Cross-bridge Turnover Research Articles

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.

Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils.

Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.

Effects of inorganic phosphate on cross‐bridge kinetics at different activation levels in skinned guinea‐pig smooth muscle.

1. The effects of inorganic phosphate (P(i)) on force, Ca(2+)-force relationship, ATPase activity, maximal shortening velocity (Vmax) and rate of tension development were investigated in chemically skinned preparations of smooth muscle from the guinea-pig taenia coli. 2. In maximally thiophosphorylated fibres, P(i) in the range 1-40 mM inhibited isometric force, with a reduction of 20% at 20 mM P(i). 3. The relative force was similar at all [Ca2+], i.e. the Ca(2+)-force relationship was not affected, when 20 mM P(i) was present. 4. After photolytic release of ATP from caged ATP in maximally thiophosphorylated fibres in the presence of 20 mM P(i), tension rose to a lower level but with a higher rate constant than in the absence of P(i). 5. Inorganic phosphate (20 mM) did not affect the ATP hydrolysis in fibres activated at intermediate [Ca2+] or by maximal thiophosphorylation. 6. Inorganic phosphate (20 mM) decreased force but did not influence Vmax in maximally activated fibres. At lower levels of activation by Ca2+, P(i) increased the Vmax and decreased force slightly without affecting the degree of myosin light chain phosphorylation. 7. We conclude that P(i) influences cross-bridge reactions associated with force generation in smooth muscle. These reactions are not rate limiting for cross-bridge turnover under isotonic or isometric conditions in maximally activated smooth muscle fibres, since P(i) did not influence Vmax or the rate of ATP turnover. 8. Since P(i) increased Vmax in submaximally activated muscles, we propose that, under these conditions, shortening velocity is rate limited by cross-bridge states, reached early after attachment, which impose a mechanical resistance to shortening.

Strain sensitivity and turnover rate of low force cross-bridges in contracting skeletal muscle fibers in the presence of phosphate

Inorganic phosphate (Pi) decreases the isometric tension of skinned skeletal muscle fibers, presumably by increasing the relative fraction of a low force quaternary complex of actin, myosin, ADP, and Pi (A.M.ADP.Pi). At the same time, Pi gives rise to a fast relaxing mechanical component as detected by oscillations at 500 Hz. To characterize the dynamic properties of this A.M.ADP.Pi complex, the effect of Pi on the tension response to stretch was investigated with rabbit psoas fibers. A ramp stretch applied in the presence of 20 mM Pi increased tension more than in the control solution (0 mM Pi) but reduced the fast relaxing component to the control level. Thus, a stretch seems to convert the low force, fast relaxing A.M.ADP.Pi complex to a high force, slow relaxing form. However, the Pi-induced enhancement of the tension response was not observed until the fibers were stretched more than 0.4% of their length, suggesting that a critical cross-bridge extension of approximately 4 nm is required for this conversion. The rate constant of the attachment/detachment of this low force complex was estimated from the velocity dependence of the enhancement. It was approximately 10 s-1, in marked contrast to the A.M.ADP.Pi complex under low salt, relaxed conditions (approximately 10,000 s-1). The enhancement of the tension response was not observed when isometric tension was reduced by lowering free calcium, implying that calcium and Pi affect different steps in the actomyosin ATPase cycle during contraction.

Stretch activation, unloaded shortening velocity, and myosin heavy chain isoforms of rat skeletal muscle fibres.

1. Contractile properties were investigated on single skinned-fibre preparations from rat leg muscles. Following the mechanical measurements, the myosin heavy chain (HC) composition of the same fibre was analysed by gradient gel electrophoresis. 2. Fibres were typed according to their myosin HC isoform composition (HCI, type I; HCIIA, type IIA; HCIID, type IID; HCIIB, type IIB). Many fibres showed the co-existence of two myosin HC isoforms (hybrid fibres). 3. A strong correlation was found between fibre type and time characteristics of stretch-induced delayed force increase (stretch activation) of fully Ca(2+)-activated fibres. 4. The maximal unloaded shortening velocity (Vmax), as measured with the slack test, was lowest in type I fibres. Within the type II group, a continuum of Vmax values was found, with large overlaps of the different fibre types. 5. The results suggest that the kinetics of stretch activation is determined by the myosin HCs whereas unloaded fibre shortening seems to be determined by other myofibrillar proteins in addition to the myosin HCs. Assuming that stretch activation represents certain steps of the cross-bridge turnover under isometric conditions and Vmax reflects cross-bridge detachment under unloaded conditions it can be deduced that different myofibrillar proteins are responsible for different steps within the cross-bridge turnover.

