ASICs are required for immediate exercise-induced muscle pain and are downregulated in sensory neurons by exercise training
Abstract
Exercise training is an effective therapy for many pain-related conditions, and trained athletes have lower pain perception compared with unconditioned people. Some painful conditions, including strenuous exercise, are associated with elevated levels of protons, metabolites, and inflammatory factors, which may activate receptors and/or ion channels, including acid-sensing ion channels (ASICs), on nociceptive sensory neurons. We hypothesized that ASICs are required for immediate exercise-induced muscle pain (IEIP) and that exercise training diminishes IEIP by modulating ASICs within muscle afferents. We found high-intensity interval training (HIIT) reduced IEIP in C57BL/6 mice and diminished ASIC mRNA levels in lumber dorsal root ganglia, and this downregulation of ASICs correlated with improved exercise capacity. Additionally, we found that ASIC3 −/− mice did not develop IEIP; however, the exercise capacity of ASIC3 −/− was similar to wild-type mice. These results suggest that ASICs are required for IEIP and that diminishment of IEIP after exercise training correlates with downregulation of ASICs in sensory neurons.
NEW & NOTEWORTHY Exercise performance can be limited by the sensations of muscle fatigue and pain transmitted by muscle afferents. It has been proposed that exercise training abrogates these negative feedback signals. We found that acid-sensing ion channels (ASICs) are required for immediate exercise-induced muscle pain (IEIP). Moreover, exercise training prevented IEIP and was correlated with downregulation of ASICs in sensory neurons.
INTRODUCTION
Regular exercise is increasingly acknowledged as an effective therapy for a variety of chronic pain conditions, as well as a means to prevent the development of chronic pain. Even in healthy people, regular exercise can increase pain tolerance, and highly-trained athletes have a higher pain threshold than unconditioned subjects (36, 47). However, pain can be a barrier to exercise. Exercise, particularly that involving high-intensity muscle contractions, can cause fatigue and pain, referred to here as immediate exercise-induced muscle pain (IEIP) (34). These aversive sensations can prevent people from adhering to regular exercise programs and act as barriers to achieving higher performance in competitive athletes. Athletes have long understood the potential benefit of training through IEIP, hence the concept of “no pain, no gain.”
So why does intensive exercise often cause immediate pain, but persistent exercise training diminishes pain? To understand this seeming paradox, we turned to our understanding of the molecules that sense muscle pain. Skeletal muscle has the capacity for high metabolic activity and is susceptible to rapid drops in pH during ischemia, hypoxia, and/or intensive exercise. Consequently, lactic acid is generated and, along with ATP hydrolysis, reduces intracellular pH, which in turn leads to acidification of the muscle interstitium. With intense exercise, the extracellular pH in human skeletal muscle can drop to the pH 6.7–7.0 range (3). Skeletal muscle is richly innervated by sensory nerves (group III and IV muscle afferents) that sense these pH changes, as well as other metabolites generated during exercise (33). Within these muscle afferents, increasing evidence suggests that acid-sensing ion channels (ASICs) are important sensors of these metabolic changes.
ASICs are H+-gated channels of the degenerin/epithelial Na+ channel (DEG/ENaC) family, expressed principally in the central nervous system and in peripheral sensory neurons. Four genes encode at least six subunits (ASIC1a, -1b, -2a, -2b, -3, and -4; ASIC1 and -2 have alternate splice transcripts) (52). In general, they seem to be highly expressed in organs that have high metabolic activity, including the brain and sensory nerves that innervate the heart and skeletal muscle (4, 35, 52). Several observations suggest the potential importance of ASICs in muscle afferents during intense exercise. First, ASICs are highly expressed in muscle afferents (formed by ASIC1, −2, and −3 subunits) (15), and are activated in the range of extracellular pH that occurs in the interstitium of exercising muscle (35). Second, ASIC currents are potentiated by other chemicals released during muscle ischemia, hypoxia, and/or exercise (e.g., lactate, ATP, arachidonic acid, and nitric oxide) (1, 6, 8, 23). Third, ASICs are required for normal exercise-mediated reflexes. Activation of muscle afferents during exercise evokes increases in blood pressure, heart rate, and ventilation (termed the “exercise pressor reflex”) (33). ASIC antagonists have been shown to attenuate the metabolic component, but not the mechanical component, of the exercise pressor reflex (19, 26). Lastly, ASICs are required for the development of some types of chronic muscle pain. Muscle inflammation, direct acid injection into muscle, and electrically induced muscle contraction all induce chronic pain in mice that is attenuated by genetic or pharmacological inhibition of ASICs (16, 44, 45).
