Rigor Stiffness Research Articles

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers.

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb-) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb- fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb- fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K 2), its rate (k 2), and the rate of the reverse force generation step (k -4) are larger in Neb+ fibers than in Neb- fibers. The rates of the force generation step (k 4) and the reversal detachment step (k -2) change in the opposite direction. These effects can be explained by Le Chatelier's principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

Effect of Antibodies to Myosin Head on the Development of Rigor Tension and Stiffness in Skinned Muscle Fibers

Using three antibodies to myosin head, attaching to (1) distal region and (2) proximal region of myosin head catalytic domain, and (3) to myosin head lever arm domain, respectively, we have shown definite differences between in vitro actin-myosin sliding and muscle contraction. In the present study, we studied the effect of these antibodies on the development of rigor tension and stiffness in single skinned muscle fibers at pCa>9. To form rigor actin-myosin linkages, myosin heads should override tropomyosin, covering myosin-binding sites on actin, and to detach antibodies from them. Despite their different attachment sites in myosin head, all these antibodies slowed down development of rigor tension and stiffness with or without changing their peak values. The rigor tension versus stiffness relation was highly variable, suggesting that the rigor tension reflects the sum of tension in individual rigor linkages, while rigor stiffness represents the total number of rigor linkages. Dummy antibody had no effect on the development of rigor state. These results indicate that the action of myosin heads overriding tropomyosin is inhibited by the antibodies, so that development of rigor state is slowed down due to gradual detachment of the antibodies from individual myosin heads. Highlights The effect of three antibodies, attaching to different regions in myosin heads on the development of rigor state was examined at pCa >9, using single skinned muscle fibers. Despite their different binding sites on myosin, all the antibodies slowed down development of rigor tension and stiffness with or without changes in their peak values. The rigor tension versus stiffness relation was highly variable, suggesting that rigor tension reflects the sum of tension generated by individual myosin heads, while stiffness serves as a measure of total number of rigor linkages. These results indicate that the antibodies inhibit myosin head movement to override tropomyosin, and detachment of the antibodies from myosin heads is necessary prerequisite for rigor linkage formation.

Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and β-myosin heavy chain with sinusoidal analysis.

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express β-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing~99% α-MHC were treated with propylthiouracil to result in 100% β-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and β-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5mM MgATP, 1mMMg2+, 8mM Pi, 200mM ionic strength, pH 7.00 at 25°C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200ms), and effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (10-15%) in β-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca2+ activation) demonstrated that, the apparent rate constants were 2-4×faster in α-MHC containing fibers. The ATP study demonstrated that, in β-MHC containing fibers, K 1 (ATP association constant) was greater (1.7×), k 2 and k -2 (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in β-MHC containing fibers, k 4 (rate constant of the force-generation step) and k -4 were smaller (0.75× and 0.25×, respectively), resulting in greater K 4 (3×). There were no differences in active tension, rigor stiffness, or K 2 (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K 5 (Pi association constant).

Cross-Bridge Kinetics in Rat Papillary Muscle Fibers that Carry α-MHC and β-MHC by Sinusoidal Analysis

In mammalian heart muscles, two isoforms of MHC were identified. Small animals carry primarily α-MHC (fast), whereas large animals (e.g., bovines and humans) carry mostly β-MHC (slow), resulting in a large difference in the heart rate. Sprague-Dawley rats, which possess 99% α-MHC in their ventricles, were treated with propylthiouracil to result in 100% β-MHC, as demonstrated by SDS-PAGE. Papillary muscles were dissected, skinned, split into small fibers (90-110 µm diameter), and mounted on experimental apparatus. To understand the functional difference between α-MHC and β-MHC, fibers were activated under intracellular ionic conditions of cardiomyocytes: 5mM ATP, 1mM Mg2+, 8mM phosphate (Pi), 200mM ionic strength, and pH 7.0. These measurements were carried out at 25°C. When steady tension developed, small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200 ms), and the effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (12%) in β-MHC than α-MHC, while cooperativity was not statistically different. Sinusoidal analysis demonstrated that, in β-MHC containing fibers, K1 (ATP association constant) was greater (1.7x), k2 and k-2 (cross-bridge detachment and its reversal rate constants) were smaller (0.6x); k4 (rate constant of the force-generation step) and k-4 (its reversal step) were smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K4 (equilibrium constant of the force generation step) than those of α-MHC. There were no differences in K2 (equilibrium constant of the cross-bridge detachment step), K5 (Pi association constant), active tension, or rigor stiffness. Our study also demonstrated that there were no differences in the above mentioned parameters between fibers obtained from left and right ventricles, indicating that there is no difference at the myofilament level in both ventricles.

