Advances in understanding the energetics of muscle contraction.

Muscle energetics encompasses the relationships between mechanical performance and the biochemical and thermal changes that occur during muscular activity. The biochemical reactions that underpin contraction are described and the way in which these are manifest in experimental recordings, as initial and recovery heat, is illustrated. Energy use during contraction can be partitioned into that related to cross-bridge force generation and that associated with activation by Ca2+. Activation processes account for 25-45% of ATP turnover in an isometric contraction, varying amongst muscles. Muscle energy use during contraction depends on the nature of the contraction. When shortening muscles produce less force than when contracting isometrically but use energy at a greater rate. These characteristics reflect more rapid cross-bridge cycling when shortening. When lengthening, muscles produce more force than in an isometric contraction but use energy at a lower rate. In that case, cross-bridges cycle but via a pathway in which ATP splitting is not completed. Shortening muscles convert part of the free energy available from ATP hydrolysis into work with the remainder appearing as heat. In the most efficient muscle studied, that of a tortoise, cross-bridges convert a maximum of 47% of the available energy into work. In most other muscles, only 20-30% of the free energy from ATP hydrolysis is converted into work.

The energetics of muscle contractions resembling in vivo performance.

Muscle energetics has expanded into the study of contractions that resemble in vivo muscle activity. A summary is provided of experiments of this type and what they have added to our understanding of muscle function and effects of compliant tendons, as well as the new questions raised about the efficiency of energy transduction in muscle.

Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula.

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (C(f)) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa(−1). This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa(−1). The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm(−1)) is smaller than that of frog (2.78 ± 0.30 pN nm(−1)). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.

Is the efficiency of mammalian (mouse) skeletal muscle temperature dependent?

Myosin crossbridges in muscle convert chemical energy into mechanical energy. Reported values for crossbridge efficiency in human muscles are high compared to values measured in vitro using muscles of other mammalian species. Most in vitro muscle experiments have been performed at temperatures lower than mammalian physiological temperature, raising the possibility that human efficiency values are higher than those of isolated preparations because efficiency is temperature dependent. The aim of this study was to determine the effect of temperature on the efficiency of isolated mammalian (mouse) muscle. Measurements were made of the power output and heat production of bundles of muscle fibres from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles during isovelocity shortening. Mechanical efficiency was defined as the ratio of power output to rate of enthalpy output, where rate of enthalpy output was the sum of the power output and rate of heat output. Experiments were performed at 20, 25 and 30◦C. Maximum efficiency of EDL muscles was independent of temperature; the highest value was 0.31}0.01 (n =5) at 30◦C. Maximum efficiency of soleus preparations was slightly but significantly higher at 25 and 30◦C than at 20◦C; the maximum mean value was 0.48±0.02 (n =7) at 25◦C. It was concluded that maximum mechanical efficiency of isolated mouse muscle was little affected by temperature between 20 and 30◦C and that it is unlikely that differences in temperature account for the relatively high efficiency of human muscle in vivo compared to isolated mammalian muscles.

Sustained performance by red and white muscle fibres from the dogfish Scyliorhinus canicula.

The mechanical performance of red and white muscle fibres from dogfish was compared during a long series of contractions with sinusoidal movement or under isometric conditions at 12 degrees C (normal in vivo temperature). Power output was measured during sinusoidal movement at 0.75 Hz and peak-to-peak amplitude about 12% L(0). Tetanus duty cycle was 33% (0.44 s) at phase -8% (first stimulus at 0.107 s before shortening started). Initially, the red fibres produced only about one third as much power as the white fibres, 6.57+/-0.63 W kg(-1) wet mass (mean +/- s.e.m.) and 18.3+/-2.3, respectively. Red fibres were better at sustaining power output; it declined rapidly to about 60% of its initial value and then remained relatively steady for up to 450 cycles of movement. Force during shortening declined, but force during stretch did not increase: force always relaxed to a low value before stretch started. By contrast, net power output by white fibres declined rapidly to zero within about 50 cycles. Two changes contributed: decline in force during shortening and an increase in force during stretch because relaxation became progressively less complete during the series of contractions. In isometric series (0.44 s stimulation every 1.33 s, cycle frequency 0.75 Hz), red and white fibres sustained peak isometric force similarly; in the 50th cycle force was 59+/-3% and 56+/-4% of initial values. The time required for force to relax to 10% of its maximum value decreased during the series for red fibres and increased for white 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 Ca(2+) 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 degrees C were sensitive to Pi, whereas fibres activated from rigor at 12 degrees C were insensitive to Pi in the range 5-25 mmol l(-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 degrees 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 degrees C, normal body temperature, the force was near to its maximum value.

