Effects of innervation state on Hsp25 content and phosphorylation in inactive rat plantaris muscles

Abstract

Previous reports suggest a role for neuromuscular activity levels and/or connectivity in modulating Hsp25 expression and phosphorylation (pHsp25) in skeletal muscles. However, pHsp25 has only been studied in denervated muscles and/or muscles exposed to high levels of residual neuromuscular activity. Spinal cord isolation (SI) provides a model in which the muscle is exposed to nearly complete inactivity with maintenance of the nerve-muscle connection. To parcel out the roles of innervation state and activity-independent neural factors, we compared Hsp25 and pHsp25 in the plantaris of control (Con), SI, and denervated (Den, inactivity without neural connectivity) rats. Hsp25 and pHsp25 protein levels (soluble and insoluble fractions) were measured with Western blot analysis after 1, 3, 8, 14, or 28 days of SI or Den. pHsp25 was normalized to non-pHsp25 at each time point. Hsp25 was unchanged (days 1, 3 and 14) or increased (days 8 and 28) in the soluble fraction, and decreased (day 1) or increased (days 3, 8 and 14) in the insoluble fraction in Den compared with Con rats. pHsp25 was reduced after 1 and 28 days of Den, but near control levels on days 3, 8, and 14 in the soluble fraction. In the insoluble fraction, pHsp25 levels were lower in Den than Con rats on all days. In both fractions, Hsp25 was lower in SI than Con rats. pHsp25 levels were lower in the soluble fraction and higher in the insoluble fraction in SI than Con rats. These results suggest that an intact innervation, even in the absence of muscle activation and/or loading, is critical for Hsp25 phosphorylation in the insoluble fraction. However, the time-dependent decrease in Hsp25 with SI suggests a role for minimal levels of muscle activation and/or loading in maintaining Hsp25 expression during sustained inactivity.