Increased Velocity Of Shortening Research Articles

Active shortening protects against stretch-induced force deficits in human skeletal muscle.

Skeletal muscle contraction results from molecular interactions of myosin "crossbridges" with adjacent actin filament binding sites. The binding of myosin to actin can be "weak" or "strong," and only strong binding states contribute to force production. During active shortening, the number of strongly bound crossbridges declines with increasing shortening velocity. Forcibly stretching a muscle that is actively shortening at high velocity results in no apparent negative consequences, whereas stretch of an isometrically (fixed-length) contracting muscle causes ultrastructural damage and a decline in force-generating capability. Our working hypothesis is that stretch-induced damage is uniquely attributable to the population of crossbridges that are strongly bound. We tested the hypothesis that stretch-induced force deficits decline as the prevailing shortening velocity is increased. Experiments were performed on permeabilized segments of individual skeletal muscle fibers obtained from human subjects. Fibers were maximally activated and allowed either to generate maximum isometric force (Fo), or to shorten at velocities that resulted in force maintenance of ≈50% Fo or ≈2% Fo For each test condition, a rapid stretch equivalent to 0.1 × optimal fiber length was applied. Relative to prestretch Fo, force deficits resulting from stretches applied during force maintenance of 100, ≈50, and ≈2% Fo were 23.2 ± 8.6, 7.8 ± 4.2, and 0.3 ± 3.3%, respectively (means ± SD, n = 20). We conclude that stretch-induced damage declines with increasing shortening velocity, consistent with the working hypothesis that the fraction of strongly bound crossbridges is a causative factor in the susceptibility of skeletal muscle to stretch-induced damage.NEW & NOTEWORTHY Force deficits caused by stretch of contracting muscle are most severe when the stretch is applied during an isometric contraction, but prevented if the muscle is shortening at high velocity when the stretch occurs. This study indicates that velocity-controlled modulation of the number of strongly bound crossbridges is the basis for the observed relationship between stretch-induced muscle damage and prevailing shortening velocity.

Abstract 068: Age-associated, Vinculin-mediated Remodeling of the Intercalated Disc Results in Localized Myocardial Stiffening but Preserved Mechanical Function

Molecular and ultrastructural remodeling of the intercalated disc occurs with advanced age and onset of cardiomyopathy, yet the precise effect of this remodeling on mechanical function remains unclear. We employed recently-developed biophysical techniques to measure passive and active cardiac mechanics to investigate how intercalated disc remodeling affects structure and mechanical function with age. Nanoindentation of intact, living Drosophila heart reveals that juvenile myocytes are mechanically homogeneous; however, aging induces mechanical heterogeneity, indicated by increased transverse passive stiffness proximal to the intercalated disc (ID). Localized stiffening up to a 111% increase correlates with an 18% age-related decline in diastolic dimension (DD) as well as increased fascia adherens expression. On the other hand, aged specimens with stiffened IDs were found to have preserved systolic function, assessed by measuring shortening velocity under acute hemodynamic load, as compared to their juvenile counterparts. Aged specimens with no change in ID stiffness experienced reduced shortening velocities at all loads. Cardiac-specific vinculin overexpression stiffened IDs and increased shortening velocity and fractional shortening at all hemodynamic loads. Cardiac specific, RNAi-mediated knockdown of vinculin softened the IDs of intact hearts and reduced fractional shortening at all loads. These data indicate that increased expression of vinculin at the ID results in a stiffer cortical cytoskeleton which acts as a more stable anchor against which the myocytes can shorten. These findings have potential implications for geriatric patients with diastolic heart failure, in whom increased vinculin expression at the intercalated disc, concomitant with increased diastolic stiffness but preserved ejection fraction, has been observed.

