Fatiguing stimulation of one skeletal muscle triggers heat shock proteins activation in several rat organs: the role of muscle innervation

Abstract

SUMMARYWe hypothesised that activation of muscle afferents by fatigue triggers a widespread activation of heat shock proteins (HSPs) in resting muscles and different organs. In anaesthetised rats, HSP25 and HSP70 levels were determined in both tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and in the diaphragm, kidney and brain by ELISA, which mostly identifies phosphorylated HSP, and western blotting. One TA muscle was electrically stimulated and tissues were sampled 10 or 60min after the stimulation had ended. The nerve supply to the stimulated TA or its counterpart in the contralateral limb was left intact or suppressed. In control rats, no muscle stimulation was performed and tissues were sampled at the same time points (10 or 60min). After TA stimulation, ELISA showed an increased HSP25 content in the contralateral TA, EDL and diaphragm at 10min but not at 60min, and HSP70 increased in all sampled tissues at 60min. Western blotting did not show any changes in HSP25 and HSP70 at 10min, while at 60min HSP25 increased in all sampled tissues except the brain and HSP70 was elevated in all tissues. Denervation of the contralateral non-stimulated limb suppressed HSP changes in TA and after denervation of the stimulated TA the widespread activation of HSPs in other organs was absent. Our data suggest that fatigue-induced activation of skeletal muscle afferents triggers an early increase in phosphorylated HSP25 in muscles and a delayed elevation of non-phosphorylated HSP25 and HSP70 in skeletal and respiratory muscles, kidney and brain.

The mechanisms of the widespread production of phosphorylated HSP25 after fatiguing muscle stimulation

“…The present study confirms our observations that fatigue of a single hindlimb muscle elicits a widespread pHSP25 response triggered by the activation of muscle afferents from the stimulated TA muscle (Jammes et al, 2012). The new findings are: (1) the groups III and IV muscle afferents from the stimulated TA muscle play key roles in the widespread pHSP25 response but the stimulation of group IV afferents is only able to induce pHSP25 activation in organs in the trunk (diaphragm, kidney) and brain; (2) the demonstration of the key role played by the adrenoreceptor simulation in the widespread pHSP25 response to TA stimulation in all samples tissues, except in the brain; (3) the absence of a glutamatergic pathway responsible for the HSP25 response.…”

“…The present study was limited to the measurements of the changes in pHSP25. Indeed, we have already shown that the pHSP25 level increased very early after the fatiguing muscle stimulation had ended, preceding the increase in HSP70 and non-phosphorylated HSP25 (Jammes et al, 2012).…”

“…Electrically induced muscle fatigue As in our previous animal studies ( Darques and Jammes, 1997;Decherchi et al, 1998;Decherchi et al, 2007;Delliaux et al, 2009;Jammes et al, 2012), muscle stimulation consisted of rhythmic contractions of the TA muscle. Two steel hook electrodes (interelectrode distance 4mm) were fixed in the belly of the muscle and contractions were produced by a neurostimulator through an isolation unit (Grass S8800, Quincy, MA, USA).…”

“…Bovine serum albumin allows the measurement of total protein content (mgproteing -1 of wet tissue). As in our previous study (Jammes et al, 2012), pHSP25 levels were determined with a high-sensitivity ELISA kit dedicated to HSP measurement in rat tissues using polyconal IgG against HSP25 phosphorylated at Ser82. The capture antibody was GWB-E2BCF (5μgml -1 in coating buffer; Genway, San Diego, CA, USA) and the detection antibody was [pSer82] HSP25, mAb (5B9) (0.5μlml -1 in assay buffer; Enzo Life Science, Villeurbanne, France).…”

“…After a fatigue bout induced in one skeletal muscle by electrical stimulation in rats, we previously showed (Jammes et al, 2012) a widespread activation of the phosphorylated and non-phosphorylated HSP25 and HSP70 in the resting skeletal muscles and the diaphragm, kidney and brain. We showed an early increase in phosphorylated HSP25 (pHSP25) measured by enzyme-linked immunosorbent assay (ELISA), confirmed by western blot analysis, and also that muscle afferents activated by the fatiguing muscle stimulation played a key role in the widespread HSP response.…”