Inferring crossbridge properties from skeletal muscle energetics.

Work is generated in muscle by myosin crossbridges during their interaction with the actin filament. The energy from which the work is produced is the free energy change of ATP hydrolysis and efficiency quantifies the fraction of the energy supplied that is converted into work. The purpose of this review is to compare the efficiency of frog skeletal muscle determined from measurements of work output and either heat production or chemical breakdown with the work produced per crossbridge cycle predicted on the basis of the mechanical responses of contracting muscle to rapid length perturbations. We review the literature to establish the likely maximum crossbridge efficiency for frog skeletal muscle (0.4) and, using this value, calculate the maximum work a crossbridge can perform in a single attachment to actin (33 x 10(-21) J). To see whether this amount of work is consistent with our understanding of crossbridge mechanics, we examine measurements of the force responses of frog muscle to fast length perturbations and, taking account of filament compliance, determine the crossbridge force-extension relationship and the velocity dependences of the fraction of crossbridges attached and average crossbridge strain. These data are used in combination with a Huxley-Simmons-type model of the thermodynamics of the attached crossbridge to determine whether this type of model can adequately account for the observed muscle efficiency. Although it is apparent that there are still deficiencies in our understanding of how to accurately model some aspects of ensemble crossbridge behaviour, this comparison shows that crossbridge energetics are consistent with known crossbridge properties.

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.

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 degrees C, the only significant effect of genotype was that the rate of heat production of OE soleus (3.04+/-0.16 mW g(-1)) was greater than for WT soleus (2.31+/-0.05 mW g(-1)). At physiological temperature (35 degrees 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 degrees C, the transgenic UCP3 was not constitutively active, but at 20 degrees 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.

The energetic cost of activation in mouse fast-twitch muscle is the same whether measured using reduced filament overlap or N-benzyl-p-toluenesulphonamide.

AIM: Force generation and transmembrane ion pumping account for the majority of energy expended by contracting skeletal muscles. Energy turnover for ion pumping, activation energy turnover (E(A)), can be determined by measuring the energy turnover when force generation has been inhibited. Most measurements show that activation accounts for 25-40% of isometric energy turnover. It was recently reported that when force generation in mouse fast-twitch muscle was inhibited using N-benzyl-p-toluenesulphonamide (BTS), activation accounted for as much as 80% of total energy turnover during submaximal contractions. The purpose of this study was to compare E(A) measured by inhibiting force generation by: (1) the conventional method of reducing contractile filament overlap; and (2) pharmacological inhibition using BTS. METHODS: Experiments were performed in vitro using bundles of fibres from mouse fast-twitch extensor digitorum longus (EDL) muscle. Energy turnover was quantified by measuring the heat produced during 1-s maximal and submaximal tetanic contractions at 20 and 30 degrees C. RESULTS: E(A) measured using reduced filament overlap was 0.36 +/- 0.04 (n = 8) at 20 degrees C and 0.31 +/- 0.05 (n = 6) at 30 degrees C. The corresponding values measured using BTS in maximal contractions were 0.46 +/- 0.06 and 0.38 +/- 0.06 (n = 6 in both cases). There were no significant differences among these values. E(A) was also no different when measured using BTS in submaximal contractions. CONCLUSION: Activation energy turnover is the same whether measured using BTS or reduced filament overlap and accounts for slightly more than one-third of isometric energy turnover in mouse EDL muscle.

Energy turnover for Ca2+ cycling in skeletal muscle.