Accumulating Evidence for Increased Velocity of Airway Smooth Muscle Shortening in Asthmatic Airway Hyperresponsiveness

It remains unclear whether airway smooth muscle (ASM) mechanics is altered in asthma. While efforts have originally focussed on contractile force, some evidence points to an increased velocity of shortening. A greater rate of airway renarrowing after a deep inspiration has been reported in asthmatics compared to controls, which could result from a shortening velocity increase. In addition, we have recently shown in rats that increased shortening velocity correlates with increased muscle shortening, without increasing muscle force. Nonetheless, establishing whether or not asthmatic ASM shortens faster than that of normal subjects remains problematic. Endobronchial biopsies provide excellent tissue samples because the patients are well characterized, but the size of the samples allows only cell level experiments. Whole human lungs from transplant programs suffer primarily from poor patient characterization, leading to high variability. ASM from several animal models of asthma has shown increased shortening velocity, but it is unclear whether this is representative of human asthma. Several candidates have been suggested as responsible for increased shortening velocity in asthma, such as alterations in contractile protein expression or changes in the contractile apparatus structure. There is no doubt that more remains to be learned about the role of shortening velocity in asthma.

Counterpoint: Alterations in airway smooth muscle phenotype do not cause airway hyperresponsiveness in asthma

We have been assigned the task of showing that “alterations in airway smooth muscle phenotype DO NOT cause the airways hyperresponsiveness of asthma.” In the following paragraphs we will briefly review the overwhelming lack of evidence that any fundamental change in the intrinsic properties of

Force Transients during Shortening of Activated Skeletal Muscle Fibers

During a ramp shortening, the force response of an active muscle shows an early change in slope (P1 transition) followed by a gradual change in slope (P2 transition), that happen at given sarcomere length (SL) extension (SL1 and SL2, respectively). In this study, we investigated the mechanisms of forces produced during an imposed shortening to active muscles, by altering either the number of crossbridges attached to actin or the crossbridge state prior to shortening. Three sets of experiments were performed with permeabilized psoas muscle fibers: (i) fibers were activated and shortened by 10% Lo, at 1.0 Lo•s−1 in different pCa2+ (4.5, 5.0, 5.5, 6.0), (ii) fibers were activated and shortened by 10% Lo at 1.0 Lo•s−1 in pCa2+ 4.5 containing either 5µM of blebbistatin (+/-) or its inactive isomer (+/+), or (iii) fibers were activated then shotened by 10% at speeds ranging from 0.125 to 2.0 Lo•s−1 in pCa2+ 4.5 or pCa2+ 6.0 and 5µM blebbistatin (+/-). All shortenings started at a SL 2.5µm. P1, P2 SL1 and SL2 were not affected by different Ca2+ concentrations; the pooled experiments provided P1 and P2 of 0.79 + 0.003 and 0.27 + 0.01 times Po, respectively, and SL1 and SL2 of 4.62 + 0.16 and 24.17+ 0.20 nm•HS−1 respectively. P1 and P2 diminished, and SL1 and SL2 augmented with increasing shortening velocity. Blebbistatin (+/-) decreased P1 (0.74 ± 0.04 times Po) when compared to control fibers (0.81 ± 0.02 times Po), but it did not change the P2. Blebbistatin did not change SL1 and SL2. The results suggest that pre-powerstroke crossbridges contributes to changes in P1, as they may be pushed into the powerstroke during shortening. Such crossbridges detach from actin at the same SL extension.

Myosin essential light chain in health and disease

The essential light chain of myosin (ELC) is known to be important for structural stability of the alpha-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH(2)-terminal approximately 40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH(2)-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.

Differential Effects of Protein Kinase C Isoform Activation in Endothelin-Mediated Myocyte Contractile Dysfunction With Cardioplegic Arrest and Reperfusion

Increased myocardial interstitial levels of endothelin (ET) occur during cardioplegic arrest (CA) and may contribute to contractile dysfunction. Endothelin receptor transduction involves the protein kinase-C (PKC) family comprised of multiple isoforms with diverse functions. Which PKC isoforms may be involved in ET-induced contractile dysfunction after CA remains unknown. Shortening velocity was measured in isolated left ventricular porcine myocytes and randomized (minimum of 30 per group): normothermia (cell culture media for 2 hours at 37 degrees C); CA (2 hours in CA solution [4 degrees C, 24 mEq K+] followed by reperfusion in cell media); ET/CA (100 pM ET incubated during CA and reperfusion). These studies were carried out in the presence and absence of PKC inhibitors (500 nM) and directed against members of the classical PKC subfamily (beta I, beta II, gamma) and the novel subfamily (epsilon, eta). Cardiac arrest reduced shortening velocity by approximately 50%, which was further reduced in the presence of ET. Inhibition of either the beta II or gamma PKC isoform significantly increased shortening velocity from ET/CA as well as CA only values. In separate studies (n = 3), total beta II and phosphorylated beta II increased by over 150% with ET/CA (p < 0.05). Taken together, these results suggest that a predominant intracellular effector for the negative contractile effects mediated by ET in the context of CA is the PKC isoform beta II. Targeted inhibition of specific PKC isoforms relieves the negative inotropic effects of ET after simulated CA. These findings provide important mechanistic support for the development of targeted inhibitory strategies with respect to ET signaling and myocyte contractile dysfunction in the context of CA and reperfusion.

