Rabbit Psoas Muscle Research Articles

Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain

Myofilament sliding in muscle is believed to result from rotation of the myosin head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to myosin head converter domain (IgG, anti-CD antibody) between in vitro actin-myosin sliding and muscle fiber contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over myosin heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated myosin head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas muscle fibers. The possibility that the antibody does not diffuse into muscle fibers can be excluded by our published results that other antibodies readily inhibit muscle fiber contraction. These findings therefore suggest that the antibody binding to the myosin head CD does not impair performance of myosin heads producing force and motion in muscle fibers.

A Comparison of Single Myofibrils and Single Muscle Fibers from Rabbit Psoas in Terms of MgATP Binding and Cross-Bridge Detachment Steps Studied with Sinusoidal Length Perturbations

Single myofibrils of 40-70 μm in length and 2-4 μm in diameter were isolated from rabbit psoas muscle bundles, and cross-bridge kinetics were studied by small amplitude (0.2%) length perturbations at 15 frequencies (1-250Hz). The experiments were performed at 15°C in the presence of 0.05-10 mM MgATP, 4 mM KH2PO4, 4 mM K2HPO4, 15 mM Na2CP (creatine phosphate), 200 mM ionic strength with KAc (acetate), pCa 4.55, and pH 7.0. Two exponential processes B and C were resolved in tension transients. Their apparent rate constants (2πb and 2πc; b < c) increased ([MgATP]=0.05-1 mM) and saturated (≥1 mM) with an increase in the MgATP concentration. However, they were close together (1.4x) at [Pi]≥4 mM, hence they were combined to deduce the accurate estimate of the kinetic constants: their sum and product were analyzed as functions of [MgATP]. These analyses yielded K1=2.91 ± 0.31 mM−1, k2=288±36 s−1, and k-2=10±21 s−1 (±95% confidence limit, N=13). These results are consistent with the cross-bridge model: AM+ATP <-> (step 1) AM.ATP <-> (step 2) A+M.ATP. These kinetic constants compare to those observed in single fibers (K1=2.35±0.31 mM−1, k2=243±22 s−1, and k-2=6±14 s−1; N=8) under the same experimental conditions and analysis methods. These values are respectively not significantly different from those of myofibrils, indicating that the same kinetic constants can be deduced from myofibril and muscle fiber studies in terms of ATP binding and cross-bridge detachments steps. The fact that in myofibrils K1 is 1.24x of that of fibers, may be explained by a small concentration gradient of ATP, ADP and/or Pi in single fibers.

The Low Angle X-Ray Diffraction Pattern from Skinned Fibers of Rabbit Psoas Muscle: Effect of Changes in [Ca++] and [Orthophosphate

Bundles of 3-5 fibers were activated isometrically at different pCa by a temperature jump from 1°C to 12°C using a mechanical apparatus (Linari et al., Biophys. J. 92:2476, 2007) modified to collect the X-ray diffraction pattern. The M3 meridional reflection from the axial repeat of the myosin heads was sampled by X-ray interference between half-sarcomeres. In relaxed fibers at 12°C, the M3 reflection had a major peak at 14.56 nm and a minor peak at 14.37 nm. The ratio of peak intensities (RM3) was 0.43±0.06 and the spacing (SM3) was 14.49±0.01 nm. In relaxed fibers the intensity of the main peak reduced with increasing temperature, so that at 36°C (the physiological temperature) the 14.37 nm peak was dominant, with small satellite peaks on either side, as in resting intact fibers from frog muscle. During activation at 12°C at saturating [Ca++], pCa 4.5, the intensity of the M3 reflection (IM3) increased to 1.9±0.4 times the relaxed value with major and minor peaks at 14.68 nm and 14.46 nm; RM3 was 0.62±0.03 and SM3 was 14.59±0.01 nm. Activation at pCa 5.5 or at pCa 4.5 with addition of 10 mM orthophosphate (Pi) had similar effects: force was reduced to 0.34±0.10 the control value and IM3 to 0.56±0.03; RM3 was 0.46±0.07 and SM3 was 14.55±0.02 nm. These results give structural support to the conclusion from mechanical experiments (Linari et al., 2007) that both decreasing [Ca++] and increasing [Pi] reduce isometric force by a decrease in the number of force generating myosin heads with no change in force per head.Supported by FIRB-Futuro in Ricerca and Ente Cassa di Risparmio di Firenze (Italy), MRC (UK).