Other chemicals released during stress conditions [including nerve growth factor (NGF), serotonin, interleukin-1β, and bradykinin] regulate the expression of ASICs (31, 32, 39). For example, ASIC3 protein is increased in rat muscle afferents after hindlimb muscle ischemia, which is dependent on the upregulation of NGF within sensory neurons (28). Similarly, ASIC2a and −3 mRNA increased 10-fold in dorsal root ganglia (DRG) in a mouse model of muscle inflammation (50). In humans, exercise is associated with altered mRNA levels of specific ASIC subunits in blood cells (48, 53). Together, these data suggest that muscle inflammation and ischemia, and possibly exercise, can selectively regulate the expression of ASICs.
Given that ASICs are important sensors of chronic muscle pain and contribute to the activation of exercise-mediated reflexes, we tested whether ASICs are required for the acute sensation of immediate exercise-induced muscle pain (IEIP). Moreover, we hypothesized that modulation of ASICs might underlie the diminishment of muscle pain associated with exercise training.
METHODS
Animals
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of University of Iowa. The generation of ASIC3 −/− mice has been previously reported (38). These mice were subsequently backcrossed for 10 generations onto a C57BL/6J background to generate a congenic line. The 7-wk-old C57BL/6J (Jackson Laboratory, Sacramento, CA) or ASIC3 −/− mice were acclimated to the new environment and room for 1 wk before the beginning of treadmill acclimation. Experimental operator was blinded to genotype. The animals were housed in a temperature-controlled room (22°C) with a 12-h light-dark cycle and had free access to standard mouse food (pellet) and water ad libitum, except for 1 h before experimentation. For the exercise training experiments, we studied female mice because the prevalence of chronic muscle pain is greater in women than in men (14), female mice exercise more readily than male mice (13), and previous work found female mice more readily develop fatigue-induced hyperalgesia than male mice (17). For the acute IEIP experiments, we studied both male and female mice.
Experimental Design
Protocol 1.
After 5 days of acclimation to the treadmill (Supplemental Table S1; see https://doi.org/10.6084/m9.figshare.11607423) and 3 days of acclimation to muscle withdrawal threshold (MWT) testing, MWT data were collected a day before (baseline) and immediately after maximal incremental treadmill exercise. Mice were then divided into sedentary and high-intensity interval training (HIIT) groups such that the mean exercise capacity and variance of each group were equal. After 8 wk of HIIT or sedentary sessions, we waited 48 h before again measuring MWT before and after maximal incremental exercise (the sedentary group was again acclimated to treadmill running before the maximal exercise test) (Fig. 1A).

Fig. 1.A and B: schematic illustration of experimental designs. HIIT, high-intensity interval training; LICT, low-intensity continuous training; MWT, muscle withdrawal threshold; NMR, nuclear magnetic resonance imaging; qPCR, quantitative polymerase chain reaction.
Protocol 2.
Separate groups of mice similarly underwent treadmill acclimation and maximal incremental treadmill exercise and were divided into sedentary, low-intensity continuous training (LICT), and HIIT groups such that the mean exercise capacity and variance of each group were equal. Mice began a 4-wk period of exercise training or sedentary sessions 24 h after maximal exercise testing. A second maximal exercise test was performed 48 h after the last training session. We measured body composition 48 h later by nuclear magnetic resonance (NMR), and then mice were euthanized and lumbar DRG were collected for measurement of mRNA by quantitative PCR (qPCR) (Fig. 1B).
Muscle Withdrawal Threshold
MWT was measured by applying a force-sensitive tweezer to the belly of the gastrocnemius muscle as previously described (42), whereby lower thresholds indicate greater sensitivity to mechanical stimuli. Briefly, mice were acclimated to the testing paradigm in two 5-min sessions for 3 consecutive days. Then, mice were placed in a gardener’s glove, the hindlimb held in extension, and the muscle squeezed with progressive force with the force-sensitive tweezers until the mouse withdrew its hindlimb. Three trials were averaged per hindlimb.
Treadmill Exercise Protocols
All mice were acclimated to a six-lane motor-driven treadmill (Columbus Instruments, Columbus, OH) for 5 days with gradually increasing velocity and incline for 30 min/day (Supplemental Table S1). Shock grids at the rear of the treadmill were set at 1 mA and 1 Hz to encourage continued ambulation on the treadmill during acclimation. Three mice were very poor runners during the acclimation period (on the last day of acclimation they spent at least 10 consecutive seconds on the shock bars) and were not added to the study. These mice were removed before we divided the mice into the various exercise training protocols, so they did not skew the results.
