Effects of cytoplasmic free Mg ²⁺ buffering on skeletal muscle fatigue in intact single mouse muscle fibers

Changes in cytoplasmic free Mg ²⁺ concentration ([Mg ²⁺ ] _cyto ) might play a key role in regulating skeletal muscle function. Mag-indo-1, a fluorescence dye that specifically binds to Mg ²⁺ , can be used both to detect and buffer [Mg ²⁺ ] _cyto . In this study, we hypothesized that buffering the fatigue-induced elevations in [Mg ²⁺ ] _cyto would mitigate fatigue by preserving sarcoplasmic reticulum (SR) Ca ²⁺ release. Intact fibers were isolated from the flexor digitorum brevis muscle, and loaded with varying concentration of Mag-indo-1-AM. The fibers were repeatedly stimulated at 70 Hz for 300 msec every 2 sec for 100 contractions (fatiguing stimulation: FS). To examine the relationship between fatigue and Mg ²⁺ buffering, we compared the force produced during the final contraction of FS with the amount of Mag-indo-1 loading before FS (Fatigue-Mg ²⁺ buffer relationship). Fatigue-Mg ²⁺ buffer relationship exhibited an inverted U-shaped curve: fatigue resistance improved when [Mag-indo-1] increased up to an optimal level, but declined when [Mag-indo-1] became excessive. Therefore, the fibers were classified into three groups: underloaded, optimal-loaded and overloaded groups. Caffeine treatment immediately after FS significantly enhanced force recovery in the underloaded group, reaching a level comparable to that of caffeine-treated, optimal-loaded group. Furthermore, mitochondrial coupled respiration was impaired, and proton leak was enhanced in the presence of nM level Mag-indo-1. These results suggest that elevated [Mg ²⁺ ] _cyto contributes to muscle fatigue due to impairments of SR Ca ²⁺ release. Conversely, excessive buffering of [Mg ²⁺ ] _cyto may also exacerbate muscle fatigue, indicating that a proper [Mg ²⁺ ] _cyto balance is critical for maintaining fatigue resistance.