Force generation induced by rapid temperature jumps in intact mammalian (rat) skeletal muscle fibres

Abstract

We examined the tension (force) responses induced by rapid temperature jumps (T-jumps) in electrically stimulated, intact fibre bundles (5-10 fibres, fibre length approximately 2 mm) isolated from a foot muscle (flexor hallucis brevis) of the rat; the muscle contains approximately 90 % type 2 fast fibres. In steady state experiments, the temperature dependence of the twitch tension was basically similar to that previously described from other fast muscles; the tetanic tension increased 3- to 4-fold in raising the temperature from approximately 2 to 35 degrees C and the relation between the tetanic tension and the reciprocal absolute temperature was sigmoidal with half-maximal tension at 9.5 degrees C. A rapid T-jump of 3-5 degrees C was induced during a contraction by applying an infrared laser pulse (lambda = 1.32 micro, 0.2 ms) to the 50 microl trough containing the fibre bundle immersed in physiological saline. At approximately 10 degrees C, a T-jump induced a large transient tension rise when applied during the rising phase of a twitch contraction, the amplitude of which decreased when the T-jump was delayed with respect to the stimulus; a T-jump probably perturbs an early step in excitation-contraction coupling. No transient increase was seen when a T-jump was applied during twitch relaxation. When applied during the plateau of a tetanic contraction a T-jump induced a tension rise to a higher steady tension level; the tension rise after a T-jump was 2-3 times faster than the corresponding phase of the initial tension rise in a tetanus. The approach to a new steady tension level after a T-jump was biphasic with a fast (phase 2b, approximately 35 s-1 at 10 degrees C) and a slow component (phase 3, < 10 s-1). The rates of both components increased (Q10 approximately 3) but their amplitudes decreased with increase of the steady temperature. These results from tetanized intact fibres are consistent with the thesis previously proposed from studies on Ca2+-activated skinned fibres, that the elementary force generation step in muscle is enhanced by increased temperature; the findings indicate that an endothermic molecular step underlies muscle force generation.

Figure 1. A schematic diagram of the trough system used in laser T-jump experiments

A, the system consists of three troughs milled in a titanium block and a front trough that was built with glass front and bottom windows and used as the experimental trough. Cooling fluid, circulated within the titanium block, acted as a heat sink. The trough system was fixed on a Teflon block (a heat insulator), that could be moved along the aluminium plate by a lever mechanism (not shown) to expose, in skinned fibre experiments, a preparation to different solutions. B, an enlarged view of the front glass experimental trough. An intact fibre bundle was attached, by aluminium foil clips, between two metal hooks, one connected to a force transducer and the other to a servomotor. The experimental trough temperature was clamped using thermoelectric modules that were under thermistor feedback control, and it was monitored by a thermocouple placed near the fibre bundle. Supramaximal voltage stimuli (0.2–0.5 ms) were delivered via two platinum foil electrodes glued to the front window and the back wall of the trough, the one in the front window being narrower to prevent fibre shadowing by the heating laser pulse. The Nd-YAG laser pulse (λ= 1.32 μ, pulse duration = 0.2 ms, elliptical cross-section ∼2 mm × 5 mm) entered through the front window, it was partially absorbed in solution and reflected by the platinum foil on the back wall. The transducer hooks and the aluminium T-clips were shadowed from the laser beam (not shown). The energy absorption of ∼50 % during the 0.2 ms pulse heated the solution and fibre bundle by 3–5 °C and the elevated temperature in the trough solution remained constant for ∼500 ms (for details see Ranatunga, 1996).

Figure 2. Twitch and tetanic contractions at different temperatures

Superimposed twitch and tetanic contractions recorded from one preparation at various temperatures. The upper series of frames shows contractions first recorded at 20 °C and then at 5 °C intervals during cooling to 5 °C, and the lower series shows those during warming to 35 °C. Contractions recorded finally at 20 °C are shown in the bottom right frame; the duration of the whole experimental procedure was ∼3 h. Note that the peak twitch tension is highest at ∼25 °C and is lower at higher and lower temperatures. The increase of twitch tension from 35 to 20–25 °C, as obtained in fast mammalian muscles, is commonly referred to as ‘cooling potentiation’. The horizontal bar below each frame shows the duration of the tetanic stimulus train; frequency was adjusted to obtain fused tetanic contractions (see Methods). The tetanic tension increases with increased temperature but the relative increase is higher at temperatures below ∼20 °C.

