Structural changes in the actomyosin cross-bridges associated with force generation.

It is generally thought that to generate active force in muscle, myosin heads (cross-bridges) that are attached to actin undergo large-scale conformational changes. However, evidence for conformational changes of the attached cross-bridges associated with force generation has been ambiguous. In this study, we took advantage of the recent observation that cross-bridges that are weakly attached to actin in a relaxed muscle are apparently in attached preforce-generating states. The experimental conditions were chosen such that there were large fractions of cross-bridges attached under relaxing and activating conditions, and high-resolution equatorial x-ray diffraction patterns obtained under these conditions were compared. Changes brought about by activation in the two innermost intensities, I10 and I11, did not follow the familiar reciprocal changes. Instead, there was almost no change in I11, whereas I10 decreased by 34%. Together with the changes found in the higher-order reflections, the results suggest that the structure of the attached force-generating cross-bridges differs from that of the weakly bound, preforce-generating cross-bridges and possibly also differs from that of the cross-bridges in rigor. These observations support the concept that force generation involves a transition between distinct structural states of the actomyosin cross-bridges.     

On the mechanism of actomyosin ATPase from fast muscle.

The labeled inorganic phosphate formed by enzymatic hydrolysis of [gamma-18O]ATP in normal water was derivatized to trimethyl phosphate and analyzed for the proportions of [18O3]Pi, [18O2]Pi, [18O1]Pi, and [18O0]Pi. The proportions observed were correlated with the kinetics of intermediate exchange by using a kinetic relationship in which it is assumed that binding of ATP and subsequent release of products are irreversible. Actomyosin and acto-heavy meromyosin catalyze intermediate exchange at a mean rate that is more than 1 order of magnitude slower than that predicted by rapid kinetic studies or implied by the essentially complete intermediate exchange observed with myosin alone. The reason for the slow apparent exchange is that there are two ATPase activities with different exchange properties. The effect of varying heavy meromyosin concentrations at a constant actin concentration shows that the two activities are interrelated and suggests further that one is due to ATP hydrolysis by the undissociated actomyosin crossbridge.     

Frequency dependent force generation correlates with sarcoplasmic calcium ATPase activity in human myocardium

Objective: In congestive heart failure both a decreased function of the sarcoplasmic Ca²⁺-ATPase and a negative force-frequency relationship have been shown. This study aimed to investigate a possible relationship between frequency potentiation, sarcoplasmic Ca²⁺-ATPase activity, and SERCA2 protein expression in human myocardium. Methods: Frequency potentiation was studied in electrically stimulated, isometric, left ventricular papillary muscle strip preparations (37°C, 0.5–3.0 Hz) from terminally failing (NYHA IV; n=5, dilated cardiomyopathy) and nonfailing (donor hearts, n=5) human myocardium. In the identical samples the Ca²⁺-ATPase activity (NADH coupled assay) and the protein expression of sarcoplasmic Ca²⁺-ATPase (SERCA2), phospholamban, and calsequestrin (western blot) were determined. The frequency dependent change in the force of contraction and V_max of the Ca²⁺-ATPase activity and the protein expression of SERCA2 were correlated with each other. Results: In terminally failing myocardium the force-frequency relationship was negative (2.0 Hz vs. 0.5 Hz: –0.2±0.1 ΔmN) contrasting a positive rate dependent potentiation of force in nonfailing tissue (2.0 Hz vs. 0.5 Hz: +0.8±0.2 ΔmN; p<0.01). In failing myocardium the corresponding maximal sarcoplasmic Ca²⁺-ATPase activity (V_max) was reduced significantly compared to nonfailing myocardium (174±24 vs. 296±31 nmol ATP/mg·min, p<0.01). The protein expression of SERCA2, phospholamban, and calsequestrin remained unchanged in failing myocardium. The maximal Ca²⁺-ATPase activity significantly correlated with the frequency dependent change in force of contraction (2 Hz vs. 0.5 Hz: r=0.88, p=0.001; 3 Hz vs. 0.5 Hz: r=0.84, p=0.004). No correlation between protein expression of SERCA2 and Ca²⁺-ATPase activity or change in force of contraction was observed. Conclusion: Due to a significant correlation between sarcoplasmic Ca²⁺-ATPase activity and frequency potentiation, the negative rate dependent force potentiation in human heart failure could be at least in part be attributed to decreased function of the sarcoplasmic Ca²⁺-ATPase. Received: 8 January 1998, Accepted: 2 June 1998     