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

Ontogenesis of myosin light chain kinase mRNA and protein content in guinea pig tracheal smooth muscle.

We previously reported in guinea pig tracheal smooth muscle that maximal shortening velocity decreases from 3 weeks of age to adulthood. It is not known whether myosin light chain kinase (MLCK), a key enzyme determining the velocity of smooth muscle contraction, undergoes maturational changes. In the present work, we investigated MLCK protein content and mRNA expression in 1-week-old, 3-week-old, and adult guinea pigs. We extracted either proteins or RNA from isolated tracheal smooth muscle. The content of MLCK was assessed by Western immunoblots. MLCK mRNA was evaluated by Northern analysis and by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). The content of MLCK increased 3-fold at 3 weeks of age and then decreased in adults, being 0.116 +/- 0.042, 0.330 +/- 0.125 (P < 0.05), and 0.153 +/- 0.054 microg/mg of total protein, respectively, in 1-week, 3-week, and adult animals. Quantitative RT-PCR revealed that MLCK mRNA increased with age to 135 +/- 35% and 177 +/- 23% (P < 0.01) in 3-week and adult animals, respectively, compared to 1-week animals. The transient increase of MLCK content in juvenile guinea pig tracheal smooth muscle may contribute to the increased shortening velocity at this age. We suggest that this increased content of MLCK is one of the mechanisms leading to maturation of airway smooth muscle contractility, which in turn contributes to the airway hyperresponsiveness reported in children and young animals.

Effects of thyroxine on myosin isoform expression and mechanical properties in guinea-pig smooth muscle.

Information on the effects of thyroid hormone on smooth muscle contractile protein expression and mechanical properties is sparse. We have addressed the following questions. (1) Can thyroxine hormone alter myosin isoform composition in smooth muscle? (2) Can a change in myosin isoform composition lead to altered mechanical properties in smooth muscle? (3) Are alterations, if occurring, equal in fast and slow smooth muscle types? Guinea-pigs were treated with thyroxine (T(4)) for 12 days. Control animals were given physiological saline solution. Maximal unloaded shortening velocity (V(max)) was measured in chemically skinned, maximally activated muscle preparations from the aorta and the taenia coli. V(max) increased following thyroxine treatment, by approximately 20 % in the taenia coli. In the aorta, no significant increase in V(max) could be detected. The sensitivity of isometric force to inorganic phosphate (P(i)) was increased in the taenia coli following thyroxine treatment. The expression of mRNA (determined with RT-PCR) for the myosin heavy chain with the seven amino acid insert increased by approximately 70 % in the aorta and about 25 % in the taenia coli following thyroxine treatment. Western blot analysis showed an increase in the inserted myosin heavy chain form in the taenia coli. Expression of mRNA for the myosin essential light chains and the corresponding proteins did not change significantly in either muscle type. No alterations in non-muscle myosin heavy chain isoforms could be detected after thyroxine treatment. In conclusion, thyroxine treatment alters the isoform composition of myosin in fast and slow smooth muscles in vivo. This change is sufficient to increase shortening velocity and sensitivity of isometric force to P(i) in the fast, but not in the slow, smooth muscle type.

Homeokinesis and short-term variability of human airway caliber.

