Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle

Abstract

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Figure 1

Effect of spontaneous contraction on rat PmFB respiratory kinetics. PmFB contraction in red (RG) and white (WG) gastrocnemius following exposure to 30°C for 30 min is prevented by myosin inhibitors blebbistatin (BLEB) and N-benzyltoluene sulfonamide (BTS) (A; RG) alone (1× amplification) and (B) following ∼30-45 min of ADP-stimulated respiration (0.6× amplification).

Figure 2

Effect of contraction on ADP-stimulated mitochondrial respiratory kinetics at 30°C in red (RG; A-B) and white gastrocnemius (WG; C-D) PmFB under air-saturated media (∼150-220 μM O₂). Data represent means ± SEM; n = 4-5; *P < 0.05 vs all other treatments at same temperature.

Figure 3

Respiratory sensitivity to ADP and Vmax are influenced by contraction in a temperature-dependent manner in rat red (RG; A-B) and white (WG; C-D) gastrocnemius PmFB under air-saturated media (∼150-220 μM O₂). Data represent means ± SEM; n = 4-5; *P < 0.05 vs all other treatments at same temperature; †P < 0.05 significant differences between all temperatures within same treatment; ^#P < 0.05 vs 30°C, or ^‡ vs 37°C within same treatment.

Figure 4

Respiratory sensitivity to ADP and Vmax are influenced by hyperoxic conditions (Hyperoxia, ∼275-450 μM O₂) compared to air-saturated media (AIR, ∼150-220 μM O₂) in a temperature-dependent manner in rat red (RG) and white gastrocnemius (WG) PmFB (A-H). Data represent means ± SEM; n = 4-5; *P < 0.05 vs all other treatments at same temperature; ^δP < 0.05 vs AIR within same treatment.

Figure 5

Effects of spontaneous contraction on human PmFB respiratory kinetics. (A) Conformation of PmFB from human male vastus lateralis following ADP-stimulated respiration at 30°C without (+/-V) or with myosin inhibitors BLEB and BTS (0.6× amplification). (B-C) Effect of contraction on ADP-stimulated respiratory kinetics 30°C and 37°C under air-saturated media (∼150-220 μM O₂). Data represent means ± SEM; n = 5; *P < 0.05 vs all other treatments at same temperature.

Figure 6

Temperature-dependent effects of contraction on respiratory sensitivity (Km) to ADP and Vmax in human PmFB under air-saturated media (∼150-220 μM O₂) (A-C). Data represent means ± SEM; n = 4; *P < 0.05 vs all other treatments at same temperature; †P < 0.05 significant differences between both temperatures within same treatment; ^§P < 0.05 vs +V within same temperature.

Figure 7

ADP-stimulated respiratory kinetics in the presence of 24 mM phosphocreatine and 12 mM creatine at 37°C (A-B), representative of “resting” in vivo conditions. Data represent means ± SEM; n = 4; *P < 0.05 vs all other treatments at same temperature; ^φP < 0.05 vs same treatment with 20mM Creatine at 37°C (Fig. 6). Experiments were conducted under air-saturated media (∼150-220 μM O₂).