Rapid dissociation and reassociation of actomyosin cross-bridges during force generation: a newly observed facet of cross-bridge action in muscle.

The force response of skinned fibers of the rabbit psoas muscle to stretches (and releases) was studied. At physiological ionic strength and low experimental temperature (5 degrees C) the force response to stretches apparently is affected neither by cross-bridges that occupy weak-binding states nor by transitions among various attached force-generating states. Plots of force vs. imposed length change (T plots) recorded during stretches suggest that cross-bridges even in force-generating states dissociate and reassociate rapidly from and to actin as had previously been proposed [Brenner, B. (1986) Basic Res. Cardiol. 81, 1-15]. Plots of fiber stiffness vs. speed of imposed length changes (stiffness-speed relations) imply rate constants for dissociation (k-) in the force-generating states ranging from 50 to 1000 s-1, while the rate constant for reassociation (k+) has to be at least an order of magnitude larger (high actin affinity). Rapidly reversible actin interaction of cross-bridges in force-generating states provides a mechanism for rapid detachment of force-generating cross-bridges during high-speed shortening which, in contrast with the hypothesis of A. F. Huxley [(1957) Prog. Biophys. 7, 255-318], and related cross-bridge models, does not require completion of the ATP-hydrolysis cycle and thus may account for the unexpectedly low ATPase activity during high-speed shortening.     

Inhibition of actomyosin ATPase by vanadate.

Actin-myosin subfragment-1 (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) under conditions such that ADP alone does not dissociate the complex. The association constant of the stable complex M.ADP.Vi, in which M indicates myosin [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. USA 76, 2620-2624] with actin is smaller than the average association constant of the intermediate states of the actin-SF-1 ATPase cycle. Actin-SF-1 ATPase activity is 90% inhibited by ADP plus vanadate. The reaction of actin with M.ADP.Vi produces a slow release of ADP and vanadate and quantitative recovery of ATPase activity. The rate of dissociation of ligands was almost linear in actin concentration; consequently, the rate constant of dissociation could only be roughly estimated as 0.5-1 sec-1. The rate of dissociation of ADP and vanadate is thus increased by a factor of 10(5) compared to M.ADP.Vi. The rate of release of ligands by regulated actin (actin-tropomyosin-troponin) was reduced to 1/10th to 1/20th by removal of calcium ion. Therefore the M.ADP.Vi complex has the properties of a more stable analogue of the myosin-ADP-phosphate complex that is generated in the normal ATPase cycle. The activation of ligand release (ratio of rate of dissociation of ADP and vanadate from actomyosin relative to myosin) is much larger than the activation of myosin ATPase by actin, whereas the actual rates of the reactions are much slower.     

ATPase activity and proteolysis of human myocardial actomyosin in ischemia and hypertrophy

Recently we have shown that actomyosin ATPase activity decreases with age when measured under appropriate conditions (Yoshida K et al, Age 12: 97-102, 1989). Many previous studies, which examined changes in ATPase activity in myosin, actomyosin, or myofibrils under pathological states, ignored the age-related changes. In this study actomyosin was isolated from myocardia of middle-aged subjects (37-49 years old) and examined for ATPase activity under various conditions and protein composition. Proteolysis of myosin and troponin was more frequently observed in ischemic heart disease (IHD) subjects than in non-IHD subjects. The proteolysis was associated with a decrease in Ca2+ sensitivity of Mg2(+)-ATPase activity and enhanced stimulation of Ca2(+)-ATPase activity with a sulfhydryl reagent, N-ethylmaleimide. Hypertrophy appeared not to significantly affect ATPase activity.     

