Early Activation of Intracellular Signaling Pathways after Denervation in Murine Skeletal Muscle

Abstract

PURPOSE: In vitro or cell culture specimens are used to study mechanotransduction; however, the effect of denervation on signaling cascades has not been examined. This study determined whether p70 and c-Jun N-terminal kinase (JNK) are influenced by denervation. METHODS: Extensor digitorum longus (EDL) muscles from 8 male mice (strain 129/SV) were assigned to either a control condition (A; no tendon or nerve injury) or one of the following: B) release of the distal tendon (unloading), C) denervation with tendons left intact, and D) release and denervation. The EDL was left either in situ (A-C) or in vitro (D) for 8 minutes before frozen in liquid nitrogen. Activation of phosphorylated p70 and JNK was measured by Western blot analysis. Student t-tests were performed between conditions with a Bonferroni correction of p<0.016 accepted for statistical significance. RESULTS: Four muscles underwent each condition (A-D: 16 muscles total). Unloading of the distal EDL tendon (condition B) did not result in activation of either p70 or JNK (p=0.8 and p=0.55, respectively; Figure 1). However, denervation resulted in a significant increase in p70 (p=0.001) but not JNK (p=0.019), and the in vitro conditions demonstrated no significant activation for either protein (p=0.15 and p=0.05, respectively; Figure 1). CONCLUSIONS: Using in vitro experimentation, p70 and JNK have been implicated in the early intracellular signaling cascades of skeletal muscle mechanotransduction. However, in this study, we demonstrate that p70 is activated early (within 8 minutes) after denervation of the muscle without any other manipulation. These results are alarming, and researchers studying early mechanotransduction signaling cascades must consider this limitation.Figure