Cross-bridge Detachment Research Articles

Regulation of Contraction by PKA Phosphorylation of Myosin Binding Protein C and Troponin I in Murine Skinned Myocardium

In skinned myocardium, cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of cardiac myosin binding protein-C (cMyBP-C) and troponin I (cTnI) leads to a decrease in myofilament Ca2+-sensitivity and an acceleration in the kinetics of cross-bridge cycling. To examine the relative roles of cTnI and cMyBP-C phosphorylation in altering contractile function, we determined the Ca2+-sensitivity of force (pCa50) and the rate of force redevelopment (ktr) in untreated and PKA-treated murine myocardium expressing: (1) phosphorylatable cTnI and cMyBP-C (WT), (2) non-phosphorylatable cTnI with serines23/24/43/45 and threonine144 residues mutated to alanines (cTnIala5), (3) phosphorylatable cTnI on a cMyBP-C null background (cMyBP-C-/-), and (4) non-phosphorylatable cTnI on a cMyBP-C null background (cTnIala5/cMyBP-C-/-). A novel aspect of this study was the use of 2,3-Butandione Monoxime (BDM) treatments to reduce the basal levels of myosin regulatory light chain (RLC) phosphorylation to near zero in order to more accurately define the functional consequences of removing cMyBP-C and/or cTnI phoshorylation in transgenic myocardium. Our results showed that in the absence of RLC phosphorylation, PKA-treatment decreased pCa50 in WT, cTnIala5, and cMyBP-C-/- myocardium by 0.13, 0.08 and 0.09 pCa units, respectively, but had no effect in cTnIala5/cMyBP-C-/- myocardium. In WT and cTnIala5 myocardium, PKA treatment increased ktr at submaximal levels of activation; however, treatment did not have an effect on ktr in cMyBP-C-/- and cTnIala5/cMyBP-C-/- myocardium. Together, these results indicate that the attenuation of the myofilament force response following PKA treatment is due to phosphorylation of both cTnI and cMyBP-C and that the reduced Ca2+-sensitivity of force mediated by phosphorylation of cMyBP-C is most likely due to an increased rate constant of cross-bridge detachment that also contributes to an acceleration of cross-bridge cycling kinetics.

The Effect of Hypertrophic Cardiomyopathy (HCM) Mutations of Tropomyosin on Force Generation and Cross-Bridge Kinetics in Thin-Filament Reconstituted Bovine Cardiac Muscle Fibers

Three HCM mutations (V95A, D175N and E180G) of Tropomyosin (Tm) were examined using the thin-filament extraction and reconstitution technique. Effects of Ca2+, ATP, phosphate and ADP concentrations on force and its transients were studied at 25°C and compared to WT. E180G showed larger isomeric tension (1.89±0.11) than WT (1.59±0.08). Tension of two other mutants (V95A 1.53±0.09, D175N 1.47±0.07) was not different from WT. pCa50 (Ca2+ sensitivity) of V95A (6.20±0.06) and E180G (6.51±0.02) was larger than WT (5.85±0.03), while that of D175N (5.88±0.05) remained the same. The cooperativity was reduced in all three mutants (V95A 1.70±0.11, D175N 1.87±0.09, E180G 1.91±0.14) compared to WT (2.79±0.25). Four equilibrium constants were deduced using sinusoidal analysis. The largest effect was on K5 (Pi association constant) which was reduced to ∼0.5X in all mutants, while K4 (force generation step) was unchanged. V95A showed significantly lower K2 (cross-bridge detachment step: 0.93±0.06) than WT (1.37±0.13). D175N and V95A showed significantly lower K1 (ATP association constant, 0.91±0.13 and 0.86±0.16, respectively) than E180G (1.84±0.33) and WT (1.60±0.35). However, the cross-bridge distribution was not significantly different among 4 Tms, indicating that force/cross-bridge in E180G is larger than WT, but it is unchanged in V95A and D175N. In conclusion, all three mutants showed significant deviations in force/cross-bridge, pCa50, cooperativity or cross-bridge kinetics; in particular, E180G had the largest effect. Because E180G and D175N are located in the Tm-Troponin (Tn) interaction region and result in the net charge increase, and E180G causes the largest hydropathy change, we infer that both electrostatic and hydrophobic interactions between Tm and Tn play vital roles in maintaining normal muscle functions.

Molecular basis of force development by skeletal muscles during and after stretch.

