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

Abstract

High intensity exercise is a 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. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) 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, and downstream gene and protein expression of Nrf2 target genes. 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), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). 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.

Keywords: High intensity exercise; Muscle contraction; Nrf2-Keap1; Redox signaling.

Figure 1.

Study Design

Figure 2.. Muscle force output and fatiguing stimulation.

A) Both low and high intensity stimulation groups had similar maximal tetanic force at 100Hz. B) The absolute force (mN-m) over time in response to either High (100Hz) or Low (50Hz) intensity stimulation frequency. C) The normalized force to maximal tetanus. Data are mean ± SD.

Figure 3.. Nrf2-ARE Binding and Keap1 Protein Expression in Response to Skeletal Muscle Stimulation

Keap1-Nrf2-ARE signaling response to high and low intensity stimulation and total glutathione content. A) Nrf2-ARE binding was low under basal unstimulated conditions (Naïve Control group and LI CON condition) but increased in response to low intensity stimulation (LI-CON vs LI-STIM, * p = 0.047). Nrf2-ARE binding was significantly higher in the HI group compared to NC group (^## p = 0.007) . B) Keap1 content was unchanged between limbs in the Naïve Control group or the LI-CON, however there was a significant decrease in Keap1 content in the LI-STIM limb (*p = 0.015). Keap1 content was significantly lower in the HI group under both conditions compared to NC (^##p = 0.008). C) Nrf2 :Keap1 ratio illustrates the same pattern as either Nrf2-ARE binding or Keap1 protein content alone (HI vs NC, ^#### p < 0.0001). D) Total glutathione content, measured in gastrocnemius muscle, was not different across groups and did not change significantly in response to stimulation. Representative western blot image of Keap1 protein is shown in Figure 5B. Keap1 was normalized to left hind limb of the NC group. Data are presented as mean ± SEM.

Fig. 4.. Gene expression of redox stress response genes to skeletal muscle stimulation.

A) Housekeeping gene average Cq change (∆ Cq) in response to skeletal muscle stimulation. B) Housekeeping gene average Cq change (∆ Cq) by group. Based on these results RPL41, RPL27, and RPL711 were selected for housekeeping genes. C) Effects of high intensity (H), low intensity (L) muscle stimulations, and control (C) for target genes HMOX1, NQ01, GCLC, GSR, and NFE2L2 fold change from unstimulated muscle (dotted line). In Nave Controls the mock stimulated muscle (right limb) was compared to the unstimulated control (left limb in all cases). There were no statistically significant increases in gene expression in response to stimulation in either group, although GCLC showed trends for increases regardless of intensity groups. RPL41, RPL27, and RPL711 were used for analysis of target gene fold change. H = High intensity stimulation group, L = Low intensity stimulation group, C = Naïve control group.

Fig. 5.. Redox stress protein response to muscle stimulation.

Redox stress protein response to muscle stimulation. A) Representative Ponceau S stain, B) Representative western blot images. C) Heme Oxygenase 1 protein is significantly elevated in both conditions of the high intensity group compared to the Naïve control group (p = 0.002). D) NQO1 protein increased significantly in response to stimulation (Stimulation condition p = 0.019), with no differences between intensities, or across groups. E) GCLC decreased slightly in stimulation conditions regardless of intensity (Stimulation condition p = 0.03) with no differences between groups. Glutathione reductase was unchanged in response to muscle stimulation or across intensity groups. All values are normalized to left limb of the NC group and set equal to 1 for western blot graphs.

Figure 6.. Model of systemic redox exerkine signaling is gated by muscle stimulation intensity

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.