Increase in vertical dimension alters mechanical properties and isometric ATPase activity in guinea pig masseter

To study the changes in mechanical and metabolic properties associated with an increase in the vertical dimension of the face, isometric tension, isometric ATPase activity, unloaded shortening velocity (V max) and the tension transients in response to step stretches in length were measured at constant levels of various Ca 2+ activations in glycerinated masseter muscles (75 to 150 μm in diameter and about 3 mm long) from normal and bite opened (6 mm increase in the vertical dimension, period of 1 week) guinea pigs. The isometric tension increased sigmoidally with an increase in Ca 2+ concentration in both preparations. However, the bite opening shifted the pCa-relative tension relationship in the direction of increasing Ca 2+ required for activation, and the pCa at Km (Ca 2+ concentration required to develop half maximum tension) was 6.40 ± 0.08 in control and 6.27 ± 0.05 in the bite opened preparations. The maximum isometric tension at the saturating Ca 2+ concentration was greater in the bite opened preparation. The ATPase activity changed almost in parallel with the tension (i.e., Ca 2+ concentration) in both preparations. However, the tension cost (ATPase activity/tension) was significantly ( p < 0.05) lower in the bite opened preparation, compared with the control. The V max at the saturating Ca 2+ concentration was lower in the bite opened preparations. The tension transients in response to step stretch in length were characterized by four distinct phases; the first phase of an immediate tension increase coincident with the stretch, the second phase of a rapid quasi-exponential tension reduction, the third phase of a minute tension increase and the fourth phase of a slow tension reduction. The time constant of the second phase (denoted by t 63%), which is believed to reflect kinetics of turnover rate of crossbridges, was 4.7 msec in control and 11.0 msec in the bite opened preparations. These results suggest that the increase in the vertical dimension for 1-week decreases the Ca 2+ sensitivity in the isometric tension development and alters the dynamic mechanical properties of masseter muscle so as to decrease the tension cost by decreasing the cycling rate of crossbridges. The economical ATP consumption in the bite opened preparation may partly contribute to the relief of muscle spasm with an increase in the vertical dimension. (A M J O RTHOD D ENTOFAC O RTHOP 1993;104:484-91.)

Unloaded shortening after a quick release of a contracting, single fibre from crayfish slow muscle.

The time course of shortening at zero load was studied by the slack test method during tetanic contractions in isolated, single, slow muscle fibres of the crayfish. In 28 of 32 shortenings (from 14 different fibres) a biphasic shortening was seen, which consisted of an initial high-velocity phase lasting 3.3-20.8 ms and a following slow-velocity phase lasting for the entire time examined (up to 89.2 ms). Provided that the shortening occurred uniformly along the fibre length, velocity in the initial phase, V1, of the biphasic shortening was 14.4 +/- 3.4 (mean +/- SD, n = 10) microns s-1 per half sarcomere at Lo, the slack length, at 20 degrees C, while that in the second phase, V2, was 7.4 +/- 1.4 microns s-1 per half sarcomere. Lowering temperature decreased both V1 and V2 with Q10 = 1.4 for V1 and 2.0 for V2. Lowering the external Ca concentration from 15 mM, the standard, to 2 mM resulted in a tetanic tension below one-third of that at 15 mM Ca and decreased both V1 (t test; p < 0.01) and V2 (p < 0.001). Prestretching the fibre to 1.5 Lo had no significant effect on V2 (p < 0.3) but increased V1 (p < 0.001). The distance shortened during the initial high-velocity phase, LV1, was 4.0 +/- 1.8% Lo (mean +/- SD, n = 10) at 20 degrees C or about 0.14 microns per half sarcomere on average. LV1 was independent of the tetanic tension level when it was changed by lowering the external Ca concentration or temperature in the same fibre. Prestretching the fibre to 1.5 Lo, at which the sum of the active and the resting tension was lower than Po at Lo in two of three fibres, increased LV1 significantly (p < 0.001). The independency of LV1 from the tension level indicates that the initial high-velocity phase was not from shortening of some inert components in the fibre. One possibility is that the initial high-velocity phase was brought about by an acceleration of shortening by a compressive force, the origin of which has been discussed. The slow-velocity phase seemed to result from the crossbridge turnover with little exogeneous stress on myofilaments. Four different fibres exhibited an unloaded shortening with a constant velocity during the entire time examined (29.9-61.8 ms). This type of shortening had a velocity between the usual V1 and V2 values, suggesting that a compressive force accelerated the shortening during the entire time.

