Role of Ca²⁺ in changing active force during intermittent submaximal stimulation in intact, single mouse muscle fibers

Fatigue of single mouse fibers during repeated high-frequency stimulation results initially from decreased Ca²⁺ sensitivity while free myoplasmic calcium concentration ([Ca²⁺]_m) increases, followed by decreasing [Ca²⁺]_m. Recovery of active force with low-frequency stimulation is slow and persistent fatigue results from low [Ca²⁺]_m. However, the consequences of intermittent submaximal contractions are not known. The aim of the present study was to investigate the changes in [Ca²⁺]_m and active force during intermittent submaximal contractions and subsequent recovery. Single fibers of mouse flexor digitorum brevis muscles at 32 °C were stimulated with 40 or 50 Hz, for 350 ms every 2 s for 2 min and then every 1 s until < 40% of initial force. Values obtained during the intermittent stimulation were compared with a control force-[Ca²⁺]_m relationship. A "P"-shaped pattern in the force-[Ca²⁺]_m relationship was observed during intermittent stimulation. Early in the intermittent stimulation, [Ca²⁺]_m increased while active force decreased. Subsequent force potentiation was accompanied by increased Ca²⁺ sensitivity. Later, as active force declined, [Ca²⁺]_m decreased significantly (p < 0.001). This was followed, in the final phase, by a significant decrease in Ca²⁺ sensitivity determined by [Ca²⁺]_m at half-maximal force (Ca₅₀) (p = 0.001). Low-frequency fatigue persisted during recovery while Ca₅₀ was not significantly different from prefatigue (p > 0.5). In conclusion, the main mechanism of fatigue is due to decreases in both [Ca²⁺]_m and Ca²⁺ sensitivity following the initial force potentiation. The intermittent submaximal contractions resulted in persistent low-frequency fatigue seen during recovery, which was explained by depressed [Ca²⁺]_m with no change in Ca²⁺ sensitivity.