Thermal pretreatment facilitates recovery from prolonged low-frequency force depression in rat fast-twitch muscle

Abstract

The aim of this study was to examine whether thermal pretreatment can accelerate recovery from prolonged low-frequency force depression. The hindlimbs of thermal treated (T-treated) rats were immersed in water heated to 42.0°C for 20 min (thermal pretreatment). The thermal pretreatment was performed once a day for 5 days before fatiguing stimulation. Intact gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected immediately [recovery 0 (REC0)] or 60 min [recovery 60 (REC60)] following the cessation of stimulation. Using skinned fiber prepared from the superficial region, the ratio of force at 1 Hz to that at 50 Hz (low-to-high force ratio), the ratio of depolarization (depol)-induced force to maximum Ca²⁺ -activated force (depol/max Ca²⁺ force ratio), the steepness of force-Ca²⁺ concentration curves, and myofibrillar Ca²⁺ sensitivity were measured. At REC0, the low-to-high force ratio and depol/max Ca²⁺ force ratio decreased in stimulated muscles from both non- and thermal-treated rats. At REC60, these two parameters remained depressed in non-treated rats, whereas they reverted to resting levels in T-treated rats. Thermal pretreatment exerted no effect on myofibrillar Ca²⁺ sensitivity. The present results reveal that thermal pretreatment can facilitate recovery of submaximum force after vigorous contraction, which is mediated via a quick return of Ca²⁺ release from the sarcoplasmic reticulum to resting levels.

Keywords: Calcium release; low-frequency muscle fatigue; reactive oxygen species; sarcoplasmic reticulum.

Figure 1

Effects of thermal pretreatment and fatiguing stimulation on low‐to‐high force ratio (1 vs. 50 Hz) in skinned fiber. Hindlimbs of thermal‐treated rats were immersed to 42.0°C for 20 min day⁻¹, which was performed five times before fatiguing stimulation. Twenty‐four hours after the last thermal pretreatment, intact gastrocnemius muscle of the left hindlimb was stimulated in vivo until the force was reduced to ~50% of the initial force (fatiguing stimulation). Immediately or 60 min after fatiguing stimulation, skinned fibers were prepared from gastrocnemius muscles. (A) Typical sample of action potential‐induced force response in stimulated (fatigued) fibers from non‐ and thermal‐treated rats that were allowed to rest for 60 min. Action potential‐induced contractions were electrically elicited using a single pulse (for twitch) and 20 pulses at 50 Hz. (B and C) ratio of twitch (1 Hz) force to force at 50 Hz. Values are means + SD. “n” denotes number of fibers and “N” the number of rats from which the fibers were obtained. ^a P < 0.05, significantly different from rested muscles; ^b P < 0.05, significantly different from stimulated muscles from non‐treated rats. Non, non‐treated; T, thermal‐treated; REC0, recovery 0 min; REC60, recovery 60 min; Stim, stimulated.

Figure 2

Effects of thermal pretreatment and fatiguing stimulation on depolarization (depol)‐induced force in skinned fiber. For the protocol of thermal pretreatment and fatiguing stimulation, see legend of Figure 1. Depol‐induced force was elicited by replacing the K‐HDTA with the Na‐HDTA solution and expressed as a percentage of maximum Ca²⁺‐activated force (depol/max Ca²⁺ force ratio). Values are means + SD. “n” denotes number of fibers and “N” the number of rats from which the fibers were obtained. *P < 0.05, significant main effect for stimulation (rested > stimulated); ^a P < 0.05, significantly different from rested muscles. Non, non‐treated; T, thermal‐treated; REC0, recovery 0 min; REC60, recovery 60 min; Stim, stimulated: HDTA, hexamethylene‐diaminetetraacetic acid.

Figure 3

Effects of thermal pretreatment and fatiguing stimulation on specific maximum (max) Ca²⁺ force in skinned fiber. For the protocol of thermal pretreatment and fatiguing stimulation, see legend of Figure 1. Max Ca²⁺‐activated force was elicited by exposing the skinned fiber to heavily buffered Ca²⁺ solution (pCa ~4.7) and normalized to the cross‐sectional area of the fiber. Values are means + SD. “n” denotes number of fibers and “N” the number of rats from which the fibers were obtained. Non, non‐treated; T, thermal‐treated; REC0, recovery 0 min; REC60, recovery 60 min; Stim, stimulated.

Figure 4

Effects of thermal pretreatment and fatiguing stimulation on myofibrillar Ca²⁺ sensitivity and Hill coefficient in skinned fiber. For the protocol of thermal pretreatment and fatiguing stimulation, see legend of Figure 1. The fiber was exposed to a sequence of solutions heavily buffered at progressively higher free [Ca²⁺] (10^−9.0–10^−4.7 mol/L). (A and B) Hill fits to force‐[Ca²⁺] data. The [Ca²⁺] was expressed as a base 10 logarithmic scale. (C and D) mean (+SD) of [Ca²⁺]₅₀ for Hill fits in A and B. (E and F) mean (+SD) of Hill coefficient for Hill fits in A and B. “n” denotes number of fibers and “N” the number of rats from which the fibers were obtained. *P < 0.05, significant main effect for stimulation (rested > stimulated). Non, non‐treated; T, thermal‐treated; REC0, recovery 0 min; REC60, recovery 60 min; Stim, stimulated; [Ca²⁺], Ca²⁺ concentration; [Ca²⁺]₅₀, [Ca²⁺] required for half‐maximum force.

Figure 5

Immunoblot analyses of heat shock protein (HSP) 70. For the protocol of thermal pretreatment and fatiguing stimulation, see legend of Figure 1. (A) immunoblot analyses of HSP70. A quantity of 20 μg of protein was applied to SDS‐PAGE. To ascertain a linear relationship between the band density and the protein amount, 5–40 μg of protein was loaded on the gel for loading control. Molecular mass of makers (M) is indicated in the left. (B and C) means + SD of HSP70 amount (N = 7 for each muscle). The HSP70 amounts were evaluated relative to the band density of GAPDH. The results are expressed as percentages of the values of rested muscles from non‐treated rats at REC0. Non, non‐treated; T, thermal‐treated; REC0, recovery 0 min; REC60, recovery 60 min; S and Stim, stimulated; R, rested; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.