When activated skeletal muscles are stretched at slow velocities, force increases in two phases: (i) a fast increase, and (ii) a slow increase. The transition between these phases is commonly associated with the mechanical detachment of cross-bridges from actin. This phenomenon is referred to as force enhancement during stretch. After the stretch, force decreases and reaches steady-state at levels that are higher than the force produced at the corresponding length during purely isometric contractions. This phenomenon is referred to as residual force enhancement. The mechanisms behind the increase in force during and after stretch are still a matter of debate, and have physiological implications as human muscles perform stretch contractions continuously during daily activity. This paper briefly reviews the potential mechanisms to explain stretch forces, including an increased number of cross-bridges attached to actin, an increased strain in cross-bridges upon stretch, the influence of passive elements upon activation and sarcomere length non-uniformities.

Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Energy turnover in relation to slowing of contractile properties during fatiguing contractions of the human anterior tibialis muscle

Slowing and loss of muscle power are major factors limiting physical performance but little is known about the molecular mechanisms involved. The slowing might be a consequence of slow detachment of cross bridges and, if this were the case, then a reduction in the ATP cost of an isometric contraction would be expected as the muscle fatigued. The human anterior tibialis muscle was stimulated repeatedly under ischaemic conditions at 50 Hz for 1.6 s with a 50% duty cycle and muscle metabolites measured by (31)P magnetic resonance spectroscopy. Over the course of 20 contractions the half-time of relaxation increased from 36.5 +/- 0.09 ms (mean +/- s.e.m.) to 113 +/- 17 ms and isometric force was reduced to 63 +/- 3% of the initial value. ATP turnover was determined from the change in high energy phosphates and lactate production, the latter estimated from the change of intracellular pH. ATP turnover over the first three contractions was 2.45 +/- 0.09 mM s(1) and decreased to 1.8 +/- 0.06 mm s(1) over the last five tetani. However, when this latter value was normalised for the decrease in isometric force, it became 2.56 +/- 0.3 mM s(1), which is the same as the turnover of the fresh muscle. The data suggest that the rate of cross bridge detachment is unaffected by fatigue and are consistent the suggestion that it is the rate of attachment which is slowed rather than the rate of detachment. The present results focus attention on stages in the cross bridge cycle concerned with attachment and the transition from low to high force states that may be influenced by metabolic changes in the fatiguing muscle.

Crossbridge Properties During The Quick Force Recovery In Single Frog Muscle Fibers

Fast stretches (∼25 nmhs−1 amplitude and ∼400 μs duration) which induced the forced rupture of the crossbridge ensemble were applied to intact muscle fibers to investigate the actomyosin bond properties during the force recovery following a step length change (release or stretch of 2 or 4 nm amplitude). Force and sarcomere length were measured with a fast force transducer (∼50 kHz natural frequency) and a striation follower device. To reduce fiber damaging by the stretches and to reduce the influence of myofilament compliance on the measurements, experiments were made on the tetanus rise at tension of about 0.5 the maximum plateau tension. Fast stretches were applied before and at progressively increasing times (up to 20 ms) after the step length change. The rupture force of the crossbridge ensemble (Pc) and the sarcomere elongation at Pc (Lc) were measured. In contrast with the data obtained previously on the tetanus rise (Bagni et al. J. Physiol., 2005; 565). The results showed that: (1) Pc was almost independent of the tension developed by the fiber and (2), Lc was not constant but increased immediately after the release and decreased after the stretch. These changes were still present 2 ms later when the quick recovery was almost complete and disappeared completely within 15-20 ms. Data analysis suggests that: 1) crossbridge number remains almost constant during the quick force recovery; 2) crossbridge detachment by the fast stretch is preceded by the reversal of the myosin head power stroke and, 3) the extent of the power stroke can be measured by the changes in Lc occurring during the quick recovery.

Effect of temperature on cross-bridge properties in intact frog muscle fibers

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 +/- 0.17 nm per half-sarcomere (nm hs(-1)) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 +/- 0.039 nm hs(-1). Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

Impact of temperature on cross‐bridge cycling kinetics in rat myocardium

The dependence of contractile properties on intracellular calcium in cardiac tissue is a highly cooperative process. Here, the temperature and calcium dependence of contractile and energetical properties in permeabilized cardiac trabeculae from rat were studied to provide novel insights into the underlying kinetic processes. Myofilament Ca(2+) sensitivity significantly increased with temperature between 15 and 25 degrees C, whereas its steepness was independent of temperature. A direct proportionality between active tension and Ca(2+)-activated rate of ATP hydrolysis was observed; the slope of this relationship (tension cost) was highly temperature dependent. The rate of tension redevelopment following a quick release-restretch manoeuvre (k(tr)) depended in a complex manner on the level of contractile activation and on temperature. At saturating calcium levels, the temperature dependence (Q(10)) of k(tr) and Ca(2+)-activated ATP hydrolysis rate were similar (Q(10) approximately 3.5), and significantly higher than the Q(10) for maximum tension (T(max); Q(10) approximately 1.3) or tension cost (Q(10) approximately 2.5). In contrast, at a low level of contractile activation ( approximately 5% of T(max)), the Q(10) of k(tr) was similar to that of tension cost, and significantly lower than the Q(10) of Ca(2+)-activated ATP hydrolysis at that level of contractile activation. Our results are consistent with the hypothesis that at high levels of contractile activation, the rates of tension redevelopment and Ca(2+)-activated ATP hydrolysis are determined by both apparent cross-bridge attachment and detachment rates, while at low levels, k(tr) is limited by cross-bridge detachment rate. Tension cost, on the other hand, is determined solely by cross-bridge detachment kinetics at all temperatures and levels of contractile activation.