Maximal Incremental Exercise Protocol
Maximal incremental exercise testing was performed to measure time to exhaustion (Fig. 2A). Mice were placed on a nonmoving treadmill (20° incline throughout) with shock bar on for 10 min, followed by a 10-min warm up at a velocity of 6 m/min. It has been shown that a 20° incline is best to achieve V̇o2max in mice (25). At the beginning of maximal exercise testing, we turned off the shock bar and instead used gentle prodding with a tongue depressor to encourage exercise as needed (10). We found this technique to be less stressful to the mice, and maximal exercise capacity was similar compared with using the shock bars (data not shown). After the warm up, the velocity was increased to 8 m/min and then increased by 2 m/min every 3 min (velocity accelerations were 2 m/min2) until the mice became exhausted. Exhaustion was defined as the point at which mice stayed at the end of treadmill and did not respond to 10 consecutive nudges with a tongue depressor in 10 s. The Vmax was defined as the velocity of the previous stage that the mouse completed.

Fig. 2.Diagrams of exercise protocols. A: maximal incremental exercise protocol. Mice were run on a treadmill at 20° incline with increasing velocity for the indicated time intervals. B: low-intensity continuous training protocol trained mice at 50% maximal velocity (Vmax) for a distance equal to the total distance of high-intensity interval training (HIIT) group in each session. C: HIIT protocol consisted of 4 bouts of 6-min intervals at 80% of Vmax, with 3 min of active rest at 50% of Vmax between bouts.
Low-Intensity Continuous Training Protocol
After a 10-min warm up at 40% of Vmax, mice ran at 50% of Vmax for a distance equal to the total distance of HIIT group in each session (Fig. 2B). Velocities were increased each week of training: by 5% (second week), by 7% (third week), and by 9% (fourth week) compared with the first week velocities.
High-Intensity Interval Training Protocol
The HIIT protocol was designed and modified from previously described protocols (25) and individualized for each mouse based on their Vmax from the initial maximal exercise test (Fig. 2C). After the 10-min warm up at 40% of Vmax, HIIT protocol consisted of 4 bouts of 6-min intervals at 80% of Vmax for the first week (20° incline), with 3 min of “active rest” at 50% of Vmax between the bouts. For the 8-wk protocol, velocities were increased each week of training by 5% (second week), 7% (third week), 9% (fourth week), and 10% (fifth–eighth weeks) compared with the first week velocities. For the 4-wk protocol, velocities were increased from the second week to the fourth week (5% → 7% → 9% greater than the first week velocities). Velocity was accelerated by 2 m/min2. Mice in the sedentary group were placed on a nonmoving treadmill for 40 min.
Nuclear Magnetic Resonance
Body composition (body fat, lean, and free fluid) was measured by nuclear magnetic resonance (NMR; Bruker LF50, Billerica, MA). Mice were weighed, placed into a polycarbonate restraint tube, and put in the scanner for ~2 minutes.
Tissue Collection and Quantitative RT-PCR
Mice were euthanized with CO2 at a rate of 3 L/min for 1 min following cessation of breathing. The right and left lumber (L4–L6) DRGs were collected and transferred to RNAlater (Thermo Fisher, Waltham, MA). Total cellular mRNA was isolated using the RNeasy mini kit (Qiagen, Germantown, MD), according to the manufacturer’s protocol. cDNA synthesis was performed using the AffinityScript QPCR cDNA Synthesis kit (Stratagene, La Jolla, CA), followed by qPCR using the Brilliant II SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA). The qPCRs were run in triplicate with cycle threshold values averaged, and the data were analyzed using the ∆∆Ct method. mRNA encoding 36B4 was used as the invariant control. qPCR primer sequences are listed in Supplemental Table S2 (see https://doi.org/10.6084/m9.figshare.11608080).
Statistical Analysis
GraphPad Prism (8.0) was used to statistically analyze data. Bar graphs represent means ± SE. One-way and two-way ANOVA and t tests were used to analyze results. If significant differences were observed, then the indicated post hoc tests as described in the figure legends were performed. Values of P < 0.05 were considered statistically significant.
