Effects of cytoplasmic free Mg2+ buffering on skeletal muscle fatigue in intact single mouse muscle fibers.

Abstract

Changes in cytoplasmic free Mg²⁺ concentration ([Mg²⁺]_cyto) might play a key role in regulating skeletal muscle function. Mag-indo-1, a fluorescence dye that specifically binds to Mg²⁺, can be used both to detect and buffer [Mg²⁺]_cyto. In this study, we hypothesized that buffering the fatigue-induced elevations in [Mg²⁺]_cyto would mitigate fatigue by preserving sarcoplasmic reticulum (SR) Ca²⁺ release. Intact fibers were isolated from the flexor digitorum brevis muscle and loaded with varying concentrations of Mag-indo-1-AM. The fibers were repeatedly stimulated at 70 Hz for 300 ms every 2 s for 100 contractions (fatiguing stimulation: FS). To examine the relationship between fatigue and Mg²⁺ buffering, we compared the force produced during the final contraction of FS with the amount of Mag-indo-1 loading before FS (fatigue-Mg²⁺ buffer relationship). Fatigue-Mg²⁺ 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 with 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 [Mg²⁺]_cyto contributes to muscle fatigue due to impairments of SR Ca²⁺ release. Conversely, excessive buffering of [Mg²⁺]_cyto may also exacerbate muscle fatigue, indicating that a proper [Mg²⁺]_cyto balance is critical for maintaining fatigue resistance.NEW & NOTEWORTHY Elevated cytoplasmic free magnesium ion may contribute to skeletal muscle fatigue. In this study, intact single fibers were loaded with various concentrations of Mag-indo-1 to buffer cytoplasmic free magnesium ions. Our results demonstrated that maintaining an appropriate cytoplasmic free magnesium ion balance is critical for resistance to muscle fatigue.