Figure 3. Temperature dependence of twitch and tetanic tensions

A, mean ±s.e.m. twitch tension (filled symbols) and tetanic tension (open symbols) from eight preparations. Tension per cross-sectional area is plotted on the ordinate and reciprocal absolute temperature on the abscissa. (Note that data for 5 °C and below are from three fibre bundles only.) The twitch tension increases linearly with temperature in the range 5–25 °C but declines at higher temperatures. Tetanic tension increases with increase of temperature, but the increase is less marked between ∼25 and 35 °C. B, tetanic tension data in A are plotted after normalisation to that at 35 °C. The relationship between tetanic tension and reciprocal temperature is approximately sigmoidal with half-maximal tension occurring at 9.5 ± 0.2 °C. The data at temperatures < 5 °C, however, are not well fitted to the curve.

Figure 4. Tension transients induced by T-jump during twitch contractions

A T-jump of ∼4 °C was induced at different times during the course of a twitch, at a steady temperature of 10 °C; a superimposed control twitch (without T-jump) is also shown in A. Records show that a T-jump (arrow) induces an increased tension relaxation. Interestingly, a T-jump also produces a transient increase of tension that is very pronounced when the T-jump is applied on the rising phase of a twitch; it is considerably diminished or absent on the relaxation phase.

Figure 5. Tension responses induced by T-jump on tetanic contractions

A, a temperature jump of ∼4 °C was induced by a laser pulse (indicated by the arrow), during the tension plateau of a tetanic contraction at 10 °C. The T-jump induces a small instantaneous drop in tension (phase 1) followed by a rise to a new steady level. B, the T-jump-induced tension rise is shown on an expanded time scale (horizontal bar is 100 ms). A bi-exponential curve is fitted to the tension transient and the exponential rates for phases 2b and 3 were 37 s⁻¹ and 9 s⁻¹, respectively. C and D, a corresponding pair of records from the same preparation but at 20 °C. The pre-T-jump tetanic tension is ∼1.5 times that at 10 °C, but the tension increment induced by the T-jump is smaller (∼0.6 times that at 10 °C). As a percentage of the pre-T-jump tension, the tension increment after a T-jump is ∼30 % at 10 °C (A) and only 10 % at 20 °C (C). Comparison of B with D shows that the tension rise to the new steady level is faster at 20 °C. The small slow creep in pre-T-jump tension as seen in D was ignored in the analyses.

Figure 6. Characteristics of T-jump-induced tension rise

A, the tension increment per °C of T-jump is plotted on the ordinate as a percentage of the pre-T-jump tetanic tension and the mid-temperature of the T-jump as 10³ K/T on the abscissa (as in Fig. 9B of Goldman et al. 1987). Note that the T-jump-induced tension enhancement decreases markedly as the temperature is increased; it is ∼10 % per °C at 10 °C whereas it is < 3 % per °C at high physiological temperatures (>30 °C). B, data from one experiment, comparing the rates of the tetanic tension rise (above ∼50 % tension level) and of the T-jump-induced tension generation; the faster of the two exponential components (from curve fitting to each trace) is plotted against pre-T-jump temperature for tetanus rise (filled symbols) and against post-T-jump temperature for T-jump tension rise (open symbols). The T-jump-induced force generation (phase 2b) is consistently (∼2.5 times) faster than the tetanic tension rise. The rate (C) and the amplitude (as percentage tension per °C of T-jump, D) of the two exponential components of the T-jump-induced transient (filled circles, phase 2b; squares, phase 3) extracted by curve fitting (as shown in Fig. 5C and D) are plotted on logarithmic ordinates against the post-T-jump reciprocal absolute temperature; lines through the points are the calculated regressions (n = 31; r > 0.8). Note that the rate constants of the two components increase with increase of temperature; for 20/10 °C, Q₁₀ is 2.3 for phase 2b and 3.5 for phase 3. Their amplitudes decrease with increase of temperature.