Background & Aims

Activity-induced muscle pain is a barrier to participation in an effective exercise program and daily activities. To address this, we developed an animal model of activity-induced muscle pain that combines fatiguing muscle contractions with a low intensity muscle insult. Fatiguing muscle contractions release fatigue metabolites including adenosine triphosphate, protons, and lactate all of which contribute to activity-induced muscle pain. Our prior studies show ASIC3-/- mice and pharmacological blockade of ASIC3 in muscle prevent development of activity-induced muscle pain. However, targeted ASIC3 deletion in primary afferents innervating muscle (HSV-miASIC) has no effect on activity-induced muscle pain. Further, macrophages in muscle mediate activity-induced muscle pain, release IL-1? in response to acidic pH, and express ASIC3. Therefore, we hypothesized that muscle fatigue metabolites activate ASIC3 on macrophages to release IL-1? to produce activity-induced muscle pain.

Methods

We developed a floxed ASIC3 mouse (ASIC3fl/fl)(expresses mcherry on deletion) to examine if ASIC3 on macrophages mediate the hyperalgesia of activity-induced muscle pain. These mice were crossed with Cx3cr1Cre mice to remove ASIC3 from macrophage/monocyte (Cx3cr1Cre:Asic3fl/fl). For comparison, ASIC3fl/fl mice were crossed with the AdvillinCre to remove ASIC3 from sensory neurons (AvilCre:Asic3fl/fl). To test if ASIC3 locally contributes to activity-induced pain, a lenti virus-expressing Cre recombinase was injected to the ASIC3fl/fl mice (LentiCre:Asic3fl/fl). ASIC3 mRNA and mCherry protein expression were examined in muscle and dorsal root ganglia neurons to confirm ASIC3 knock down. Muscle pain was induced by 2, 20?l pH 5.0 injections, separated by 5 days, in the gastrocnemius muscle combined with 6 minutes of electrically stimulated fatiguing muscle contractions. Muscle withdrawal thresholds (MWT) of the gastrocnemius muscle were measured before and after induction of the model.

Results

Reduction of ASIC3 in macrophages (Cx3cr1Cre: Asic3fl/fl) prevented development of hyperalgesia after induction of the model. Reduction of ASIC3 mRNA in peritoneal macrophages and mCherry in F4/80+ macrophages in muscle confirmed reduction of macrophage ASIC3. In contrast, reduction of ASIC3 in sensory neurons (AvilCre:Asic3fl/fl) had no effect on muscle hyperalgesia after induction of the model when compared to genetic controls. For comparison, AvilCre:Asic3fl/fl injected with carrageenan into the muscle did not develop hyperalgesia. Reduction ASIC3 mRNA and mCherry expression in L4–L6 DRG confirmed reduction of sensory neuron ASIC3. Reduction of ASIC3 at the site of induction prevented hyperalgesia in the activity-induced pain model when compared to genetic controls. Reduction in ASIC3 mRNA in muscle and transfected macrophages, and expression of mCherry in F4/80+ muscle macrophages confirmed reduction of ASIC3 in local macrophages.

Conclusions

Since reduction of ASIC3 in monocytes/macrophages (Cx3cr1Cre:Asic3fl/fl) and local knockdown of ASIC3 in muscle (LentiCre:Asic3fl/fl) prevented hyperalgesia, while knockdown in sensory neurons had no effect on hyperalgesia, our data suggests that activity-induced pain is mediated by activation of ASIC3 in muscle tissue and not sensory neurons. This effect occurs equally in male and female mice, and thus is not sex-dependent. We further showed that acidic pH combined with ATP promotes release of IL-1? from isolated macrophages, suggesting a synergistic effect of P2X and ASIC receptors. We further show a role for the fatigue-metabolite receptors P2X4 and P2X7 receptors on muscle macrophages in the generation of activity-induced pain. We thus suggest ASIC3 and P2XR in muscle macrophage work together to release IL-1? and promote development of activity-induced muscle pain.

References

Gregory NS, Gibson-Corley K, Frey-Law L, Sluka KA. Fatigue-enhanced hyperalgesia in response to muscle insult: induction and development occur in a sex-dependent manner. Pain. 2013; 154:2668–2676.
Gregory NS, Brito RG, Fusaro MCGO, Sluka KA. ASIC3 Is Required for Development of Fatigue-Induced Hyperalgesia. Mol Neurobiol. 2016; 53:1020–1030.
Lesnak JB, Inoue S, Lima L, Rasmussen L, Sluka KA. Testosterone protects against the development of widespread muscle pain in mice. Pain. 2020; 161:2898–2908.
Lesnak JB, Hayashi K, Plumb AN, Janowski AJ, Chimenti MS, Sluka KA. The impact of sex and physical activity on the local immune response to muscle pain. Brain Behav Immun. 2023; 111:4–20.
Hayashi K, Lesnak JB, Plumb AN, Rasmussen LA, Sluka KA. P2X7-NLRP3-Caspase-1 signaling mediates activity-induced muscle pain in male but not female mice. Pain. 2023; 164:1860–1873.

Presenting Author

Kazuhiro Hayashi

Poster Authors

Kazuhiro Hayashi

PhD

Kyoto University, Kyoto, Japan

Lead Author

E-mail

Joseph B. Lesnak

Lead Author

E-mail

Ashley Plumb

Lead Author

E-mail

Adam Janowski

DPT

Lead Author

E-mail

Angela Smith

University of Iowa

Lead Author

E-mail

Lynn A. Rasmussen

Lead Author

E-mail

Kathleen Sluka

PT

University of Iowa, Carver College of Medicine

Lead Author

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Topics

  • Models: Musculoskeletal