Force produced by isolated sarcomeres and half-sarcomeres after an imposed stretch

When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres (n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5-10% of initial SL, at a speed of 0.3 μm·s(-1)·SL(-1)) after which they were maintained isometric for at least 5 s before deactivation. Single sarcomeres showed two patterns: 31 sarcomeres showed a small level of force enhancement after stretch (10.46 ± 0.78%), and 28 sarcomeres did not show force enhancement (-0.54 ± 0.17%). In these preparations, there was not a strong correlation between the force enhancement and half-sarcomere length nonuniformities. When three or more sarcomeres arranged in series were stretched, force enhancement was always observed, and it increased linearly with the degree of half-sarcomere length nonuniformities. The results show that the residual force enhancement has two mechanisms: 1) stretch-induced changes in sarcomeric structure(s); we suggest that titin is responsible for this component, and 2) stretch-induced nonuniformities of half-sarcomere lengths, which significantly increases the level of force enhancement.

Sarcomere length-dependent Ca2+ activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca2+ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca2+ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca2+ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca2+ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca2+ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca2+ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.Electronic supplementary materialThe online version of this article (doi:10.1007/s12576-011-0173-8) contains supplementary material, which is available to authorized users.

Electron Microscopy and X-Ray Diffraction Evidence for Two Z-Band Structural States

In vertebrate muscles, Z-bands connect adjacent sarcomeres, incorporate several cell signaling proteins, and may act as strain sensors. Previous electron microscopy (EM) showed Z-bands reversibly switch between a relaxed, “small-square” structure, and an active, “basketweave” structure, but the mechanism of this transition is unknown. Here, we found the ratio of small-square to basketweave in relaxed rabbit psoas muscle varied with temperature, osmotic pressure, or ionic strength, independent of activation. By EM, the A-band and both Z-band lattice spacings varied with temperature and pressure, not ionic strength; however, the basketweave spacing was consistently 10% larger than small-square. We next sought evidence for the two Z-band structures in unfixed muscles using x-ray diffraction, which indicated two Z-reflections whose intensity ratios and spacings correspond closely to the EM measurements for small-square and basketweave if the EM spacings are adjusted for 20% shrinkage due to EM processing. We conclude that the two Z-reflections arise from the small-square and basketweave forms of the Z-band as seen by EM. Regarding the mechanism of transition during activation, the effects of Ca 2+ in the presence of force inhibitors suggested that the interconversion of Z-band forms was correlated with tropomyosin movement on actin.

Sarcomere overextension reduces stretch-induced tension loss in myofibrils of rabbit psoas

Stretch-induced damage to skeletal muscles results in loss of isometric tension. Although there is no direct evidence, loss of tension has been implicitly assumed to be the consequence of permanent loss of myofilament overlap in some sarcomeres (‘sarcomere overextension’). Using isolated myofibrils of rabbit psoas muscle (n=38; 6 control and 32 test specimens) at 12–15°C, we directly tested the idea that loss of tension following stretch is caused by sarcomere overextension. Experimental myofibrils were maximally activated at the edge of the descending limb (sarcomere length ∼2.9μm) of the sarcomere length-tension relationship and then stretched by 1μmsarcomere–1 at a constant speed of 0.1μms–1sarcomere–1 to result in an average strain of 33.6±0.9% (mean±1SE). Myofibrils were immediately returned to the original lengths and relaxed. Isometric tension measured in a subsequent re-activation 3–5min later was reduced by 24.6±1.5% from its original value. In 22 out of the 32 test specimens, all sarcomeres maintained myofilament overlap, while in 10 myofibrils one or two sarcomeres were stretched permanently beyond myofilament overlap (>4.0μm), and thus exhibited overextended sarcomeres. Loss of tension following stretch was significantly smaller in myofibrils with overextended sarcomeres compared to myofibrils with no overextended sarcomeres (19.5±2.3% and 27.1±1.8%, respectively; p=0.017). Combined, these results suggest that the loss of tension associated with stretch-induced damage can occur in the absence of sarcomere overextension and that sarcomere overextension limits rather than causes stretch-induced tension loss.