Characterization of actomyosin bond properties in intact skeletal muscle by force spectroscopy

Force generation and motion in skeletal muscle result from interaction between actin and myosin myofilaments through the cyclical formation and rupture of the actomyosin bonds, the cross-bridges, in the overlap region of the sarcomeres. Actomyosin bond properties were investigated here in single intact muscle fibers by using dynamic force spectroscopy. The force needed to forcibly detach the cross-bridge ensemble in the half-sarcomere (hs) was measured in a range of stretching velocity between 3.4 x 10(3) nm.hs(-1).s(-1) or 3.3 fiber length per second (l(0)s(-1)) and 6.1 x 10(4) nm.hs(-1).s(-1) or 50 l(0).s(-1) during tetanic force development. The rupture force of the actomyosin bond increased linearly with the logarithm of the loading rate, in agreement with previous experiments on noncovalent single bond and with Bell theory [Bell GI (1978) Science 200:618-627]. The analysis permitted calculation of the actomyosin interaction length, x(beta) and the dissociation rate constant for zero external load, k(0). Mean x(beta) was 1.25 nm, a value similar to that reported for single actomyosin bond under rigor condition. Mean k(0) was 20 s(-1), a value about twice as great as that reported in the literature for isometric force relaxation in the same type of muscle fibers. These experiments show, for the first time, that force spectroscopy can be used to reveal the properties of the individual cross-bridge in intact skeletal muscle fibers.     

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.     

Enhanced force generation by smooth muscle myosin in vitro.

To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was estimated by measuring the density of biochemically active myosin adhered to the surface; with this estimate, the average force per cross-bridge head of smooth and skeletal muscle myosins is 0.6 pN and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately three times greater average force per cross-bridge head than does skeletal muscle myosin.     

Age-related activity changes in actomyosin ATPase and arginine phosphokinase in male Drosophila melanogaster Meig

The specific activity of each of the two enzymes primarily concerned with the energy metabolism of flight muscle contraction was determined in male Drosophila melanogaster, from emergence through 5 weeks postemergence. Ca++-activated actomyosin ATPase activity rose rapidly from a minimum at emergence to a peak level at 4-5 days, falling gradually thereafter to a minimum level at 10-11 days postemergence, with no change thereafter. Arginine phosphokinase activity rose more gradually to a maximum at 11-12 days postemergence and then fell slowly to a minimum at 18-19 days. These data represent a sequential pattern of biochemical changes similar to those previously observed in other dipteran species, confirming the primary role of a genetic program for maturation and senescence of flight ability in holometabolous adult insects at the functional and underlying cellular, biochemical levels.     

Effect of sulfhydryl group modification on age-associated alteration of actomyosin ATPase activity in human myocardium

Summary Cardiac ventricular actomyosin was prepared from autopsy samples from humans ranging in age from one to 83 years, and its Ca²⁺-ATPase and K⁺-EDTA-ATPase activities were determined in the presence or absence of a sulfhydryl reagnet, N-ethylmaleimide (NEM). The Ca²⁺-ATPase activity increased in the presence of appropriate concentrations of NEM. The extent of the stimulation of Ca²⁺-ATPase activity by NEM decreased significantly with age. The ratio of K⁺-EDTA-ATPase activity to Ca²⁺-ATPase activity also decreased with age. This suggests that there is an age-related modification of sulfhydryl groups in the myosin molecule.     