We hypothesized that short-term variation in airway caliber could be quantified by frequency distributions of respiratory impedance (Zrs) measured at high frequency. We measured Zrs at 6 Hz by forced oscillations during quiet breathing for 15 min in 10 seated asthmatic patients and 6 normal subjects in upright and supine positions before and after methacholine (MCh). We plotted frequency distributions of Zrs and calculated means, skewness, kurtosis, and significance of differences between normal and log-normal frequency distributions. The data were close to, but usually significantly different from, a log-normal frequency distribution. Mean lnZrs in upright and supine positions was significantly less in normal subjects than in asthmatic patients, but not after MCh and MCh in the supine position. The lnZrs SD (a measure of variation), in the upright position and after MCh was significantly less in normal subjects than in asthmatic patients, but not in normal subjects in the supine position and after MCh in the supine position. We conclude that 1) the configuration of the normal tracheobronchial tree is continuously changing and that this change is exaggerated in asthma, 2) in normal lungs, control of airway caliber is homeokinetic, maintaining variation within acceptable limits, 3) normal airway smooth muscle (ASM) when activated and unloaded closely mimics asthmatic ASM, 4) in asthma, generalized airway narrowing results primarily from ASM activation, whereas ASM unloading by increasing shortening velocity allows faster caliber fluctuations, 5) activation moves ASM farther from thermodynamic equilibrium, and 6) asthma may be a low-entropy disease exhibiting not only generalized airway narrowing but also an increased appearance of statistically unlikely airway configurations.

Measurement of Nucleotide Exchange Rate Constants in Single Rabbit Soleus Myofibrils during Shortening and Lengthening Using a Fluorescent ATP Analog

The kinetics of displacement of a fluorescent nucleotide, 2′(3′)- O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5′-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (Chaen et al., 1997, Biophys. J. 73:2033–2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K m for Cy3-EDA-ATP and the K d for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 μM and 3.8 μM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 μM and 50 μM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 ± 0.007 s −1 ( n = 32) at 10°C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s −1 (isometric) to 0.14 s −1 at 20°C with increasing shortening velocity up to 0.1 myofibril length/s ( V max, the shortening velocity under no load was ∼0.2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.

Work-dependent deactivation of a crustacean muscle.

Active shortening of respiratory muscle L2B from the crab Carcinus maenas results in contractile deactivation, seen as (1) a decline of force during the course of isovelocity shortening, (2) a reduction in the rate of force redevelopment following shortening, (3) a depression of the level of isometric force reached following shortening, and (4) an accelerated relaxation at the end of stimulation. The degree of deactivation increases with increasing distance of shortening, decreases with increasing shortening velocity, and is approximately linearly related to the work done during shortening. Deactivation lasts many seconds if stimulation is maintained, but is largely although not completely removed if the stimulation is temporarily interrupted so that the force drops towards the resting level. Deactivation for a given distance and velocity of shortening increases with increasing muscle length above the optimum length for force production. Stimulating muscle L2B at suboptimal frequencies gives tetanic contractions that are fully fused but of less than maximal amplitude. The depression of force following shortening, relative to the force during an isometric contraction, is independent of the stimulus frequency used to activate the muscle, indicating that deactivation is not a function of the background level of stimulus-controlled muscle activation upon which it occurs. Deactivation reduces the work required to restretch a muscle after it has shortened, but it also lowers the force and therefore the work done during shortening. The net effect of deactivation on work output over a full shortening/lengthening cycle is unknown.

Scaling of intrinsic contractile properties and myofibrillar protein composition of fast muscle in the fish myoxocephalus scorpius L

Contractile properties were determined for fast muscle fibres from short-horn sculpin (Myoxocephalus scorpius L.) ranging from 5 to 35 cm total body length (L) and from 2.0 to 758 g body mass. Maximum unloaded shortening velocity (V0) decreased with increasing body size, scaling as 19.5L-0.34, and isometric twitch activation and tetanus relaxation times became longer, scaling as 12.0L0.31 and 19. 5L0.42 respectively. Myofibrillar Mg2+/Ca2+-ATPase activity scaled as 2.51L-0.28. In order to investigate the mechanisms underlying the scaling of contractile properties, myofibrillar protein composition and I filament lengths were determined. One-dimensional SDS-PAGE and two-dimensional isoelectric focusing/non-equilibrium isoelectric focusing-PAGE revealed no differences in the myofibrillar protein isoforms of myosin light chains, actin, tropomyosin, troponin-T and troponin-C in fish of differing body size. Peptide maps of purified myosin heavy chains digested with eight different proteolytic enzymes were also similar in all fish examined. Three isoforms of troponin-I were present in fish less than 20 cm in total length with relative molecular masses of 17 (TnIf3), 22 (TnIf2) and 23 (TnIf1). The ratio of TnI isoforms varied with body length, and only TnIf3 was present in fish greater than 28 cm total length. The length of I filaments was independent of body length. Thus, although the expression of TnI isoforms changes during growth, the underlying mechanism responsible for increased shortening velocity with decreased body size remains unknown. <P>

Measurement of ATP turnover during shortening and lengthening of rabbit psoas myofibrils using a fluorescent ATP analog.