Alternative Versions of the Myosin Converter Vary Cross-Bridge Stiffness and Muscle Force Generation

The converter has been proposed to be the myosin elastic element that sets cross-bridge stiffness. According to most cross-bridge models, the elastic element's stiffness should directly influence muscle force generation. When the elastic element is stretched by the power stroke, a stiffer element should increase force generation. We tested these hypotheses by using the natural variation in converter regions found in different Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative versions of exon 11. We modified the gene to express alternative versions in Drosophila indirect flight muscle (IFM) fibers instead of the wild-type 11a version. Using 0.125% muscle length sinusoidal oscillations at 500 Hz, we found that two of the four alternative versions, 11b (found in larval body wall muscles) and 11c (jump muscle) significantly decreased fiber rigor stiffness by 28% and 20%, respectively, indicating a decrease in cross-bridge stiffness. The changes in rigor stiffness (elastic modulus) roughly correlated (r2 = 0.57) with force production by the fibers because 11b and 11c fibers also produced the lowest isometric tension. The correlation improved (r2 = 0.71) when it was corrected for the influence of cross-bridge duty ratio on isometric tension. Relative duty ratio was estimated from the frequency at which maximum power was generated (fmax), and the muscle apparent rate constant 2πc. Converters 11b, 11d, and 11e decreased fmax by 26%, 34% and 43%, respectively. Thus, we conclude that the converter is at least part of the elastic element that sets cross-bridge stiffness, and that this element helps set natural variation in force production between muscle types. Supported by NIH grant R01 AR055611 to D.M.S.

Myofilament dysfunction contributes to impaired myocardial contraction in the infarct border zone.

After myocardial infarction, a poorly contracting nonischemic border zone forms adjacent to the infarct. The cause of border zone dysfunction is unclear. The goal of this study was to determine the myofilament mechanisms involved in postinfarction border zone dysfunction. Two weeks after anteroapical infarction of sheep hearts, we studied in vitro isometric and isotonic contractions of demembranated myocardium from the infarct border zone and a zone remote from the infarct. Maximal force development (Fmax) of the border zone myocardium was reduced by 31 ± 2% versus the remote zone myocardium (n = 6/group, P < 0.0001). Decreased border zone Fmax was not due to a reduced content of contractile material, as assessed histologically, and from myosin content. Furthermore, decreased border zone Fmax did not involve altered cross-bridge kinetics, as assessed by muscle shortening velocity and force development kinetics. Decreased border zone Fmax was associated with decreased cross-bridge formation, as assessed from muscle stiffness in the absence of ATP where cross-bridge formation should be maximized (rigor stiffness was reduced 34 ± 6%, n = 5, P = 0.011 vs. the remote zone). Furthermore, the border zone myocardium had significantly reduced phosphorylation of myosin essential light chain (ELC; 41 ± 10%, n = 4, P < 0.05). However, for animals treated with doxycycline, an inhibitor of matrix metalloproteinases, rigor stiffness and ELC phosphorylation were not reduced in the border zone myocardium, suggesting that doxycycline had a protective effect. In conclusion, myofilament dysfunction contributes to postinfarction border zone dysfunction, myofilament dysfunction involves impaired cross-bridge formation and decreased ELC phosphorylation, and matrix metalloproteinase inhibition may be beneficial for limiting postinfarct border zone dysfunction.

Optical Trapping Shows a 3-Fold Increase in Myosin Head Stiffness by the FHC-Mutation R723G in the β-Cardiac Myosin Heavy Chain

Missense mutations in the β-cardiac myosin heavy chain (β-MyHC) are the most frequent cause of Familial Hypertrophic Cardiomyopathy (FHC). For these mutations mutant and wildtype β-MyHC are co-incorporated in the contractile apparatus, and direct functional effects of such mutations presumably trigger development of the FHC-phenotype. In M. soleus biopsies of affected patients we previously found increased isometric force generation for the converter mutation R723G, most likely due to increased stiffness of the myosin head domain. Quantification of the increase in head stiffness from fiber studies, however, was complicated by filament compliance, as well as non-equal and variable co-expression of mutant and wildtype protein in the tissue samples.In the present work we directly determined stiffness of individual myosin heads isolated from M. soleus tissue samples of affected patients using the three-bead optical trapping assay. Since each assay contained mutant and wildtype head domains, stiffness of individual heads with and without the R723G mutation could directly be compared. We determined head stiffness by imposing triangular stage displacements and recording bead displacements during binding events, taking into account compliance of the actin-bead link determined by the variance-Hidden-Markov method (Smith et al., BJ 2001). We found two populations of head domains, one with a head stiffness of 0.39 ± 0.24 pN/nm the other with an about 3-fold higher stiffness. The low value is close to our previous optical trapping with β-MyHC (0.38 ± 0.06 pN/nm; Brenner et al., JMRCM 2012), the about 3-fold increase in head stiffness agrees well with our previous estimate from rigor stiffness in fibers with the same mutation (Seebohm et al., BJ 2009) while the absolute value of head stiffness from fibers was about 25% lower than determined here.

