) motor endplates. Panels a and b depict 30‐day‐old and 16‐week‐old NP offspring respectively; panels c and d depict 30‐day‐old and 16‐week‐old LP offspring respectively. A, muscle fibre‐associated axon terminal; DP, dense points; F, junctional fold; M, mitochondria; MVB, multivesicular body; N, myonuclei; S, sarcomere; SV, synaptic vesicles. The scale bars are 0.58, 0.23, 0.43 and 0.23 μm in a, b, c and d respectively.