Calcium-dependence of Donnan potentials in glycerinated rabbit psoas muscle in rigor, at and beyond filament overlap; a role for titin in the contractile process

In glycerinated rabbit psoas muscle, Donnan potential measurements demonstrated that the net electric charge on the actin–myosin matrix undergoes a sharp switch-like transition at pCa 50 = 6.8. The potentials are 2 mV less negative at the lower pCa 2+ ( P < 0.001). If ATP is present, the muscle contracts and breaks the microelectrode. Therefore the rigor state was studied. There is no reason to suppose a priori that a similar voltage switch does not occur during contraction, however. Calcium dependence is still apparent in muscles stretched beyond overlap (sarcomere length > 3.8 μm) and is also seen in the gap filaments between the A- and I-band ends; further stretching abolishes the dependence. These experiments strongly suggest that calcium dependence is controlled initially by the titin component, and that this control is lost when titin filaments break. We suppose that that effect is mediated by the titin kinase in the M-line region and may involve the extensible PEVK region of titin. There is great interest in the electric charge on proteins in muscle within the structural system. We suggest how changes in these charges may control the calcium activation process. We also suggest some simple experimental approaches that could clarify these effects.

Tropomyosin is in a reduced state in rabbit psoas muscle

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.

Orientation of the Calcium Sensitizing Agent dfbp-o, when Bound to Troponin in a Muscle Fiber as Determined by Solid-State NMR Spectroscopy

Heart failure is characterized by the inability of the heart to supply the body with oxygenated blood. There is a large range of treatment strategies employed by physicians; however, many of these options are limited in their ability to improve mortality during acute heart failure. A more recently identified therapeutic option involves a class of pharmaceuticals termed “calcium sensitizers”. These molecules enhance the contractile apparatus's response to calcium, instead of increasing the concentration of cytosolic calcium. Levosimendan is the most widely used calcium sensitizer, but since it is unstable, its exact mode of action has yet to be identified. Recently, we have determined that the stable structural analog of levosimendan, dfbp-o, functions as a calcium sensitizer in a similar manner as levosimendan [Robertson et al., J Mol Cell Cardiol. 2010 Aug 27. [Epub ahead of print]]. Two fluorine atoms are present on dfbp-o which can be used to elucidate useful structural information via 19F-NMR spectroscopy. 19F is a particularly attractive nucleus for study by NMR spectroscopy because like 1H it is ∼100% naturally abundant, has a spin of 1/2, and possesses a large gyromagnetic ratio. We have used 19F solid-state NMR spectroscopy to study the orientation of dfbp-o in demembranated rabbit psoas muscle fibers. Upon incubating the muscle fiber in relaxing solution containing dfbp-o, the length of the fiber decreased approximately three fold, presumably due to the calcium sensitizing nature of dfbp-o. This approach will allow us to establish the orientation of the dfbp-o:troponin complex with respect to the thin filament axis. In addition, the results give us further evidence that dfbp-o binds muscle fibers to elicit its calcium sensitization effect.