The myosin converter region influences cross‐bridge stiffness and muscle kinetics

The converter has been proposed to be the myosin elastic element that sets cross‐bridge stiffness. According to most cross‐bridge models, the elastic element's stiffness should directly influence muscle fiber type force and power generation levels. We tested these hypotheses by using the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative exon versions. We expressed four non‐native versions in Drosophila indirect flight muscle (IFM) and found that two of the three versions, 11b and 11e (found in larval and adult body wall muscles) decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is the critical elastic element of myosin. The converters also influenced IFM fiber and myosin kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased myosin attachment kinetics, however, 11d and 11e also increased detachment kinetics. Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in myosin duty ratio. With little to no force differences, changes in muscle power almost directly correlated with fmax. All three lines displayed reduced wing beat frequency, 11 to 15% less than control lines, with flight performance better correlating with fiber fmax than cross‐bridge stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross‐bridge kinetics and muscle power generation. Supported by NIH grant R01 AR055611 to D.S.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca2+-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM−1·s−1 in F764L/+, 483±74mM−1·s−1 in S532P/+ and 303±18mM−1·s−1 in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s−1 in F764L/+, 32.9±4.5s−1 in S532P/+ and 18.2±1.7s−1 in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

N-Terminal Truncation of Flightin Reduces Oscillatory Power Output without Affecting Cross-Bridge Kinetics in Drosophila Indirect Flight Muscles

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive stiffness and exceptionally fast myosin kinetics, two attributes that enhance power output to sustain flight. Flightin is an IFM-specific, 20kDa myosin rod-binding protein required for normal thick filament stiffness, sarcomere integrity, and flight. Previously, we showed that a COOH-terminal truncation of flightin (flnDeltaC44) decreased myofilament lattice order and myosin kinetics, resulting in lower oscillatory power output and flightlessness. Here, we investigate the function of the flightin N-terminal 62 amino acids by creating transgenic Drosophila (flnDeltaN62) expressing a truncated flightin. flnDeltaN62 flies were flight impaired (flight index: 2.8±0.1 vs. 4.2±0.4 for flnDeltaN62 vs. fln+ rescued null control) despite having a normal wing-beat frequency (195±4 vs. 198±2 Hz for fln+). Mechanical analysis of skinned IFM fibers showed that the flightin N-terminal truncation reduced passive, active, and rigor stiffness without affecting cross-bridge kinetics (frequency of maximum power: 205±7 vs. 217±7 Hz for fln+). flnDeltaN62 fibers produced approximately half the isometric tension (passive: 0.9±0.1 vs. 1.7±0.3 kN/m2, active: 0.8±0.1 vs. 1.5±0.2 kN/m2, rigor: 1.1±0.2 vs. 3.1±0.4 kN/m2) and maximum oscillatory power output (38.0±4.6 vs. 89.5±9.6 W/m3) as fln+ fibers. Moreover, about 60% of the flnDeltaN62 fibers tore in rigor, demonstrating mechanical failure near isometric tension values that were sustained by fln+ fibers. Fourier transform analysis of cross-sectional electron micrographs revealed that the flightin N-terminal truncation compromised myofilament lattice crystallinity and reduced inter-thick filament spacing by 10% (44.1±1.3 vs. 49.7±0.4 nm). These results indicate that the flightin N-terminal region enhances myofilament lattice order and mechanical integrity, which in turn is required for effective force transmission, normal oscillatory power output, and flight.