In order to study ATP turnover during shortening and lengthening of rabbit psoas myofibrils, we have used fluorescence microscopy in which the displacement of a fluorescent nucleotide analog, 2'(3')-O-[N-[2-[[Cy3] amido] ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) bound to cross-bridge on flash photolysis of caged ATP was measured [Chaen et al. (1997) Biophys. J. 73, 2033-2042]. In the previous paper, we reported that when a myofibril was imposed to shorten with a constant velocity by a piezo-electric actuator, the nucleotide displacement rate constant initially increased to 0.7 s-1 with increasing shortening velocity and then declined with a further increase in shortening velocity. The rate constant during lengthening measured in the present experiment was found to be not significantly affected. These results suggest that the cross-bridge kinetics show a asymmetrical dependence on the mechanical strain in the cross-bridges, namely, the rate constants are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening.

Force-Velocity Characteristics of Individual Human Skeletal Muscles: TBCIat and TBCIong. Outline of the Method

The aim of the work is to outline a procedure of finding force-velocity (F–V) characteristics (F = f(V)) of individual skeletal muscles of the human locomotor system. The presentation is based on an example concerning extensors of the elbow joint: the lateral and long heads of triceps brachii (TBCIat and TBCIong). The experimental part of the procedure involves a natural movement of using the upper extremity to push an external object of variable, adjustable load, engaging both the elbow and shoulder joint.Five men aged 23 took part in the experiment. Their task was to push the handle of a physical pendulum whose moment of inertia could be adjusted within the range of 58 kg m2-450 kg m2, so as to give it maximum angular velocity. During each trial the movement of the trunk, of the upper extremity and of the pendulum was video recorded and the force applied with the hand to the handle of the pendulum was measured.In order to find the F–V characteristics a simulation model SHOULDER was used, which is capable of solving the synergy problem for muscles of the arm and the shoulder girdle.It was found that despite considerable dispersion of experimental points the respective regression lines revealed a clear tendency of decreasing muscle force for increased shortening velocity of the monoarticular head (TBCIat) and of increasing muscle force for increased lengthening velocity of the biarticular head (TBCIong) of the triceps brachii muscle.

Measurement of nucleotide release kinetics in single skeletal muscle myofibrils during isometric and isovelocity contractions using fluorescence microscopy

Rabbit psoas muscle myofibrils, in the presence of the fluorescent nucleotide analog 2'(3')-O-[N-[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP), showed selective fluorescence staining of the A-band with a reduced fluorescence at the M-line. Addition of Cy3-EDA-ATP to a myofibril in the presence of Ca2+ caused auxotonic shortening against a compliant glass microneedle. These results indicate that Cy3-EDA-ATP is a substrate for myosin in the myofibril system. The kinetics of nucleotide release from a single myofibril, held isometrically between two needles, were measured by the displacement of prebound Cy3-EDA-ATP on flash photolysis of caged ATP. The A-band fluorescence of the myofibril decayed exponentially with a rate constant of 0.3 s(-1) at 8 degrees C, an order of magnitude faster than that for isolated thick filaments in the absence of actin. When a myofibril was imposed to shorten with a constant velocity by a piezoelectric actuator, the nucleotide displacement rate constant initially increased to 0.7 s(-1) with increasing shortening velocity and then declined with a further increase in shortening velocity. These results demonstrate that the displacement rates of Cy3-EDA-nucleotides bound to the cross-bridges in the contracting myofibril reflect a process that shows strain dependence.

Increase in ATP consumption during shortening in skinned fibres from rabbit psoas muscle: effects of inorganic phosphate.