RESULTS
Exercise Training Diminished Immediate Exercise-Induced Muscle Pain
To test the effect of exercise training on immediate exercise-induced muscle pain (IEIP), we used an 8-wk high-intensity interval training (HIIT) or sedentary (control) protocols. We found that mice having undergone HIIT had a significant improvement in maximal exercise capacity after the 8-wk period compared with the sedentary group (Fig. 3A). Prior to the 8-wk training period, we measured muscle pain in mice using muscle withdrawal threshold (MWT) before and immediately after exhaustive maximal treadmill running. We found that a single bout of exhaustive exercise induced an immediate decrease in MWT. This finding indicates that exercise causes acute muscle hyperalgesia, or immediate IEIP (Fig. 3B). After the 8-wk training period, the baseline MWT (before exhaustive exercise) was similar between HIIT and sedentary groups and unchanged from their baseline data before the 8-wk training period. Thus, exercise training did not alter baseline muscle pain sensitivity. On the other hand, HIIT abolished the decrease in MWT induced by exhaustive exercise, whereas MWT was still significantly decreased after exhaustive exercise in the sedentary group (Fig. 3B). In conclusion, exercise training did not alter baseline muscle pain perception; however, it abolished the acute muscle hyperalgesia, or IEIP, that occurred immediately after exhaustive exercise, and this was associated with enhanced exercise capacity.

Fig. 3.Effect of exercise training on exercise performance and immediate exercise-induced pain (IEIP). A: time to exhaustion (s) was measured for maximal incremental (exhaustive) exercise before and after an 8-wk period of sedentary (SED) or high-intensity interval training (HIIT) protocols. Two-way repeated measures ANOVA found a significant interaction effect between time and groups [F(1,10) = 10.83, P < 0.01, n ≥ 6]. Sidak’s test showed a significant difference between SED and HIIT groups after the 8-wk period (*P < 0.05). B: MWT was measured before (baseline) and immediately after exhaustive exercise. This was then repeated after an 8-wk period of sedentary (SED) (left panel) or HIIT (right panel) protocols. protocols. Two-way repeated measures ANOVA showed that there was a significant time effect [F(3,30) = 7.15, P < 0.001, n ≥ 6]. Fisher’s least significant difference test showed a significant decrease in MWT immediately after exhaustive exercise compared with baseline for both SED (*P < 0.05) and HIIT groups (**P < 0.01) before the 8-wk period. A decrease in MWT was also observed after exhaustive exercise in the SED group after the 8-wk period (*P < 0.05). On the other hand, after 8 wk of HIIT, MWT was unchanged after exhaustive exercise compared with baseline (P > 0.05). ns, not significant.
Exercise Training Altered the Expression of ASICs in Sensory Ganglion
We hypothesized that alterations in ASICs might, in part, underlie the diminished IEIP and improved exercise capacity seen after exercise training. To be able to distinguish the effects of acute exercise from the more chronic adaptive changes associated with exercise training, we first did a pilot study to test the effect of a single bout of exhaustive exercise on the expression of ASICs in lumbar DRG (which contain the cell bodies of muscle afferents that innervate the hindlimb muscles). For comparison, we also measured the expression of another sensory receptor, transient receptor potential cation channel subfamily V member 1 (TRPV1), which is also purported to contribute to muscle pain and the exercise pressor reflex (46, 49). Because native ASIC channels in muscle afferents are formed by the heteromeric combination of multiple different ASIC subunits (15), we measured the mean mRNA level of all ASICs together and found they were significantly diminished 40% (P < 0.001) 7 h following exercise when compared with a control group of mice that did not exercise (Fig. 4). However, in a separate group of mice tested 48 h after exercise, the mRNA levels were similar to control values (Fig. 4). Thus, strenuous exercise induced a transient reduction in mRNA levels within lumbar DRG.

Fig. 4.Dorsal root ganglia (DRG) mRNA levels after a single bout of exhaustive exercise. Acid-sensing ion channel (ASIC)1a, ASIC1b, ASIC2, ASIC3, and transient receptor potential cation channel subfamily V member 1 (TRPV1) mRNA levels in lumbar DRG as measured by quantitative PCR within 7 h (n = 6) or 48 h after exhaustive exercise (n = 10), compared and normalized to the levels in a control group that did not undergo exercise (n = 14). A two-way ANOVA revealed a significant difference between groups [F(2,135) = 14.90, P < 0.0001]. Dunnett’s test found a significant difference between the means of all ASIC subunits combined at 7 h (0.60 ± 0.04) compared with control (P < 0.001) but not at 48 h (1.18 ± 0.14) compared with control (P > 0.05). Also, mRNA levels of ASIC1a and ASIC1b were significantly downregulated 7 h after exhaustive exercise [#P < 0.05, Fisher’s least significant difference (LSD)], and ASIC1b was upregulated at 48 h compared with control (#P < 0.01, Fisher’s LSD).