Cross-bridge dynamics in human ventricular myocardium. Regulation of contractility in the failing heart.

To investigate whether altered cross-bridge kinetics contribute to the contractile abnormalities observed in heart failure, we determined the mechanical properties of cardiac muscles from control and myopathic hearts. Muscle fibers from normal (n = 5) and dilated cardiomyopathy (n = 6) hearts were obtained and chemically skinned with saponin. The muscles were then maximally activated at saturating calcium concentrations. Unloaded shortening velocities (V0) were determined in both groups. V0 in control was 0.72 +/- 0.09 Lmax/sec, whereas in myopathic muscles, V0 was 0.41 +/- 0.06 Lmax/sec at 22 degrees C. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. The dynamic stiffness of the muscles was calculated from the ratio of force response amplitude to length oscillation amplitude. At low frequencies (< 0.2 Hz) the stiffness was constant but was larger in myopathic muscles. In the range of 0.2-1 Hz, there was a drop in the magnitude of dynamic stiffness to approximately one quarter of the low-frequency baseline. This range reflects cross-bridge turnover kinetics. In control muscles, the frequency of minimum stiffness was 0.78 +/- 0.06 Hz, whereas it was 0.42 +/- 0.07 Hz in myopathic muscles. At higher frequencies, the dynamic stiffness increased and reached a plateau at 30 Hz. There were no differences in the plateau reached between control and myopathic muscles. Because myopathic hearts have a markedly diminished energy reserve, the slowing of the cross-bridge cycling rate plays an important adaptational role in the observed contractility changes in human heart failure. Although the potential to generate maximal Ca(2+)-activated force is similar in normal and myopathic hearts, alterations in contractile protein composition could explain the diminished cross-bridge cycling rate in failing hearts.

Reduction of myocardial cross-bridge turnover rate in presence of EMD 53998, a novel Ca(2+)-sensitizing agent.

We have studied the effect of EMD 53998 (5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl)-6-me thyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one) on cross-bridge turnover rate at varying Ca2+ concentrations. Cross-bridge cycling rate was estimated both by adenosine triphosphatase measurements and determination of mechanical characteristics of constantly activated fibres, which is assumed to reflect cross-bridge kinetics. The results indicate that the turnover rate of myocardial cross-bridges was reduced in the presence of EMD 53998 at low Ca2+ concentrations (pCa greater than or equal to 6.25), but not at higher Ca2+ concentrations (pCa less than or equal to 5.85).

Influence of myosin isoforms on ATPase activity and crossbridge turnover kinetics in skinned rat cardiac muscle : J.F.Y. Hoh, G.H. Rossmanith and A.M. Hamilton. Department of Physiology, University of Sydney, NSW, 2006 and School of Mathematics, Physics, Computing and Electronics, Macquarie University, NSW, 2109, Australia

Influence of myosin isoforms on ATPase activity and crossbridge turnover kinetics in skinned rat cardiac muscle : J.F.Y. Hoh, G.H. Rossmanith and A.M. Hamilton. Department of Physiology, University of Sydney, NSW, 2006 and School of Mathematics, Physics, Computing and Electronics, Macquarie University, NSW, 2109, Australia

The role of calcium in the energetics of contracting skeletal muscle.

