High intensity muscle stimulation activates a systemic Nrf2-mediated redox stress response

Introduction High intensity exercise is an increasingly popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. Methods An intra-animal design was used to control for variations in individual responses to muscle stimulation by using a stimulated limb (STIM) and comparing to the contralateral unstimulated control limb (CON). High Intensity (HI – 100Hz), Low Intensity (LI – 50Hz), and Naïve Control (NC – Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein content, and downstream gene and protein expression of Nrf2 target genes. Results Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019). Conclusions Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.


CON CON
to determine which were the best three genes to use for internal controls ( Table 1). The geometric 147 mean for the three most stable housekeeping genes were used to quantify changes in target gene mRNA expression in response to stimulation [29]. All primer sequences are listed in Table 1.

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assay. An equal volume of 200mM 5-sulfosalicylic acid dehydrate (SSA) was mixed in. After 158 resting on ice for 15 minutes the samples were centrifuged at 12,000 x g for 3 minutes at 4 °C.

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The supernatant was plated into a 96 well plate in duplicate. Standards with known GSH 160 concentrations (0-50µM) were also plated in duplicate. 0.2 M NEM/0.02 M KOH were added to 161 each well followed by 10 mM TCEP. After a 20-minute incubation at room temperature, 0.5 N 162 NaOH was added followed by 10mM NDA. After a 30-minute incubation the plate was read at 163 fluorescence intensity using 472 (excitation) and 528 (emission). GSH levels were assessed using 164 the standard curve and normalized to protein concentration.

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The results from the muscle stimulation protocol in HI and LI are shown in figure 2. The force 200 output of a tetanic contraction at 100Hz was not different between groups (12.1 mN-m ± 0.9 and 201 11.9 mN-m ± 1.7 for LI-STIM and HI-STIM respectively). The fatigue protocol at HI-STIM led to a 202 final force output that was 27.7% ± 5.6 of initial 100Hz tetanus, and LI-STIM led to a final force 203 output that was 12.8% ± 5.6 of a 100Hz tetanus and 37.3% ± 2.8 of the initial 50Hz contraction.  0.015) with no changes between CON vs STIM conditions in HI or differences between the two 220 NC-CON. There were no differences in total glutathione content across groups, or in response to  was a significant effect of stimulation condition on NQO1 protein ( Figure 5D, p = 0.019) with no differences between high and low intensity stimulus and no differences between stimulation 245 groups. There was a significant main effect of stimulation on GCLC protein content where the 246 stimulation condition decreased GCLC protein slightly ( Figure 5E, p = 0.03) with no differences 247 between groups. There were no significant differences between groups or in response to muscle 248 stimulation for GR protein ( Figure 5F).   . High intensity muscle contraction induces release of a redox active exerkine that travels through the blood stream and acts on unstimulated skeletal muscle. This effect is not seen in low intensity contralateral control muscle, suggesting the redox exerkine release from skeletal muscle is intensity gated. In other words, this redox exerkine is only released upon stimuli above a certain stress threshold or is released at lower intensities but not at sufficient concentrations to cause signaling effects in other tissues.
Nrf2. However, we are unaware of any study that has directly investigated the redox status of 372 Keap1 protein thiols in response to exercise specifically. Furthermore, in studies measuring Nrf2 373 in tissues other than muscle tissue, it is still unclear how redox signaling mechanisms are being  However, this is the first report that we are aware of demonstrating Nrf2 activation in response to 391 high intensity but not low intensity exercise in a contralateral non-exercised muscle. These findings provide exciting future directions to unveil the novel signaling mechanisms highlighted 393 here.