Next, we tested the effects of 4 wk of low-intensity continuous training (LICT) or HIIT exercise training on expression of ASICs and TRPV1 compared with a sedentary group. Because mRNA levels after acute exhaustive exercise returned to baseline at 48 h, we measured mRNA levels of each training group at 48 h after the last maximal incremental exercise test. We found that mean expression of all ASIC subunits together was significantly reduced 49% (P < 0.0001) in the lumbar DRG in mice that underwent HIIT compared with the sedentary group (Fig. 5A). On the other hand, in the LICT group, mean ASICs mRNA in the lumbar DRG was slightly increased 27% (P < 0.05).

Fig. 5.Effect of exercise training on acid-sensing ion channels (ASICs) mRNA expression and exercise capacity. A: ASIC1a, ASIC1b, ASIC2, ASIC3, and transient receptor potential cation channel subfamily V member 1 (TRPV1) mRNA levels in lumbar dorsal root ganglia (DRG) as measured by quantitative PCR after either 4 wk of low-intensity continuous training (LICT) or high-intensity interval training (HIIT), compared and normalized to the levels in a sedentary (SED) group that did not undergo exercise training. A two-way ANOVA revealed significant differences between groups [F(2,135) = 28.25, P < 0.0001, n ≥ 8]. Dunnett’s test detected a significant difference between the means of all ASIC subunits combined for the LICT (1.27 ± 0.11, P < 0.05) and HIIT groups (0.51 ± 0.03, P < 0.0001) compared with SED. Also, the mRNA levels of ASIC1b, ASIC2, ASIC3, and TRPV1 were lower in the HIIT group, and ASIC1a and ASIC3 were increased in LICT group, compared with SED group (#P < 0.05, Fisher’s least significant difference). B: time to exhaustion (TTE; s) on maximal incremental exercise testing before and after 4 wk of exercise training for SED, LICT, and HIIT groups. Two-way ANOVA revealed a significant effect of exercise training within groups [F(1,26) = 32.77, P < 0.0001] and between groups [F(2,26) = 9.04, P < 0.001], as well as significant interaction between time and groups [F(2,26) = 18.17, P < 0.0001]. Sidak’s post hoc test detected no pretraining differences between the groups; however, the HIIT group showed a significant increase in TTE posttraining compared with the pretraining baseline (***P < 0.0001), and the HIIT group showed greater posttraining exercise capacity compared with LICT and SED groups (###P < 0.001). Mean mRNA levels of all ASIC subunits together (C) and individual ASIC subunits and TRPV1 (D–H) plotted against the TTE; TTE for individual mice in each group (SED, HIIT, and LICT). Pearson correlation testing revealed significant negative relationships between TTE and the mean of all mRNA levels of ASICs, as well as the individual ASIC subunits ASIC1a, ASIC1b, ASIC2, ASIC3, and TRPV1 [lines in C–H are best-fit regressions ± 95% confidence intervals; correlation coefficients (r) and P values are shown on each graph].
Similar to 8 wk of HIIT, we found that 4 wk of HIIT was sufficient to improve maximal exercise capacity of mice, and their performance was significantly greater than that of mice in the LICT or sedentary groups (Fig. 5B). The improvement in exercise capacity was not associated with changes in body mass; weights (g) were not different between groups before (sedentary 18.64 ± 0.39; LICT 19.21 ± 0.33; HIIT 18.05 ± 0.38) and after the 4-wk (sedentary 20.60 ± 0.48; LICT 20.34 ± 0.39; HIIT 19.91 ± 0.36) training period. Additionally, there were no differences between groups in the percentage of body fat (sedentary 8.52 ± 0.67; LICT 8.5 ± 0.70; HIIT 7.5 ± 0.99) and lean (sedentary 67.61 ± 0.52; LICT 67.59 ± 0.66; HIIT 68.42 ± 0.87) at the end of the 4-wk training period.
We also examined the possible correlation between the mRNA levels of ASICs and TRPV1 in lumbar DRG and exercise capacity for all groups (Sedentary, LICT, and HIIT). We found a significant negative relationship between mRNA levels of all ASICs averaged together and time to exhaustion during maximal exercise testing (Fig. 5C), as well as for each individual ASIC subunit and TRPV1 (Fig. 5, D–H).
