R. C. Woledge

Temperature change as a probe of muscle crossbridge kinetics: a review and discussion

Following the ideas introduced by Huxley (Huxley 1957, Prog. Biophys. Biophys. Chem. 7, 255–318), it is generally supposed that muscle contraction is produced by temporary links, called crossbridges, between myosin and actin filaments, which form and break in a cyclic process driven by ATP splitting. Here we consider the interaction of the energy in the crossbridge, in its various states, and the force exerted. We discuss experiments in which the mechanical state of the crossbridge is changed by imposed movement and the energetic consequence observed as heat output and the converse experiments in which the energy content is changed by altering temperature and the mechanical consequences are observed. The thermodynamic relationship between the experiments is explained and, at the first sight, the relationship between the results of these two types of experiment appears paradoxical. However, we describe here how both of them can be explained by a model in which mechanical and energetic changes in the crossbridges occur in separate steps in a branching cycle.

Changes in crossbridge and non-crossbridge energetics during moderate fatigue of frog muscle fibres.

1. The effect of sarcomere length (SL) during a fatiguing series of isometric tetani of frog muscle fibres was investigated. Tetani at 2.3 microns SL were more fatiguing than tetani at 3.2 microns SL, in that force declined twice as much as relaxation became much slower. 2. In a second set of experiments the force and heat production were measured during a series of fatiguing tetani. Heat was separated into two components: (a) crossbridge heat which is dependent on filament overlap and interaction, and (b) non‐crossbridge heat which is independent of filament overlap and due to Ca2+ turnover. 3. In a series of fifty tetani, force, crossbridge heat and non‐crossbridge heat each declined by 25‐30% of its initial value. 4. The 25% reduction in non‐crossbridge heat occurred completely during the first few tetani of the fatiguing series while force declined by less than 3%. This may be due to a reduction in Ca2+ binding to parvalbumin and to Ca2+ remaining bound during the remainder of the fatigue series. 5. After the first few tetani of the fatigue series the non‐crossbridge heat hardly changed as force declined by a further 25% of its initial value. Continuing reduction of force with constant Ca2+ turnover indicates a reduction in the Ca2+ sensitivity of the filaments, and/or a reduction in the average force per attached crossbridge. 6. At the start of the fatiguing series, as force declines by about 7.5% there is a much larger decline of crossbridge heat (17%). The reason for this is unknown. Later in the series, force declined more rapidly than heat. This is probably due to a progressive accumulation of inorganic phosphate which acts by depressing force more than it depresses ATP breakdown.

Mechanical and energetic properties of papillary muscle from ACTC E99K transgenic mouse models of hypertrophic cardiomyopathy

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca²⁺ jump in myofibrils. The EC₅₀ for Ca²⁺ activation of force in myofibrils was 0.39 ± 0.33 μmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 μmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca²⁺ transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca²⁺ sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%.

Power output of skinned skeletal muscle fibres from the cheetah ( Acinonyx jubatus )

SUMMARY Muscle samples were taken from the gluteus, semitendinosus and longissimus muscles of a captive cheetah immediately after euthanasia. Fibres were ‘skinned’ to remove all membranes, leaving the contractile filament array intact and functional. Segments of skinned fibres from these cheetah muscles and from rabbit psoas muscle were activated at 20°C by a temperature-jump protocol. Step and ramp length changes were imposed after active stress had developed. The stiffness of the non-contractile ends of the fibres (series elastic component) was measured at two different stress values in each fibre; stiffness was strongly dependent on stress. Using these stiffness values, the speed of shortening of the contractile component was evaluated, and hence the power it was producing. Fibres were analysed for myosin heavy chain content using gel electrophoresis, and identified as either slow (type I) or fast (type II). The power output of cheetah type II fibre segments was 92.5±4.3 W kg−1 (mean ± s.e., 14 fibres) during shortening at relative stress 0.15 (the stress during shortening/isometric stress). For rabbit psoas fibre segments (presumably type IIX) the corresponding value was significantly higher (P<0.001), 119.7±6.2 W kg−1 (mean ± s.e., 7 fibres). These values are our best estimates of the maximum power output under the conditions used here. Thus, the contractile filament power from cheetah was less than that of rabbit when maximally activated at 20°C, and does not account for the superior locomotor performance of the cheetah.

Effects of UCP3 genotype, temperature and muscle type on energy turnover of resting mouse skeletal muscle

Uncoupling protein 3 (UCP3) is a mitochondrial transporter protein which, when over-expressed in mice, is associated with increased metabolic rate, increased feeding and low body weight. This phenotype probably reflects the increased levels of UCP3 partially uncoupling mitochondrial respiration from cellular ATP demands. Consistent with that, mitochondria isolated from muscles of mice that over-express UCP3 are less tightly coupled than those from wild-type mice but the degree of uncoupling is not modulated by likely physiological regulatory factors. To determine whether this also applies to intact muscle fibres, we tested the hypothesis that UCP3 constitutively (i.e. in an unregulated fashion) uncouples mitochondria in muscles from mice that over-expressed human UCP3 (OE mice). The rate of heat production of resting muscles was measured in vitro using bundles of fibres from soleus and extensor digitorum longus muscles of OE, wild-type (WT) and UCP3 knock-out mice. At 20°C, the only significant effect of genotype was that the rate of heat production of OE soleus (3.04u2009±u20090.16xa0mW g−1) was greater than for WT soleus (2.31u2009±u20090.05xa0mW g−1). At physiological temperature (35°C), the rate of heat production was independent of genotype and equal to the expected in vivo rate for skeletal muscles of WT mice. We conclude that at 35°C, the transgenic UCP3 was not constitutively active, but at 20°C in slow-twitch muscle, it was partially activated by unknown factors. The physiological factor(s) that activate mitochondrial uncoupling by UCP3 in vivo was either not present or inactive in resting isolated muscles.

Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l−1 (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P3‐1‐(2 nitrophenyl) ethyl ester of ATP (NPE‐caged ATP), and time‐resolved phosphate (Pi) release was detected with the fluorescent protein MDCC‐PBP, N‐(2[1‐maleimidyl]ethyl)‐7‐diethylamino‐coumarin‐3‐carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of Pi release at standard and high IS values were not significantly different. A five‐step reaction mechanism was used to account for the observed time courses of force and Pi release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and Pi with the contractile proteins varied with IS, thus suggesting that the actin–myosin interactions are largely non‐ionic. Our reaction scheme also fits previous results for intact fibres.

Effect of phosphate and temperature on force exerted by white muscle fibres from dogfish.

Effects of Pi (inorganic phosphate) are relevant to the in vivo function of muscle because Pi is one of the products of ATP hydrolysis by actomyosin and by the sarcoplasmic reticulum Ca2+ pump. We have measured the Pi sensitivity of force produced by permeabilized muscle fibres from dogfish (Scyliorhinus canicula) and rabbit. The activation conditions for dogfish fibres were crucial: fibres activated from the relaxed state at 5, 12, and 20°C were sensitive to Pi, whereas fibres activated from rigor at 12°C were insensitive to Pi in the range 5–25xa0mmolxa0l−1. Rabbit fibres activated from rigor were sensitive to Pi. Pi sensitivity of force produced by dogfish fibres activated from the relaxed state was greater below normal body temperature (12°C for dogfish) in agreement with what is known for other species. The force-temperature relationship for dogfish fibres (intact and permeabilized fibres activated from relaxed) showed that at 12°C, normal body temperature, the force was near to its maximum value.

Skinned fibres produce the same power and force as intact fibre bundles from muscle of wild rabbits

ABSTRACT Skinned fibres have advantages for comparing the muscle properties of different animal species because they can be prepared from a needle biopsy taken under field conditions. However, it is not clear how well the contractile properties of skinned fibres reflect the properties of the muscle fibres in vivo. Here, we compare the mechanical performance of intact fibre bundles and skinned fibres from muscle of the same animals. This is the first such direct comparison. Maximum power and isometric force were measured at 25°C using peroneus longus (PL) and extensor digiti-V (ED-V) muscles from wild rabbits (Oryctolagus cuniculus). More than 90% of the fibres in these muscles are fast-twitch, type 2 fibres. Maximum power was measured in force-clamp experiments. We show that maximum power per volume was the same in intact (121.3±16.1u2005Wu2005l−1, mean±s.e.m.; N=16) and skinned (122.6±4.6u2005Wu2005l−1; N=141) fibres. Maximum relative power (power/FIM Lo, where FIM is maximum isometric force and Lo is standard fibre length) was also similar in intact (0.645±0.037; N=16) and skinned (0.589±0.019; N=141) fibres. Relative power is independent of volume and thus not subject to errors in measurement of volume. Finally, maximum isometric force per cross-sectional area was also found to be the same for intact and skinned fibres (181.9u2005kPa±19.1; N=16; 207.8u2005kPa±4.8; N=141, respectively). These results contrast with previous measurements of performance at lower temperatures where skinned fibres produce much less power than intact fibres from both mammals and non-mammalian species. Summary: Maximum isometric power and force from skinned and intact muscle fibres (wild rabbits, 25°C) match within experimental error, strengthening confidence in use of skinned fibres when intact fibres cannot be obtained.

Rate of actomyosin ATP hydrolysis diminishes during isometric contraction.

The aim of the experiments reported here was to measure the time course of ATP hydrolysis by actomyosin during isometric contraction. It is known from previous studies of energy produced as heat and work that most, but not all of this energy, is due to ATP hydrolysis stoichiometrically coupled to the creatine kinase reaction (Curtin and Woledge, 1979). Furthermore energy is produced at a much higher rate at the start of contraction than later. These facts raise the question of whether the rate of ATP hydrolysis by actomyosin, which is the major source of energy, changes during isometric contraction.

General Discussion Part III

Woledge: I will start this session by showing some diagrams describing energy conversion in a manner similar to the description in Dr Tawada’s presentation (this volume, p. 363). I think these diagrams can help us to understand under what conditions we can expect to see a high efficiency of energy conversion, and therefore to understand why in muscle the efficiency of energy conversion is usually not very high, in contrast to the special situation in Dr Sugi’s experiments (this volume, p. 603) which I shall also refer to.