Cross-bridge kinetics of fast and slow fibres of cat jaw and limb muscles: correlations with myosin subunit composition

Mechanical properties of the jaw-closing muscles of the cat are poorly understood. These muscles are known to differ in myosin and fibre type compositions from limb muscles. This work aims to correlate mechanical properties of single fibres in cat jaw and limb muscles with their myosin subunit compositions. The stiffness minimum frequency, f(min), which reflects isometric cross-bridge kinetics, was measured in Ca(2+)-activated glycerinated fast and slow fibres from cat jaw and limb muscles for temperatures ranging between 15 and 30 degrees C by mechanical perturbation analysis. At 15 degrees C, f(min) was 0.5 Hz for limb-slow fibres, 4-6 Hz for jaw-slow fibres, and 10-13 Hz for limb-fast and jaw-fast fibres. The activation energy for f(min) obtained from the slope of the Arrhenius plot for limb-slow fibres was 30-40% higher than values for the other three types of fibres. SDS-PAGE and western blotting using highly specific antibodies verified that limb-fast fibres contained IIA or IIX myosin heavy chain (MyHC). Jaw-fast fibres expressed masticatory MyHC while both jaw-fast and jaw-slow fibres expressed masticatory myosin light chains (MLCs). The nucleotide sequences of the 3' ends of the slow MyHC cDNAs isolated from cat masseter and soleus cDNA libraries showed identical coding and 3'-untranslated regions, suggesting that jaw-slow and limb-slow fibres express the same slow MyHC gene. We conclude that the isometric cross-bridge cycling kinetics of jaw-fast and limb-fast fibres detected by f(min) are indistinguishable in spite of differences in MyHC and light chain compositions. However, jaw-slow fibres, in which the same slow MyHCs are found in combination with MLCs of the jaw type, show enhanced cross-bridge cycling kinetics and reduced activation energy for cross-bridge detachment.

Tropomyosin in the groove? Molecular insights into an inherited myopathy

Tropomyosin in the groove? Molecular insights into an inherited myopathy

Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

The mechanics of mouse skeletal muscle when shortening during relaxation

The dynamic properties of relaxing skeletal muscle have not been well characterised but are important for understanding muscle function during terrestrial locomotion, during which a considerable fraction of muscle work output can be produced during relaxation. The purpose of this study was to characterise the force–velocity properties of mouse skeletal muscle during relaxation. Experiments were performed in vitro (21 °C) using bundles of fibres from mouse soleus and EDL muscles. Isovelocity shortening was applied to muscles during relaxation following short tetanic contractions. Using data from different contractions with different shortening velocities, curves relating force output to shortening velocity were constructed at intervals during relaxation. The velocity component included contributions from shortening of both series elastic component (SEC) and contractile component (CC) because force output was not constant. Early in relaxation force–velocity relationships were linear but became progressively more curved as relaxation progressed. Force–velocity curves late in relaxation had the same curvature as those for the CC in fully activated muscles but V max was reduced to ∼50% of the value in fully activated muscles. These results were the same for slow- and fast-twitch muscles and for relaxation following maximal tetani and brief, sub-maximal tetani. The measured series elastic compliance was used to partition shortening velocity between SEC and CC. The curvature of the CC force–velocity relationship was constant during relaxation. The SEC accounted for most of the shortening and work output during relaxation and its power output during relaxation exceeded the maximum CC power output. It is proposed that unloading the CC, without any change in its overall length, accelerated cross-bridge detachment when shortening was applied during relaxation.

Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Change in contractile properties of human muscle in relationship to the loss of power and slowing of relaxation seen with fatigue.