Genetic Deletion of ASIC3 Abolished Immediate Exercise-Induced Pain but Had No Effect on Exercise Capacity
Because HIIT diminished IEIP and improved exercise capacity, and these results correlated with diminished ASICs expression in sensory neurons, we hypothesized that genetic deletion of ASICs might also diminish IEIP and improve exercise capacity. We studied ASIC3 −/− mice because ASIC3 is a required subunit in essentially all ASIC mouse muscle afferents, and genetic deletion of ASIC3 produces the most significant changes in the biophysical properties of ASIC channels in mouse DRG compared with deletion of other ASIC subunits (5, 15). We found that ASIC3 −/− mice had similar baseline MWT as wild-type mice before exhaustive exercise (Fig. 6A), suggesting that ASIC3 is not required for normal muscle mechanical pain threshold. However, unlike wild-type mice, ASIC3 −/− mice did not develop reduced MWT after exhaustive exercise (Fig. 6A). This finding indicates that ASIC3 is required for IEIP. On the other hand, the exercise capacity of ASIC3 −/− was similar to that of wild-type mice (Fig. 6B).

Fig. 6.Effect of genetic deletion of ASIC3 on immediate exercise-induced pain (IEIP) and exercise capacity. A: muscle withdrawal threshold (MWT) was measured before (baseline) and immediately after maximal incremental (exhaustive) exercise in male and female wild-type (WT) and ASIC3 −/− mice. Two-way ANOVA found no sex difference for MWT (P = 0.20) or time to exhaustion (P = 0.52), and so sexes were combined for analysis. Two-way repeated measures ANOVA showed a significant time effect [F(1,23) = 35.27, P < 0.0001, n ≥ 12]. Sidak’s multiple comparison test found no difference in baseline MWT before exhaustive exercise (P > 0.05). On the other hand, there was a significant decrease in MWT immediately after exercise compared with baseline for WT (***P < 0.0001) but not ASIC3 −/− mice (P > 0.05). B: time to exhaustion (s) was measured during maximal incremental exercise for the data in A. Student’s t test revealed no difference between groups. ns, not significant.
DISCUSSION
ASICs are expressed in sensory nerves that innervate skeletal muscle, where they sense the accumulation of metabolites and other chemicals during muscle inflammation, ischemia, injury, and strenuous exercise and therefore purportedly contribute to the sensations of muscle pain and fatigue. Here, we show that ASICs are required for immediate exercise-induced muscle pain (IEIP); ASIC3 −/− mice did not develop IEIP. Additionally, because exercise training is increasingly recognized as a therapeutic means to improve pain conditions, we tested whether exercise training is associated with alterations of ASICs in sensory neurons. Compared with mice that were sedentary or underwent low-intensity continuous training (LICT), mice that underwent high-intensity interval training (HIIT) had improved maximal exercise capacity and exhibited significantly lower ASICs and TRPV1 mRNA levels in their lumber DRG. Moreover, mice that underwent HIIT did not develop IEIP. Together, these data show that 1) ASICs are required for IEIP and 2) exercise training diminished IEIP, improved exercise capacity, and lowered expression of ASICs in sensory neurons.
Exercise Training Abolished Immediate Exercise-Induced Muscle Pain, and ASICs Are Required for IEIP
We found that a single bout of exhaustive exercise caused IEIP in mice and that HIIT abolished this muscle mechanical hypersensitivity precipitated by strenuous exercise. Given that ASICs have been shown to mediate chronic muscle pain in different rodent models and intense exercise is associated with accumulation of protons and other metabolites in skeletal muscle, we hypothesized that ASICs would be required for IEIP and that the loss of IEIP with exercise training might be due to modulation of ASICs. To test the first hypothesis, we studied ASIC3 null mice. ASIC3 subunits are necessary components of ASIC channels in mouse muscle afferents, and genetic deletion of ASIC3 produces profound alterations of ASIC-evoked currents recorded from muscle afferents (15). We found that genetic loss of ASIC3 completely abolished IEIP. Similar to mice that underwent HIIT, ASIC3 null mice had normal baseline muscle mechanosensation but did not develop hyperalgesia after intense exercise.