The majority of energy consumed by contracting muscle can be accounted for by two ATP-dependent processes, cross-bridge cycling and Ca(2+) cycling. The energy for Ca(2+) cycling is necessary for contraction but is an overhead cost, energy that cannot be converted into mechanical work. Measurement of the energy used for Ca(2+) cycling also provides a means of determining the total Ca(2+) released from the sarcoplasmic reticulum into the sarcoplasm during a contraction. To make such a measurement requires a method to selectively inhibit cross-bridge cycling without altering Ca(2+) cycling. In this review, we provide a critical analysis of the methods used to partition skeletal muscle energy consumption between cross-bridge and non-cross-bridge processes and present a summary of data for a wide range of skeletal muscles. It is striking that the cost of Ca(2+) cycling is almost the same, 30-40% of the total cost of isometric contraction, for most muscles studied despite differences in muscle contractile properties, experimental conditions, techniques used to measure energy cost and to partition energy use and in absolute rates of energy use. This fraction increases with temperature for amphibian or fish muscle. Fewer data are available for mammalian muscle but most values are similar to those for amphibian muscle. For mammalian muscles there are no obvious effects of animal size, muscle fibre type or temperature.

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 degrees 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 P(3)-1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (P(i)) 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 P(i) 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 P(i) release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and P(i) 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.

Actomyosin energy turnover declines while force remains constant during isometric muscle contraction.

Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish (Scyliorhinus canicula) at 12 degrees C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (Pi) release by permeabilized fibres was recorded using the fluorescent protein MDCC-PBP, N-(2-[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3 carboxamide phosphate binding protein. The steady-state ADP release rate was measured using a linked enzyme assay. The rates decreased five-fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 +/-s.e.m. 28 microW mg(-1) (n = 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the Pi release rate decreased from 5.00 +/- 0.39 mmol l(-1) s(-1) at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady-state ADP release rate at 15 s was 21% of the Pi rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and Pi release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing Pi concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres.

Energy storage during stretch of active single fibres from frog skeletal muscle.

Heat production and force were measured during tetani of single muscle fibres from anterior tibialis of frog. During stimulation fibres were either kept under isometric conditions, or were stretched or allowed to shorten (at constant velocity) after isometric force had reached its plateau value. The energy change was evaluated as the sum of heat and work (work = integral of force with respect to length change). Net energy absorption occurred during stretch at velocities greater than about 0.35 L0 s-1 (L0 is fibre length at resting sarcomere length 2.10 microm). Heat produced by 1 mm segments of the fibre was measured simultaneously and separately; energy absorption is not an artefact due to patchy heat production. The maximum energy absorption, 0.092 +/- 0.002 P0L0 (mean +/- S.E.M., n = 8; where P0 is isometric force at L0), occurred during the fastest stretches (1.64 L0 s-1) and amounted to more than half of the work done on the fibre. Energy absorption occurred in two phases. The amount in the first phase, 0.027 +/- 0.003 P0L0 (n = 32), was independent of velocity beyond 0.18 L0 s-1. The quantity absorbed in the second phase increased with velocity and did not reach a limiting value in the range of velocities used. After stretch, energy was produced in excess of the isometric rate, probably from dissipation of the stored energy. About 34 % (0.031 P0L0/0.092 P0L0) of the maximum absorbed energy could be stored elastically (in crossbridges, tendons, thick, thin and titin filaments) and by redistribution of crossbridge states. The remaining energy could have been stored in stretching transverse, elastic connections between myofibrils.

Excess recovery heat production by isolated muscles from mice overexpressing uncoupling protein-3.

Contractile and energetic performance of bundles of muscle fibres from the soleus of mice overexpressing uncoupling protein 3 (UCP-3tg) were compared with the performance of bundles from wild-type mice. Force and heat production were measured during a series of thirty 0.2 s isometric tetani at L(o), the length optimal for force. UCP-3tg fibres were as strong as the wild-type and maintained force in the series equally well; in the first tetanus force was 116.9 +/- 15.1 and 133.3 +/- 19.7 mN x mm(-2) respectively (all values means +/- S.E.M., n = 6 for UCP-3tg and n = 5 for wild-type). Heat production was partitioned into initial heat (due to contractile ATPases and the creatine kinase reaction) and recovery heat (due to other ATP-supplying processes) and expressed relative to the first cycle total heat. Initial heat production was similar for the UCP-3tg and wild-type fibres, decreasing during the series from 0.799 +/- 0.052 to 0.661 +/- 0.061 relative units (UCP-3tg), and from 0.806 +/- 0.024 to 0.729 +/- 0.039 relative units (wild-type). In both types the recovery heat was small at the start of the series and increased as the series progressed. At the end of the series, recovery heat production by UCP-3tg fibres, 1.575 +/- 0.246 relative units, was twice that of the wild-type fibres, 0.729 +/- 0.072 relative units. The extra recovery heat represents inefficient recovery in UCP-3tg fibres. This is the first direct evidence of enhanced energy dissipation as heat when UCP-3tg is overexpressed.