In its second messenger role in skeletal muscle, calcium coordinates the function of muscle (contractile activity) with its overall energetics, thereby controlling the provision of ATP in a time of need. Not only is ATP required for crossbridge turnover in the myofibrils, but it is also needed for the maintenance of ion pumps, nuclear activity, and so forth. When oxygen is limiting, the sustained contractions of both fast and slow muscle (after the immediate burst of activity) is primarily supported by glycogenolysis and the glycolytic pathway (anaerobic). Calcium is important to this process, and the compartmentation of the glycogen particle and some of the enzymes associated with the glycolytic pathway in the terminal cisternae of the sarcoplasmic reticulum ensures that the provision of glucose-6-phosphate to the glycolytic pathway for the generation of the needed ATP proceeds rapidly. The activation of phosphorylase and glycogenolysis by calcium-troponin-C is another example of the tight control of cellular energetics deemed possible by compartmentation within the cell. The regulation by calcium, therefore, is only dependent on the diffusion of calcium rather than diffusion of substrate. When oxygen is not limiting (i.e. when a new steady-state is reached), the aerobic metabolism of pyruvate and fatty acids may be regulated in part by calcium at least in slow skeletal muscle. Oxidative phosphorylation, where ADP is phosphorylated to ATP, is though to be controlled by the concentration of ADP in skeletal muscle. However, because of the obvious compartmentation of the mitochondria within the slow muscle fibre and the higher free calcium required for peak force development (5 mumol/L), the kinetics are theoretically favourable for the calcium cycle in slow muscle mitochondria to play an important role in the regulation of aerobic substrate oxidation, as it does in the heart. Although this hypothesis is attractive based on the available data, the direct demonstration of a major role for calcium as a regulator of substrate oxidation in slow muscle awaits experimentation.

Parallel inhibition of active force and relaxed fiber stiffness in skeletal muscle by caldesmon: implications for the pathway to force generation.

In recent hypotheses on muscle contraction, myosin cross-bridges cycle between two types of actin-bound configuration. These two configurations differ greatly in the stability of their actin-myosin complexes ("weak-binding" vs. "strong-binding"), and force generation or movement is the result of structural changes associated with the transition from the weak-binding (preforce generating) configuration to strong-binding (force producing) configuration [cf. Eisenberg, E. & Hill, T. L. (1985) Science 227, 999-1006]. Specifically, in this concept, the main force-generating states are only accessible after initial cross-bridge attachment in a weak-binding configuration. It has been shown that strong and weak cross-bridge attachment can occur in muscle fibers [Brenner, B., Schoenberg, M., Chalovich, J. M., Greene, L. E. & Eisenberg, E. (1982) Proc. Natl. Acad. Sci. USA 79, 7288-7291]. However, there has been no evidence that attachment in the weak-binding states represents an essential step leading to force generation. It is shown here that caldesmon can be used to selectively inhibit attachment of weak-binding cross-bridges in skeletal muscle. Such inhibition causes a parallel decrease in active force, while the kinetics of cross-bridge turnover are unchanged by this procedure. This suggests that (i) cross-bridge attachment in the weak-binding states is specific and (ii) force production can only occur after cross-bridges have first attached to actin in a weakly bound, nonforce-generating configuration.

Modulation of crossbridge kinetics by myosin isoenzymes in skinned human heart fibers.

Skinned fibers from the normal human heart with the beta-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2+ sensitivity than skinned fibers with the alpha-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (g(app)), myosin heavy chain isoenzymes seem to have different crossbridge turnover kinetics. Modulation of g(app) by myosin heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. g(app) was independent of Ca2+.

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres

The effect of Ca2+ activation on the apparent rate constant governing the dissociation of force generating myosin crossbridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21 +/- 1 degree C. Simultaneous measurements of Ca2(+)-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnCDANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnCDANZ. The Ca2+ activation of isometric force occurred at approximately two times higher Ca2+ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnCDANZ fluorescence and ATPase activity occurred over approximately the same Ca2+ concentration range at substantially lower concentrations of Ca2+ than did force, this data suggests that the TnCDANZ fluorescence is associated with the Ca2+ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constant gapp for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure of gapp shows approximately a fivefold decrease during Ca2+ activation of isometric force. This change in gapp is responsible for separation of the Ca2+ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change in gapp is reduced and consequently the difference in Ca2+ sensitivity between normalized steady state ATPase and force is also reduced.