How might loss of a pH-sensitive ion channel subunit diminish the response from a mechanical stimulus? First, because ASIC3 is only expressed in rodent peripheral sensory neurons and not in the central nervous system, the mechanism most certainly involves the peripheral rather than central nervous system (9, 51). Additionally, there is evidence that ASICs might serve a mechanosensitive function in particular situations; however, other data do not support this role (24, 27). Moreover, ASICs are reported to participate in the chemical component of the exercise-pressor reflex and not the mechanical component (19, 26). In support of this, we found that baseline MWT (before exhaustive exercise) was the same in ASIC3 null mice compared with wild-type mice. Therefore, we speculate that ASICs are functioning to sensitize mechanoreceptors. Previous work has shown that metabolites and other chemicals can potentiate the response of mechanoreceptive muscle afferents (20, 41). Hotta et al. (21) found that acidosis increased the response magnitude and lowered the mechanical threshold of Aδ- and C-fiber in a rat muscle-nerve preparation and lowered the activation threshold of mechanically activated currents recorded from cultured small-diameter DRG neurons. Thus, although we cannot rule out the possibility that ASICs themselves could be functioning as mechanoreceptors, we suspect it is more likely that they are playing a role in the chemical sensitization of muscle afferent mechanoreceptors.
ASIC Expressions Are Downregulated by Exercise
To test whether modulation of ASICs might underlie the loss of IEIP after exercise training, we first did a pilot study to test the effect of a single bout of strenuous exercise on their expression in lumbar DRG. We found that ASICs and TRPV1 mRNA levels were reduced at 7 h after a single bout of exhaustive exercise, and these levels returned to baseline at 48 h after exercise. Similar transient reductions in sensory receptor mRNA have also been described in humans after a single bout of exercise. White et al. (53) found reduced leukocyte mRNA levels of ASIC3 and TRPV1 in healthy humans 30 min after sustained moderate exercise, with normalization of mRNA to preexercise levels at 24 h postexercise. In contrast, patients with chronic fatigue syndrome did not have a transient decrease in sensory receptor gene expression acutely after exercise, and instead showed an increase in expression at 24 h, which correlated with an increased perception of postexercise pain and fatigue. A similar study performed on trained cyclists found a similar transient reduction in gene expression of metabolite-detecting receptors at 0.5 and 8 h after an intense time-trial exercise, with normalization of expression at 24 h (48). We speculate that these transient exercise-induced reductions in sensory receptor expression in healthy subjects might represent adaptive negative feedback to the enhanced receptor activation.
Given that receptor mRNA levels returned to baseline at 48 h postexercise, we chose this time postexercise to measure mRNA in DRG after 4 wk of exercise training. We found that HIIT, but not LICT, produced a sustained decrease in sensory receptor mRNA level. In future studies, it would be interesting to see how long these changes in mRNA persist after cessation of exercise training. Previous work has shown that exercise training can interact with disease processes to modulate expression of sensory receptors. For example, in a diabetic rat model, exercise training prevented the diabetes-induced increase in TRPV1 and TRM8 protein in DRG (55). Ours was the first study to demonstrate that exercise training can induce a persistent downregulation of TRPV1 and ASIC mRNAs in sensory neurons. However, it should be mentioned that male mice do not exercise as readily as female mice (13), and so our results here may not necessarily extend to exercise training in male mice. The differences we found with HIIT compared with LICT are not surprising. To achieve the full benefits of exercise training, it is well recognized that exercise intensity level and duration are critical factors (12). In skeletal muscle of endurance-trained athletes, alterations in gene expression are dependent on the intensity of exercise (37). Our data demonstrate that these exercise intensity-dependent changes in gene expression extend to sensory neurons. In summary, the effect of exercise on expression of sensory receptors appears to be complex and dynamic: some of the dependent variables include the intensity, frequency, and duration of exercise and the health of the subjects. Additionally, our data provide evidence that diminished IEIP after HIIT could, in part, be due to alterations in muscle afferents. Although descending central inhibition may certainly play a role in exercise-induced hypoalgesia (43), our results showing that ASIC3 in muscle afferents is required for IEIP lend support for a novel adaptive mechanism at the level of the primary muscle afferent.
ASIC3 −/− Mice Had Similar Exercise Capacity as Wild-Type Mice
Compared with sedentary mice or those that underwent LICT, mice that underwent HIIT had an improvement in maximal exercise capacity. Interestingly, this improvement in exercise capacity correlated with downregulation of ASICs expression in lumber DRG. We speculated that lower expression of ASICs in muscle afferents, and the resultant abrogation of IEIP, might have contributed to the improvement in exercise capacity in mice after HIIT. Thus, we anticipated that ASIC3 −/− mice would have diminished IEIP and, consequently, higher exercise capacity. Indeed, deletion of ASIC3 abolished IEIP. Then why did ASIC3 −/− mice not show higher exercise capacity compared with wild-type mice?