Isometric and isovelocity contractile performance of red muscle fibres from the dogfish Scyliorhinus canicula.

Maximum isometric tetanic force produced by bundles of red muscle fibres from dogfish, Scyliorhinus canicula (L.), was 142.4+/-10.3 kN m(-2) (N=35 fibre bundles); this was significantly less than that produced by white fibres 289.2+/-8.4 kN m(-2) (N=25 fibre bundles) (means +/- S.E.M.). Part, but not all, of the difference is due to mitochondrial content. The maximum unloaded shortening velocity, 1.693+/-0.108 L(0) s(-1) (N=6 fibre bundles), was measured by the slack-test method. L(0) is the length giving maximum isometric force. The force/velocity relationship was investigated using a step-and-ramp protocol in seven red fibre bundles. The following equation was fitted to the data: [(P/P(0))+(a/P(0))](V+b)=[(P(0)(*)/P(0))+(a/P(0))]b, where P is force during shortening at velocity V, P(0) is the isometric force before shortening, and a, b and P(0)(*) are fitted constants. The fitted values were P(0)(*)/P(0)=1.228+/-0.053, V(max)=1.814+/-0.071 L(0) s(-1), a/P(0)=0.269+/-0.024 and b=0.404+/-0.041 L(0) s(-1) (N=7 for all values). The maximum power was 0.107+/-0.005P(0)V(max) and was produced during shortening at 0.297+/-0.012V(max). Compared with white fibres from dogfish, the red fibres have a lower P(0) (49%) and V(max) (48%), but the shapes of the force/velocity curves are similar. Thus, the white and red fibres have equal capacities to produce power within the limits set by the isometric force and maximum velocity of shortening of each fibre type. A step shortening of 0.050+/-0.003L(0) (N=7) reduced the maximum isometric force in the red fibres' series elasticity to zero. The series elasticity includes all elastic structures acting in series with the attached cross-bridges. Three red fibre bundles were stretched at a constant velocity, and force (measured when length reached L(0)) was 1.519+/-0.032P(0). In the range of velocities used here, -0.28 to -0.63V(max), force varied little with the velocity.

Mechanical properties of red and white swimming muscles as a function of the position along the body of the eel Anguilla anguilla.

The way in which muscles power steady swimming depends on a number of factors, including fibre type and recruitment, muscle strain, stimulation pattern and intensity, and the intrinsic mechanical properties of the muscle fibres. For a number of undulatory swimming fish species, in vivo studies have shown that muscles at different positions along the body are stimulated during different phases of the strain cycle. Moreover, some intrinsic contractile properties of the muscles have been found to vary according to their position along the body. We report the first results on the mechanical properties of the red and white muscles of an anguilliform swimmer, Anguilla anguilla. Small preparations (0.147-1.335 mg dry mass) were dissected from positions at fractions of 0.2, 0.4, 0.6 and 0.8 of total body length (BL). We determined the time to 50% and 100% peak force and from the last stimulus to 50% relaxation for isometric contractions; we measured the sarcomere lengths that coincided with in situ resting length. None of these quantities varied significantly with the longitudinal position from which the fibres were taken. We also measured power and work output during contractions under conditions approximating those used in vivo (cycle frequency, 1Hz; strain amplitude, +/- 10%L(0), where L(0) is the length giving maximum isometric force). During these experiments, work output was affected by stimulation phase, but did not depend on the longitudinal position in the body from which the muscles were taken. Our results indicate that red and white eel muscles have uniform properties along the body. In this respect, they differ from the muscle of most non-anguilliforms, in which muscle kinetics varies in a systematic way along the body. Uniform properties may be beneficial for anguilliform swimmers, in which the amplitude of the travelling wave can be pronounced over the entire body length.