Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition

Abstract

Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature, resulting in a loss of contractile function in isolated diaphragm (thermal tolerance), and then tested the hypotheses 1) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and 2) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43 degrees C. Between 40 and 42 degrees C, there were no effects of hyperthermia, but at 43 degrees C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox, did not inhibit the loss of contractile force at 43 degrees C. Furthermore, treatment with dithiothreitol, a thiol (-SH) reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43 degrees C and caused a significant loss of thermal tolerance at 42 degrees C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42 degrees C. Blockage of phospholipase with phospholipase A(2) inhibitors, bromoenol lactone or arachidonyltrifluoromethyl ketone failed to significantly prevent the loss of force at 43 degrees C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.

Fig. 1.

A: force frequency relationships obtained at the end of 30 min exposure to 37°C (n = 6), 40°C (n = 9), 41°C (n = 9), 42°C (n = 8), and 43°C (n = 8). Force is expressed as a % of preheat, pretreatment, tetanic force at 150 Hz. Peak twitch force is represented as zero Hz. Temperature-induced increases in passive tension are also shown (inset). B: Force-frequency relationships obtained after 30 min heat exposure, followed by 30 min of recovery at 37°C. ‡‡P < 0.001 vs. 37°C.

Fig. 2.

Change in %max force (150 Hz) of diaphragm strips exposed to 43°C with antioxidants. Trolox (n = 6) and Tiron (n = 6) data are after 30 min. at 43°C. Dose-responses are shown with time-matched controls. *P < 0.05 vs. control 43°C.

Fig. 3.

Change in %max force (150 Hz) of diaphragm strips exposed to 43°C with dithiothreitol (DTT). DTT (n = 8) data are shown after 30 min of recovery at 37°C (n = 8), shown with control recovery after 43°C and 5 mM DTT 37°C control. No statistical significance was seen with DTT exposure vs. control recovery following 43°C.

Fig. 4.

Change in %max force (150 Hz) of tissues exposed to 42°C with LOX or COX inhibitor treatment. A: general LOX inhibitors, baicalein (n = 4) and ETYA (n = 4) reduced force in a dose-dependent manner. B: general COX inhibitors, indomethacin (n = 4) and ibuprofen (n = 4) decreased function in a dose-dependent manner. C: passive tension increased in LOX inhibited tissues. D: passive tension increased in COX-inhibited tissues, *P < 0.05, **P < 0.01, ‡P < 0.005, ‡‡P < 0.001 vs. controls.

Fig. 5.

Change in %max force (150 Hz) of tissues exposed to 43°C and PLA₂ inhibition. A: iPLA₂ inhibition by BEL. BEL (20 μM) showed a significant reduction in force compared with control, whereas 1 μM and 10 μM had no effect. B: cPLA₂ and iPLA₂ inhibition by AACOCF₃. AACOCF₃ caused no change in the loss of force production at 43°C ‡P < 0.005 vs. control (n = 6). @P < 0.05 vs. 37°C control.

Fig. 6.

Membrane permeability evaluated using procion orange. Baicalein and indomethacin addition at 43°C caused a significant increase in the number of procion orange-positive cells. Procion orange diaphragm tissue: control 37°C (A), control 43°C (B), and baicalein and indomethacin 43°C (C). ‡‡P < 0.001 vs. control (n = 6).