A-Band Shortening in Isolated Myofibrils of Rabbit Psoas Muscles

Muscle contraction is explained by cyclical interactions between actin and myosin filaments. These filaments are typically assumed to be rigid and thus are not thought to contribute to muscle contraction and force production. However, previous works have observed reduction in A-band width, an indicator of myosin length, in activated single fibers. As individual sarcomeres and A-bands cannot be resolved accurately in single fibers the purpose of this study was to determine A-band widths in a preparation that allows such resolution i.e., isolated myofibrils. Isolated myofibrils (n=13, 10-15 sarcomeres long) of rabbit psoas muscles were placed in a bath containing relaxing solution under an inverted microscope. Myofibrils were secured in two segments with two glass micro needles and a silicon nitrate nano lever. Mean sarcomere length (SL) of the control segment between the glass needle and the cantilever was set to ∼2.4µm and the mean SL of the experimental segment between the needles was varied between 2.0µm and 3.2µm. Upon Ca2+-activation, myofibrillar forces were calculated in the control segment from the cantilever deflection (186±112nN). A-band widths and SL were measured from video continually for 5min following activation. Reduction in A-band width (range=0.0-0.6µm) correlated (p=-0.7884) with the post-activation mean SL (range=2.1-3.2µm) of the experimental segment in a bi-linear fashion with an inflection at ∼2.8µm. A-bands shorten upon activation at SL below 2.8µm, but remain constant at SL above 2.8µm. This result contradicts long held beliefs about myosin filament rigidity and may challenge our current thinking about the molecular mechanisms of contraction. However, the slow speed of A-band shortening raises questions about its functional relevance in everyday muscle activity and its occurrence at short SL only may suggest a role in adjusting the length of the molecular spring titin.

Temperature jump induced force generation in rabbit muscle fibres gets faster with shortening and shows a biphasic dependence on velocity.

We examined the tension responses to ramp shortening and rapid temperature jump (<0.2 ms, 3-4 degrees C T-jump) in maximally Ca(2+)-activated rabbit psoas muscle fibres at 8-9 degrees C (the fibre length (L(0)) was approximately 1.5 mm and sarcomere length 2.5 microm). The aim was to investigate the strain sensitivity of crossbridge force generation in muscle. The T-jump induced tension rise was examined during steady shortening over a wide range of velocities (V) approaching the V(max) (V range approximately 0.01 to approximately 1.5 L(0) s(1)). In the isometric state, a T-jump induced a biphasic tension rise consisting of a fast (approximately 50 s(1), phase 2b) and a slow (approximately 10 s(1), phase 3) component, but if treated as monophasic the rate was approximately 20 s(1). During steady shortening the T-jump tension rise was monophasic; the rate of tension rise increased linearly with shortening velocity, and near V(max) it was approximately 200 s(1), approximately 10x faster than in the isometric state. Relative to the tension reached after the T-jump, the amplitude increased with shortening velocity, and near V(max) it was 4x larger than in the isometric state. Thus, the temperature sensitivity of muscle force is markedly increased with velocity during steady shortening, as found in steady state experiments. The rate of tension decline during ramp shortening also increased markedly with increase of velocity. The absolute amplitude of T-jump tension rise was larger than that in the isometric state at the low velocities (<0.5 L(0) s(1)) but decreased to below that of the isometric state at the higher velocities. Such a biphasic velocity dependence of the absolute amplitude of T-jump tension rise implies interplay between, at least, two processes that have opposing effects on the tension output as the shortening velocity is increased, probably enhancement of crossbridge force generation and faster (post-stroke) crossbridge detachment by negative strain. Overall, our results show that T-jump force generation is strain sensitive and becomes considerably faster when exposed to negative strain. Thus the crossbridge force generation step in muscle is both temperature sensitive (endothermic) and strain sensitive.

Changes in the Conformation of Troponin C on Activation of Skeletal Muscle

Skeletal muscle contraction is regulated by calcium-dependent changes in the structure and thin-filament location of troponin and tropomyosin. The structural changes in the isolated calcium-binding subunit of troponin (TnC) are well characterized, but those of TnC in the native thin filament are much less clear. We measured the in situ orientation of the C-terminal lobe of TnC (CTnC) by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteines at TnC residues 96-103, 116-123, 132-139, and 119-135. Each BR-labeled TnC was exchanged into single permeablized fibers from rabbit psoas muscle, and polarized fluorescence from the BR-TnCs was measured during relaxation and maximal calcium activation. The orientation distribution of CTnC with respect to the thin filament axis was calculated by maximum entropy analysis using the in vitro structure of CTnC in the troponin core complex (Vinogradova et al. (2005) PNAS102:5038-5043). The peak angle between E helix of CTnC and the filament axis was 49° in relaxed muscle and 64° during maximal activation. Comparison with the results of our previous study of the orientation of the N-terminal lobe of TnC (Ferguson et al. (2003) Mol. Cell11: 865-874) suggests that the central D/E helix of TnC is bent by about 30° in relaxed muscle and becomes straight during maximal activation.Supported by MRC (UK).