Myocardial force production and energy turnover in anoxia

ATP turnover of isolated rabbit papillary muscles, contracting isometrically at 20 degrees C, was determined in oxygen and during 40 min of exposure to nitrogen (anoxia). Stimulus frequency was 0.2 hertz (Hz) in oxygen and 0.2 or 1.0 Hz in nitrogen. In oxygen, ATP turnover was determined from oxygen consumption using a P/O2 ratio of 6.3. The time-dependent rate of ATP turnover in nitrogen was found from the production of lactate, and the changes in adenine nucleotides and phosphocreatine, measured in rapidly frozen preparations at different time-points during the anoxic period. A P/lactate ratio of 1.5 was used. In muscles stimulated at 0.2 Hz, twitch force dropped during the anoxic period to 33% while force production of muscles stimulated at 1.0 Hz stopped completely. However, in the latter muscles, resting force rose to 19% of the twitch force in oxygen. The rate of ATP hydrolysis in anoxia depended strongly on stimulus frequency, indicating that it is not solely determined by the glycolytic capacity. In the 0.2 Hz-stimulated muscles the decrease in energy turnover occurred in parallel with the drop in force. However, the rise in resting force in muscles stimulated at 1.0 Hz occurred when ATP turnover was close to zero. It was concluded that anoxia hardly affects the energy required for twitch force production, but that the rise of resting force measured when twitch force had disappeared occurred when the rates of cross-bridge cycling and calcium turnover were very low.

Influence of temperature on mechanics and energetics of muscle contraction

Results gleaned from use of temperature as a probe to study skeletal muscle performance and mechanisms of activation and contraction are reviewed. Steady-state and non-steady-state responses to changes in temperature are considered. Temperature sensitivities, Q10 values, of mechanical and energetic parameters range from nearly 1 to greater than 5 in frog skeletal muscle. Factors that are less temperature sensitive (Q10 less than or equal to 1.5) are peak tetanic force, instantaneous stiffness, curvature of force-velocity relation, magnitude of labile heat, and mechanical efficiency. Rates with intermediate temperature sensitivities (Q10 greater than 2 but less than 3) include rate of isometric force development, maximum shortening velocity, and relaxation from a brief tetanus. Rates with high temperature sensitivities (Q10 greater than 3) include cross-bridge turnover during an isometric tetanus, isometric economy, maximum power output, Ca2+ sequestration by sarcoplasmic reticulum, relaxation from a prolonged tetanus, and recovery metabolism. The observation that the Q10 for relaxation rate depends on tetanic duration can be explained in terms of the possible role of parvalbumin as a soluble relaxing factor.

Electrically evoked isometric and isokinetic properties of the triceps surae in young male subjects.

The relationship between electrically evoked isometric and isokinetic properties of the triceps surae have been studied in 11 healthy male subjects. The results showed that the time to peak tension (TPT) and half relaxation time (1/2 RT) of the maximal twitch were 110 +/- 11 ms and 82 +/- 11 ms respectively, and the peak rates of rise of contraction (delta P50, delta P200) and relaxation (delta PR50, delta PR200) at 50 and 200 Hz were 0.36 +/- 0.07, 0.48 +/- 0.08 and 1.27 +/- 0.33, 1.25 +/- 0.27% Po ms-1 respectively. The decline in force during a fatigue test was significantly (P less than 0.02) associated with the decrease in maximal relaxation rate (r = 0.79). The TPT was significantly (P less than 0.05) and inversely related to delta P50 and delta P200. The mean angle specific torque-velocity relationship for the 11 subjects was adequately described by the empirical exponential equation of the form: V = 16.5 (e-P/30.8-e-84.3/30.8) where V = velocity (rads s-1) and P = torque (Nm). The only significant association found between the isometric and isokinetic properties of the muscle was between delta PR200 and the torque expressed at a given velocity of 4 rads s-1. This lack of association between the two variables is difficult to explain with certainty but it is suggested that it may be due to the differential effects of Ca2+ release and uptake and cross-bridge turnover rate in the two situations.

Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction.