Myosin Stiffness and Cross-Bridge Kinetics are Influenced by Five Drosophila Alternative Converters

The converter has been proposed to be the myosin elastic element that sets cross-bridge stiffness. According to most cross-bridge models, the elastic element's stiffness should influence muscle fiber type force and power generation levels. We tested these hypotheses by exploiting the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative versions. We forced the four non-native versions to be expressed in Drosophila indirect flight muscle (IFM) and found that two of the three versions tested to date, 11b and 11e (found in larval and adult body wall muscles) significantly decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is at least part of the elastic element of myosin. The converters influenced more than myosin stiffness, as all three converters slowed the super fast IFM fiber kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased apparent rate constant 2πb (set by myosin attachment and Pi release kinetics). However, 11d and 11e increased 2πc (set by detachment kinetics). Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in duty ratio. With little to no force differences, power almost directly correlated with fmax, and the optimal percent ML amplitude for power inversely correlated with fmax. All three lines showed reduced wing beat frequency, with flight performance better correlating with fiber fmax than rigor stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross-bridge kinetics and power generation.

Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation

Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction, providing a model system to examine MLP's mechanism of action. Mlp84B-null flies that survive to adulthood are not able to fly or beat their wings. Transgenic expression of the mlp84B gene in the Mlp84B-null background rescues flight ability and restores wing beating ability. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production and a slight increase in the frequency at which maximum power is generated for fibers lacking Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25% decrease in passive, active, and rigor stiffness compared with rescued fibers, but no significant decrease in isometric tension generation was observed. Muscle ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null fibers have a slight decrease, 11%, in thick filament number per unit cross-sectional area. Our data indicate that MLP contributes to muscle stiffness and is necessary for maximum work and power generation.

Quality Characterization of Farmed Atlantic Halibut During Ice Storage

ABSTRACT:A quality index method (QIM) was developed for farmed Atlantic halibut and together with instrumental, chemical, sensory, and bacteriological analysis, quality changes of halibut stored on ice for 26 d was evaluated. Two groups of fish were fed diets that differed only in the source of lipid, where 1 diet contained only marine oil sources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored on ice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effect on freshness, (ATP) degradation (K‐value), texture, color, or liquid‐holding capacity. The QIM scores increased with storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut. The K‐value was strongly correlated with storage time (r= 0.99), while total bacterial counts increased after 7 to 8 d of ice storage. The texture, liquid‐holding capacity, and color were significantly affected by storage time during the early period of storage, probably due to rigor stiffness and rigor resolution. The texture, liquid‐holding capacity, and color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.

Salmon Fillet Texture is Determined by Myofiber‐Myofiber and Myofiber‐Myocommata Attachment

ABSTRACT: Atlantic salmon (Salmo salar) fillets were stored on ice for a maximum of 14 d to determine which muscle structures are associated with changes in texture and rigor. Texture was measured as shear force and the changes correlated with structural alterations. Texture decreased significantly within 24 h in parallel with loss of attachment of muscle fibers. Loss of rigor stiffness by 5 d was associated with myofiber detachment from the myocommata. These results show that fillet texture depends on several distinct structures and events, initially including breaks in the cell cytoskeleton and loss of fiber‐fiber attachment and later breaks in the connective tissue and fiber detachment from the myocommata.

Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella

Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420–431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 μm of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (±1.4) × 10-7 dyne × cm (2.6 × 10-14 N × m) and the apparent stiffness was 4.3 (±1.3) × 10-10 dyne × cm2 (4.3 × 10-19 N × m2). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions. Cell Motil. Cytoskeleton 49:33–40, 2001. © 2001 Wiley-Liss, Inc.

Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella.

Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420-431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 microm of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (+/-1.4) x 10(-7) dyne x cm (2.6 x 10(-14) N x m) and the apparent stiffness was 4.3 (+/-1.3) x 10(-10) dyne x cm(2) (4.3 x 10(-19) N x m(2)). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions.

Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2.

Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on beta-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca(2+)/force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca(2+)-activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca(2+) sensitivity of active force (leftward shift of the Ca(2+)/force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development. Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a "tether," in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly.

Thiophosphorylation of myosin light chain increases rigor stiffness of rabbit smooth muscle.

1. The effect of thiophosphorylation of the regulatory myosin light chain (MLC20) on rigor stiffness was determined in permeabilized rabbit bladder smooth muscle. 2. Rigor stiffness of alpha-toxin-permeabilized smooth muscle was significantly increased by thiophosphorylation of MLC20. This increase may have been due to partial shortening (melting) in the proximal rod region and/or stiffening of the regulatory domain of the myosin head. 3. We suggest that phosphorylation of MLC20, by increasing the stiffness of the S1 lever arm and/or S2 hinge regions of the myosin molecule, favours separation of the two phosphorylated heads and consequent deinhibition of motor domain activity.

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Electron Tomography of Insect Flight Muscle in Rigor and AMPPNP at 23°C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23°C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23°C reveal many double-headed and some single-headed “lead” bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situcrossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar compari son using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.