1. The influence of inorganic phosphate (P(i)) on the relationship between ATP consumption and mechanical performance under isometric and dynamic conditions was investigated in chemically skinned single fibres or thin bundles from rabbit psoas muscle. Myofibrillar ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH. NADH absorbance at 340 nm was determined inside a miniature (4 microliters) measuring chamber. 2. ATP consumption due to isovelocity shortenings was measured in the range between 0.0625 and 1 L0 s-1(L0: fibre length previous to shortening, corresponding to a sarcomere length of 2.64 microns), in solutions without added P(i) and with 30 mM P(i). To get an estimate of the amount of ATP utilized during the shortening phase, quick releases of various amplitudes were also performed. 3. After quick releases, sufficiently large that force dropped to zero, extra ATP was hydrolysed which was largely independent of the amplitude of the release and of the period of unloaded shortening. This extra amount, above the isometric ATP turnover, corresponded to about 0.7 and 0.5 ATP molecules per myosin head at 0 and 30 mM P(i), respectively. 4. ATP turnover during the isovelocity shortenings was higher than isometric turnover and increased with increasing shortening velocity up to about 2.7 times the isometric value. At low and moderate velocities of shortening (< 0.5 L0 s-1), P(i) reduced ATP turnover during isovelocity shortening and isometric ATP turnover to a similar extent, i.e. a decrease to about 77% between 0 and 30 mM added P(i). 5. The extra ATP turnover above the isometric value, resulting from isovelocity shortenings studied at different speeds, was proportional to the power output of the preparation, both in the absence and presence of added [P(i)]. 6. The effect of shortening velocity and [P(i)] on energy turnover can be understood in a cross-bridge model that consists of a detached, a non- or low-force-producing, and a force-producing state. In this model, mass action of P(i) influences the equilibrium between the force-producing and the non-or-low-force-producing cross-bridges, and shortening enhances cross-bridge detachment from both attached states.

Supramaximal Activation Increases Motor Unit Velocity of Unloaded Shortening

It has been shown that the rate of tension generation (dP/dt) continues to increase with increasing stimulation rates, even after maximal tetanic tension has been achieved. Since dP/dt is directly proportional to unloaded shortening velocity, it was questioned whether supramaximal stimulation rates would increase shortening velocity. To test the relationship of velocity and stimulation rate, slack tests were performed on motor units isolated in the rat soleus muscles. For each motor unit tested, two slack tests were performed at two different stimulation rates: one rate yielded a maximal tetanic tension with a "slow" dP/dt (PO) and the other rate yielded a maximal tetanic tension with a "fast" dP/dt (RG). The two stimulation rates (PO and RG) had significantly different effects (p &lt; .05) on motor unit shortening velocity, with the RG rate yielding a shortening velocity greater than that of PO by an average of 13 ± 6%. This suggests that rate coding could be used to grade motor unit power production by grading force production and/or shortening velocity.

Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles

The increased shortening velocity and capacity of airway smooth muscle (ASM) from ragweed pollen-sensitized dogs, which may be responsible for its in vivo airway hyperresponsiveness, have been shown to be associated with higher actomyosin adenosinetriphosphatase activity and greater level of phosphorylation of the 20-kDa myosin light-chain (MLC20) at rest and during contraction. Current studies show that the elevated level of phosphorylation may be the result of an increased myosin light-chain kinase (MLCK) activity due to excessive quantity of MLCK. There were no significant changes in total activity of calmodulin, a protein that binds and activates MLCK, in sensitized dog ASM (SASM) compared with control ASM (CASM). When normalized to the relative calmodulin content in the tissues, the specific calmodulin activities (means +/- SE) in sensitized tracheal smooth muscle (STSM) and sensitized bronchial smooth muscle (SBSM) and in their controls were not different (STSM 0.359 +/- 0.117, CTSM 0.339 +/- 0.136. SBSM 0.201 +/- 0.098, and control bronchial smooth muscle 0.213 +/- 0.056 nmol Pi.calmodulin content-1.min-1, respectively). Intracellular Ca2+ levels indicated by fura 2 fluorescent dye remained unaltered in SASM. We conclude that airway hyperresponsiveness may result from higher MLCK content in SASM rather than from changes in Ca(2+)-calmodulin activities, which is an example of alteration in Ca2- sensitivity of ASM.