Slow relaxation from an isometric contraction is characteristic of acutely fatigued muscle and is associated with a decrease in the maximum velocity of unloaded shortening (V(max)) and both these phenomena might be due to a decreased rate of cross bridge detachment. We have compared the change in relaxation rate with that of various parameters of the force-velocity relationship over the course of an ischaemic series of fatiguing contractions and subsequent recovery using the human adductor pollicis muscle working in vivo at approximately 37 degrees C in nine healthy young subjects. Maximal isometric force (F(0)) decreased from 91.0 +/- 1.9 to 58.3 +/- 3.5 N (mean +/- s.e.m.). Maximum power decreased from 53.6 +/- 4.0 to 17.7 +/- 1.2 (arbitrary units) while relaxation rate declined from -10.3 +/- 0.38 to -2.56 +/- 0.29 s(-1). V(max) showed a smaller relative change from 673 +/- 20 to 560 +/- 46 deg s(-1) and with a time course that differed markedly from that of slowing of relaxation, showing very little change until late in the series of contractions. Curvature of the force-velocity relationship increased (a/F(0) decreasing from 0.22 +/- 0.02 to 0.11 +/- 0.02) with fatigue and with a time course that was similar to that of the loss of power and the slowing of relaxation. It is concluded that for human muscle working at a normal physiological temperature the change in curvature of the force-velocity relationship with fatigue is a major cause of loss of power and may share a common underlying mechanism with the slowing of relaxation from an isometric contraction.

Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling

Sudden Ca2+ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152-2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (approximately 10 ms) after Ca2+ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (approximately 24 ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca2+ removal until troponin C is switched off. However, this switch off occurs approximately 5 times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.

Can activation account for 80% of skeletal muscle energy use during isometric contraction?

presence of a submaximal dose of dantrolene (an inhibitor of the skeletal muscle Ca 2 -release channel), tetanic force was reduced to 36% of its “control” value. Nevertheless, activation heat still accounted for only 42% of the total heat liberated. iv) The idea that 20% of isometric energy turnover is due to cross-bridge cycling is incompatible with the known mechanical performance of fast-twitch muscles. If cross bridges account for only 20% of energy use, then the rate of ATP splitting per cross bridge must be one-quarter the value previously assumed. Maintenance of a high isometric force, in any cross-bridge scheme that requires a low rate of turnover, can be achieved only by reducing the rate of cross-bridge detachment. However, a low detachment rate is inconsistent with the rapid force dynamics and the flat force-velocity curve characteristic of EDL muscles (2). Zhang et al. (5) offer no mechanistic explanation for their result but suggest that it differs from previous data due to an effect of species or temperature or as a result of using submaximal activation. Points i and iii above argue against an effect of either species or temperature, and the dantrolene experiments (point iii) counter the idea that activation costs are higher at submaximal levels of activation.

Cross-Bridge-Dependent Change of the Ca2+ Sensitivity During Relaxation in Aequorin-Injected Tetanized Ferret Papillary Muscles

BACKGROUND The aim of the present study was to indicate the cross-bridge-dependent change in the Ca2+ affinity of troponin-C (TnC) during relaxation in an intact preparation, because the intracellular mechanism of relaxation is not fully understood, although several methods of evaluating global diastolic function have been reported. The aequorin method was used with intact ferret papillary muscles and a tetanic contraction was induced by a repetitive electrical stimulation in the presence of ryanodine. The extra-Ca2+, the transient increase in the intracellular Ca2+ concentration in response to a rapid reduction in muscle length, which reflects the change in the Ca2+ affinity of TnC because of cross-bridge detachment, was measured, and the cross-bridge-dependent change in the Ca2+ affinity of TnC was estimated by observing the change in the slope of the extra-Ca2+ -tension relation. The extra-Ca2+ -tension relation measured during relaxation became steeper than that during contraction in all cases. The extra-Ca2+ -tension relation became steeper in the presence of 20 mmol/L caffeine during contraction in all cases. During relaxation, the downstream-dependent change in the Ca2+ affinity of TnC was enhanced, compared with that during contraction, because of a decrease in the number of attached cross-bridges.

DESIGN AND FUNCTION OF SUPERFAST MUSCLES: New Insights into the Physiology of Skeletal Muscle

Superfast muscles of vertebrates power sound production. The fastest, the swimbladder muscle of toadfish, generates mechanical power at frequencies in excess of 200 Hz. To operate at these frequencies, the speed of relaxation has had to increase approximately 50-fold. This increase is accomplished by modifications of three kinetic traits: (a) a fast calcium transient due to extremely high concentration of sarcoplasmic reticulum (SR)-Ca2+ pumps and parvalbumin, (b) fast off-rate of Ca2+ from troponin C due to an alteration in troponin, and (c) fast cross-bridge detachment rate constant (g, 50 times faster than that in rabbit fast-twitch muscle) due to an alteration in myosin. Although these three modifications permit swimbladder muscle to generate mechanical work at high frequencies (where locomotor muscles cannot), it comes with a cost: The high g causes a large reduction in attached force-generating cross-bridges, making the swimbladder incapable of powering low-frequency locomotory movements. Hence the locomotory and sound-producing muscles have mutually exclusive designs.