Effect of Inorganic Phosphate on the Rate of ADP Release During Ramp Shortening in Activated Permeabilized Fibers from Rabbit Psoas Muscle

A coumarin-labeled recombinant phosphorylated nucleoside diphosphate kinase (P∼NDPK-IDCC; West et al., 2009; Biophys.J. 96:3281-3294), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during steady shortening of single permeabilized rabbit psoas fibers at 12°C (pCa 4.5, pH 7.1, ATP 5.7mM). A fiber contracted from the relaxed state by immersion into a Ca2+ activation solution at 0°C. Temperature activation was then initiated by immersion of the fiber into silicone oil at 12°C. The activation solutions were prepared with either zero added Pi or with 10 mM added Pi at constant ionic strength (0.15 M). The decline in fluorescence intensity emission (470nm) associated with MgADP-dependent dephosphorylation of P∼NDPK-IDCC (60 μM) was monitored during activation and during a period of isovelocity shortening. Fluorescence emission (580 nm) of a rhodamine dye was measured simultaneously to correct the P∼NDPK-IDCC signal for the effects of fiber movements and volume changes. The rate of MgADP release in the absence of added Pi increased from 0.7 ± 0.07 mM·s−1 at 0.2 fiber-lengths·s−1 to approximately 3.4 ± 0.25 mM·s−1 for shortening velocities between 1 and 2 fiber-lengths·s−1. When 10 mM Pi was added, the rate of ADP release at 0.2 fiber-lengths·s−1 was 0.48 ± 0.05 mM·s−1 and 2.6 ± 0.4 mM·s−1 at 1-2 fiber-lengths·s−1. In the absence of added Pi, the rate of ATP hydrolysis calculated from the appearance of ADP is similar to that calculated previously from the appearance of Pi using MDCC-labeled phosphate binding protein, over the same range of fiber shortening speeds (He et al., 1999, J. Physiol. 517: 839-854). Thus Pi slows the ATPase rate by 25-30%, both in the isometric and isotonic state. The energetic consequences will be discussed.

A Second Look at the Two Phases of Ca2+ Binding to Fast Skinned Fibers

Experiments that replace native TnC with TnCdanz in skinned rabbit psoas muscle fibers generate pCa/Ca2+ binding curves that are left of and roughly parallel to the pCa/force curve (Guth & Potter, 1987). The fluorescence curve best fits two binding phases, #1 with a slope of about one and #2 with a slope of about 3 or more, a cooperative binding that is associated with major force development (Allen et al, 1992, Huang et al, 2001). Phase 1 is also accompanied with subtle increments in force. The force, fluorescence, phase 1 & 2 curves synthesized from mean fitted parameters for 22 experiments are shown. To demonstrate the difference between force and binding, we subtract the force from the fluorescence. The difference curve indicates that phase 1 Ca2+ binding dominates at high pCa then decreases as cooperative binding increases. We argue that the regulatory sites are shifting from normal to cooperative binding and this is why the phase 2 parameters can only approximate force. Because phase 2 binding begins on top of phase 1 the fluorescence curve is shifted left away from force.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Correlation between Myofibrillar Biochemistry and Muscle Fiber Mechanics using Rabbit Psoas Muscle Preparations Indicates that Phase 2 of Step Analysis Represents the Cross-Bridge Detachment Step