The effect of Ca2+ upon the rate constant of force redevelopment following a period of isotonic shortening with immediate restretch to the starting sarcomere length was studied in rabbit psoas fibers at 5 degrees C. Control experiments support the assumption that the rate constant of force redevelopment represents isometric cross-bridge turnover kinetics (fapp + gapp), where fapp and gapp are the rate constants characterizing the transitions from the non-force-generating states to the force-generating states and back to the non-force-generating states, respectively. Parallel measurements of the rate constant of force redevelopment and of force, stiffness, and fiber ATPase during isometric contraction allow the effect of Ca2+ upon fapp and gapp to be determined. Analysis reveals that Ca2+ has a marked effect upon fapp, while gapp remains approximately unchanged. Furthermore, in the range above 25-30% of maximum Ca2+ activation, regulation of force, stiffness, and ATPase is mediated through changes in fapp. Below this range, however, it cannot be ruled out that, in addition, cross-bridges are also switched in and out of the turnover process ("recruitment"). As a consequence of regulation through turnover kinetics, both Ca2+ sensitivity and the slope of force-pCa (-log[Ca2+]) relations are shown to be affected by the ratio fapp/gapp, which may represent an important mechanism of modulation of contractile function in addition to modulation through changes within the regulatory protein system.

Epinephrine infusion enhances muscle glycogenolysis during prolonged electrical stimulation.

To determine the effects of epinephrine (EPI) infusion on muscle glycogenolysis and force production, the quadriceps muscles of both legs in six subjects were intermittently stimulated for 30 min. Contractions lasted 1.6 s (20 Hz) and were separated by 1.6 s of rest. EPI was infused (approximately 0.14 micrograms.kg body wt-1.min-1) in one leg during the last 15 min and the vastus lateralis was biopsied at rest (control leg only) and after 15, 18 (EPI leg only), and 30 min of stimulation. EPI infusion doubled the mole fraction of phosphorylase a (22.5 +/- 4.1 to 44.8 +/- 9.0%) and glycogenolysis (2.16 +/- 0.72 to 5.45 +/- 0.81 mmol glucosyl U.kg dry muscle wt-1.min-1) during stimulation. Muscle glucose 6-phosphate increased from 3.04 +/- 0.17 to 6.43 +/- 0.20 mmol/kg dry muscle wt, and lactate increased from 25.8 +/- 4.4 to 34.3 +/- 4.6 mmol/kg after 3 min of EPI infusion. Isometric force production was unaltered by EPI infusion. These results demonstrate a strong glycogenolytic effect of EPI infusion during prolonged electrical stimulation and suggest that the extra pyruvate formed was converted mainly to lactate. Exclusive anaerobic metabolism of the extra substrate would provide only a 10% increase in total ATP production, possibly accounting for the lack of improvement in force production. We suggest that the decrease in force production during prolonged electrical stimulation is related to decreased excitation of the contractile mechanism rather than inhibition of cross-bridge turnover caused by a shortage of energy or accumulation of hyproducts.

ATP utilization and force during intermittent and continuous muscle contractions

Energy utilization and force generation under anaerobic conditions were studied in electrically stimulated quadriceps femoris muscle of four volunteers. To investigate the effects of intermittent vs. continuous stimulation one leg was stimulated intermittently and the other continuously during 50 s. The same initial force was produced, and biopsy samples were obtained before the stimulation and after 10, 20, and 50 s and analyzed for energy-rich phosphagens, glycolytic intermediates, and phosphorylase. The ATP utilization and glycolysis were greater during intermittent contraction, but glycogenolysis was equal. ATP content decreased to lower values after intermittent contraction (16.4 compared with 19.6 mmol/kg dry muscle after continuous contraction). Force generation was well preserved during continuous contraction but successively decreased after 20 s of intermittent stimulation down to 50% of initial at end of work. The energy cost per unit work was greater during intermittent stimulation and increased with contraction time, whereas it decreased with time during continuous stimulation. The decrease in force generation in intermittent exercise is suggested to be due to the higher energy cost for contraction resulting in greater changes in the intracellular environment with lower ATP and increased H+ and Pi. These changes would decrease both activation of the contractile system and the cross-bridge turnover rate resulting from activation.