The contribution of pH-dependent mechanisms to fatigue at different intensities in mammalian single muscle fibres

Abstract

1. The contribution of intracellular pH (pHi) to the failure of Ca2+ release and inhibition of contractile proteins observed during fatigue was assessed in single intact mouse muscle fibres at 22 C. Fatigue was induced by repeated tetani at intensities designed to induce different levels of intracellular acidosis. Force and either intracellular free Ca2+ concentration ([Ca2+]i; measured using indo-1) or pHi (measured using SNARF-1) were recorded in fibres fatigued at two different intensities. 2. Intensity was varied by the repetition rate of tetani and quantified by the duty cycle (the fraction of time when the muscle was tetanized). Stimulation at the low intensity (duty cycle approximately 0.1) reduced force to 30 % of initial values in 206 +/- 21 s (60 +/- 7 tetani); at the high intensity (duty cycle approximately 0.3) force was reduced to 30% in 42 +/- 7 s (43 +/- 7 tetani) (P < 0.05; n = 14). 3. When force was reduced to 30 % of initial values, tetanic [Ca2+]i had fallen from 648 +/- 87 to 336 +/- 64 nM (48% decrease) at the low intensity but had only fallen from 722 +/- 84 to 468 +/- 60 nM (35% decrease) at the higher intensity (P < 0.05 low vs. high intensity; n = 7). 4. Fatigue resulted in reductions in Ca2+ sensitivity of the contractile proteins which were greater at the high intensity (pre-fatigue [Ca2+]i required for 50 % of maximum force (Ca50) = 354 +/- 23 nM; post-fatigue Ca50 = 421 +/- 48 nM and 524 +/- 43 nM for low and high intensities, respectively). Reductions in maximum Ca2+-activated force (Fmax) were similar at the two intensities (pre-fatigue Fmax = 328 +/- 22 microN; post-fatigue Fmax = 271 +/- 20 and 265 +/- 19 microN for low and high intensities, respectively). 5. Resting pHi was 7.15 +/- 0.05. During fatigue at the low intensity, pHi was reduced by 0.12 +/- 0.02 pH units and at the high intensity pHi was reduced by 0.34 +/- 0.07 pH units (P < 0.05; n = 5). 6. Our results indicate that the more rapid fall in force at a high intensity is due to a reduction in Ca2+ sensitivity of the contractile proteins, probably related to the greater acidosis. Our data also indicate that the failure of Ca2+ release and reduced maximum Ca2+-activated force observed during fatigue are not due to reductions in intracellular pH.

Figure 1. Force and [Ca²⁺]_i traces from a single fibre fatigued at low and high intensities

Continuous force records (upper panel) and [Ca²⁺]_i and force transients on an expanded time scale (middle and bottom panels, respectively) are shown during fatigue at low and high intensities. Fatigue 1 was induced by tetani repeated initially every 4 s and increased to a rate of 1/3 s after 2 min (low intensity). Following 60 min of recovery Fatigue 2 was induced by tetani repeated every 1 s (high intensity). Tetani were continued until force reached 30 % of initial values. The fibre was perfused with a 5 mm caffeine solution towards the end of fatigue to assess E-C coupling failure. For Fatigue 1, force and [Ca²⁺]_i, transients are shown for the first tetanus (a), the tetanus at 30 % (b) and a tetanus after exposure to caffeine (c). For Fatigue 2 force and [Ca²⁺]_i, transients represent the first tetanus (d), the tetanus at 30 % (e) and a tetanus after exposure to caffeine (f).

Figure 2. Average force and [Ca²⁺]_i in response to fatigue at low and high intensities

Force (upper panel) and [Ca²⁺]_i, (lower panel) for the first tetanus (pre-fatigue; □), when force reached 30 % (fatigue; ), minimum force () and after 5 mm caffeine exposure at the end of fatigue (). Minimum force was defined as the force immediately before a response to caffeine was observed. Changes in force and [Ca²⁺]_i during fatigue at both the low intensity (1/4 s, etc.) and high intensity (1/1 s) are shown. Values represent mean ± s.e.m. (n = 7). *P < 0.05 vs. pre-fatigue; †P < 0.05 vs. fatigue; ‡P < 0.05 vs. low intensity.

Figure 3. Force-Ca²⁺ relationship in response to fatigue at low and high intensities

Pre-fatigue force and [Ca²⁺]_i are plotted at 30, 50, 70 and 100 Hz and maximum force obtained at 100 Hz in the presence of 5 mm caffeine. Force and [Ca²⁺]_i are plotted for pre-fatigue (Fatigue 1, •; Fatigue 2, ▴) and during the final phase of fatigue, when force and Ca²⁺ are decreasing, for the low intensity (▪) and high intensity (▾). Data points representing force and [Ca²⁺]_i after 5 mm caffeine exposure at the end of fatigue are included. All points are fitted to a Hill equation (see Methods). The rightward shift of the force-Ca²⁺ curve, representing a decrease in Ca²⁺ sensitivity, is greater at the high vs. low intensity. The plateau, representing maximum Ca²⁺-activated force, is reduced during fatigue but to a similar extent at the two intensities.

Figure 4. Force and pH_i traces from a single fibre fatigued at low and high intensities

Force (upper panel) and pH_i (lower panel) were recorded simultaneously at both the high and low intensity. pH_i was measured using SNARF-1. Fatigue 1 was induced by tetani repeated every 1 s (high intensity). Following 20 min of recovery, Fatigue 2 was induced by tetani repeated initially every 4 s then increased to 1/3 s, 1/2.5 s, etc. every 2 min (low intensity). Tetani were continued until force reached 30 % of initial values. Change in pH_i induced by 30 % CO₂ exposure (∼0.5 pH unit change) is shown relative to the pH_i changes at high and low intensities.

Figure 5. Force and [Ca²⁺]_i after 60 min recovery demonstrate low frequency fatigue

Force (top panel) and [Ca²⁺]_i (bottom panel) after 60 min of recovery from fatigue at low and high intensities ( and , respectively). Reductions in force and [Ca²⁺]_i, shown as % of initial values, were of similar magnitude at both intensities. Values represent mean ± s.e.m. (n = 6–7). * Significant difference (P < 0.05) compared with initial force or [Ca²⁺]_i at the same frequency or condition.