Most certainly the role of ASICs within muscle afferents on exercise capacity is complicated and multifactorial. Neural reflexes activated by muscle afferents have dual and opposing effects on human exercise performance. On the one hand, activation of group III/IV muscle afferents increases central fatigue, which in turn inhibits motor neuronal output, thus attenuating exercise performance (7). On the other hand, activation of group III/IV muscle afferents also leads to ventilatory and cardiovascular reflex responses that serve to increase oxygen delivery to exercising muscle (i.e., exercise pressor reflex), thus improving exercise performance (2). As evidence of these opposing pathways, Hureau et al. (22) recently showed that endurance performance in cyclists was improved after pharmacological attenuation of III/IV muscle afferents, but only when oxygen delivery to locomotor muscle was held constant. ASICs within group III/IV muscle afferents likely contribute to both of these opposing neural circuits. Therefore, we suspect that the exercise capacity of ASIC3 null mice reflects the balance of its role in these two opposing mechanisms.
Altered Expression of ASICs Might Contribute to the Benefits of Exercise Training
Although much of the research focus on the benefits of exercise training on chronic pain has been on the endogenous opioid and other systems within the central nervous system, our results provide novel mechanistic insight at the level of the primary afferent neuron. Although we found that exercise caused downregulation of ASICs and TRPV1, these receptors are, in fact, upregulated within sensory neurons in models of chronic muscle pain. In a mouse model of muscle inflammation, ASIC2a and ASIC3 mRNA levels increased 10-fold in DRG (50). Similarly, hindlimb ischemia causes ASIC3 protein levels to increase in rat muscle afferents (28). These changes in ASIC gene expression are regulated by neurotrophins and other signaling molecules, including NGF, serotonin, IL-1, bradykinin (31, 32), and possibly TNF-α and IL-6 (29). NGF regulates ASIC3 expression through tropomyosin receptor kinase A (TrkA) receptor-activated phospholipase C/protein kinase C pathway (31, 32) and probably upregulates TRPV1 via a similar mechanism (54).
Exercise training has been shown to reduce neurotrophic and inflammatory signaling pathways, and these changes are associated with diminishment of pain (11, 43). For example, exercise training after sciatic nerve injury in rat reduces NGF and brain-derived neurotrophic factor expression in DRG and spinal cord, thereby preventing the associated hyperalgesia (30). In various models of diabetic neuropathy, exercise alleviates hindpaw mechanical hypersensitivity, reduces NGF protein levels and TrkA receptor expression in the hindpaw (18), and lowers the levels of IL-6, IL-1β, and TNF-α (55). Most pertinent to our study, the Jankowski group (39) showed that myalgia in a mouse ischemia/reperfusion model of muscle injury is dependent on IL-1β-induce upregulation of ASIC3. Moreover, 2 days of volunteer wheel running before muscle injury prevents the muscle injury-induced increase in IL-1β and associated muscle pain behavior (40). Therefore, there is substantial evidence that exercise training leads to a reduction, or prevention of upregulation, in neurotrophic and inflammatory signals that regulate expression of ASICs and TRPV1 in sensory neurons, and this may, in part, underlie the beneficial effects of exercise on chronic pain.
In conclusion, we found that HIIT, but not LICT, improved exercise capacity and prevented exercise-induced pain immediately after strenuous exercise (IEIP). These results correlated with a decrease in ASICs and TRPV1 expression within sensory neurons. Moreover, we demonstrated that ASICs are required for IEIP. These results further our understanding of the role of ASICs in exercise physiology and lend mechanistic insight into the benefits of exercise training.
GRANTS
This study was supported by the Department of Veterans Affairs Merit Award (5I01BX000776).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
T.K., M.J., H.R., K.A.S., and C.J.B. conceived and designed research; T.K., A.M.S.H., M.E.-G., and C.J.B. performed experiments; T.K. analyzed data; T.K., P.M.S., and C.J.B. interpreted results of experiments; T.K. prepared figures; T.K. and C.J.B. drafted manuscript; T.K., A.M.S.H., M.J., H.R., P.M.S., K.A.S., and C.J.B. edited and revised manuscript; T.K., A.M.S.H., M.J., H.A.-A., H.R., P.M.S., K.A.S., and C.J.B. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Alan Ryan (University of Iowa) and Mahdieh Molanouri Shamsi (University of Tarbiat Modares) for helpful advice and Lynn Rasmussen and the Iowa Institute of Human Genetics for technical assistance.
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