Our goal is to correlate results obtained from myofibrillar suspensions and muscle fibers. For myofibrils, tryptophan fluorescence with stopped-flow apparatus was used; for fibers, tension transients with small amplitude sinusoidal length perturbations were used. Experiments were performed in identical solutions with 0.2M ionic strength at pH 7.00. The concentration of MgATP was varied to detect kinetic constants of the ATP binding step 1 (K1: dissociation constant), the cross-bridge detachment step 2 (k2, k-2: rate constants), and the ATP cleavage step 3 (k3, k-3). By following the fast rate constant at 20°C, we found in myofibrils: k2/K1=1.0∈μM−1s−1, K1=0.3mM, k2=300s−1, and k-2≈0; in fibers: k2/K1=0.23∈μM−1s−1, K1=1.58mM, k2=363s−1, k-2=180s−1. From these results we conclude that (1) ATP binding is ∼5X stronger in myofibrils than in fibers, (2) cross-bridge detachment rate is just about the same, and (3) its reversal step is almost absent in myofibrils, but it is finite in fibers. Consequently, we found a good agreement in the results obtained from myofibrils and fibers, indicating that phase 2 of tension transients from step analysis in fibers (Huxley and Simmons, 1971) represents the cross-bridge detachment step. We also studied actin-myosin cross-linked myofibrils and found no difference, indicating that cross-linking does not significantly modify steps 1-3 kinetics. We further studied the Pi effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP with the inhibitory dissociation constant of 7-8mM. To deduce the kinetic constants of the ATP cleavage step, we measured the slower rate constant in fluorescence in myofibrils and found that k3+k-3=10.7s−1 at 4°C. From the Pi burst experiments using radioactive ATP, we found that K3=6.1 at 4°C. From these, k3=9.2s−1 and k-3=1.50s−1 were deduced.

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

The goal of this study was to develop a system to experiment with sarcomeres mechanically isolated from skeletal muscles. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (22 degrees C or 15 degrees C) experimental chamber, and sarcomeres were isolated using precalibrated glass microneedles that were pierced externally, adjacent to the Z-lines. The force produced during activation was measured by tracking the displacement of the microneedles, and the sarcomere and half-sarcomere changes were measured by continuously tracking the Z-lines and A-bands position during the experiments. Sarcomeres produced a stress (force/cross-sectional area) of 112.75 +/- 4.96 nN/microm(2) (15 degrees C) and 128.47 +/- 5.58 nN/microm(2) (22 degrees C) at lengths between 2.0 microm and 2.4 microm. The descending limb was fitted with linear regression for length between 2.4 microm and 3.5 microm, which provided an abscissa extrapolating to 3.87 microm. The force-length relation was remarkably similar to a theoretical curve based on the degree of filament overlap. During sarcomere activation, we tracked the distance between the center of the A-band and the Z-lines. At lengths below 1.6 microm, movements of A-band were not detected. A-band movements increased with length to achieve a maximum displacement of 59.40 +/- 10.1 nm from the center at 2.0 microm-2.4 microm. A-band displacement decreased linearly in sarcomere lengths between 2.6 microm and 3.6 microm. A technique for monitoring force and length in single sarcomeres isolated from myofibrils represents a reliable technique to evaluate contractile mechanisms at the most basic, intact level of muscle organization, opening the possibility to clarify long-standing issues in the field of muscle contraction.

Cardiomyopathy Mutations Reveal Variable Region of Myosin Converter as Major Element of Cross-Bridge Compliance

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the β-myosin heavy chain, 723Arg → Gly (R723G) and 736Ile → Thr (I736T), in comparison to 719Arg → Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S∗rigor R723G = 0.84 pN/nm S∗rigor WT = 0.29 pN/nm); and ii), a significant increase in force per head (F∗10°C, 1.99 pN vs. 1.49 pN = 1.3-fold increase; F∗20°C, 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S∗active10°C, 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S∗rigor; 1.8-fold increase in F∗10°C, 1.6-fold in F∗20°C; twofold increase in S∗active10°C). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous findings that for the slow myosin isoform S∗ and F∗ are significantly lower than for fast myosin e.g., of rabbit psoas muscle. The data indicate that two mutations, R723G and R719W, are associated with an increase in resistance to elastic distortion of the individual mutated myosin heads whereas mutation I736T has essentially no effect. The data strongly support the notion that major elastic distortion occurs within the converter itself. Apparently, the compliance depends on specific residues, e.g., R719 and R723, presumably located at strategic positions near the long α-helix of the light chain binding domain. Because amino acids 719 and 723 are nonconserved residues, cross-bridge stiffness may well be specifically tuned for different myosins.

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.

Protein kinase A–dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during β-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.