Effect of pH and temperature on age-associated alteration of actomyosin ATPase activity in human myocardium

The K⁺-EDTA-ATPase and Ca²⁺-ATPase activities of cardiac ventricular actomyosin prepared from autopsy samples from subjects of various ages were determined. We could not detect differences in the K⁺-EDTA-ATPase or Ca²⁺-ATPase activity of actomyosin prepared from the hearts of young (0–30 yrs.), middle-aged (31–60 yrs.) and old (61-yrs.) subjects at pH 7.0 and at 30°C conditions widely used in previous studies. However, there were significant decreases in both ATPase activities of actomyosin isolated from old and middle.aged subjects, compared with young subjects at pHs 6.0, 8.0 and 9.0 (at 30°C or at 37°C (at pH 7.0). The alkaline lability of the ATPase activities also increased significantly with age. There has been much debate as to whether or not there is a relationship between age and myocardial myosin ATPase activity. In many studies regarding myocardial myosin ATPase activity of hypertrophied or failed hearts, the age of the subjects was ignored. Our results draw attention to the effect of aging on the myocardial actomyosin ATPase activity and the importance of choosing appropriate assay conditions.     

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin–actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure–function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin–actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

Many physiological processes are driven by mechanical force generated through myosin–actin interactions, such as muscle contraction, vesicle trafficking, and membrane deformation (1, 2). It is remarkable that actomyosin carries out these diverse functions through a conserved mechanochemical cycle, in which the chemical energy from ATP hydrolysis is used to generate force via myosin motor domain conformational changes (3, 4).In muscles, the basic contractile apparatus is formed primarily by myosin (thick) and actin (thin) filaments, which slide past each other to contract the muscle fiber (7). The interaction between myosin head and actin powered by ATP results in the cross-bridge formation (8). Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), two leading causes of cardiac death, can often be due to mutations in sarcomeric proteins (9). HCM mutations cause a hypercontractile state of the sarcomere, whereas DCM mutations are linked to ventricular dilatation and loss of systolic function. Studies have shown that HCM and DCM mutations in myosin give rise to changes in the basic physical parameters of the mechanochemical cycle (10, 11) or the number of available myosin heads to interact with actin (12, 13). Several small molecules targeting the myosin motor domain (14–16) have been developed to treat these cardiac diseases, but clear molecular bases for the drug effects remain to be established (17).The actomyosin functional cycle includes two major stages: a force-generating stage in which myosin swings its lever arm and stays engaged with actin (Fig. 1A) and a recovery stage in which myosin returns the lever arm to a primed configuration and stays detached from the actin filament. The force generation stage involves a few key processes, i.e., formation of a tight myosin–actin interface, actin-binding cleft closure, lever arm swing, and ATP hydrolysis product (phosphate and ADP) release. Despite extensive structural, biochemical, and single-molecule studies, see reviews (6, 18), the causality and ordering of these events are difficult to characterize. Recent cryo-EM structures provide atomistic views of different myosin–actin isoforms at the strongly bound rigor state (19–22), in which no nucleotide is bound to the myosin active site. Fewer mechanistic details are known for the early binding events and the powerstroke transition. So far, no actomyosin structures have been solved for the prepowerstroke (PPS) state due to its weak binding nature. Although it is generally believed that myosin attachment to actin leads to phosphate (Pi) release and powerstroke, the timing of Pi release and lever arm motion is still equivocal. A FRET study on myosin V (23) suggested that the initial contact triggers a fast lever arm motion, followed by a slower stroke swing after Pi release and before ADP release. Later on, a high-resolution optical tweezers experiment on cardiac myosin (24) demonstrated that the powerstroke rate is much faster than the estimated Pi release rate.Open in a separate windowFig. 1.Modeling of multiple actomyosin states. (A) Simplified scheme for the force-generation stage, during which the myosin motor transitions from the prepowerstroke (PPS, orange) to the rigor (red) states. (B) The flowchart of comparative modeling with Rosetta. (C) A specific case in which structural models for actin-bound human cardiac myosin at the PPS state are obtained by using two templates—5N69 (bovine cardiac muscle) and 5H53 (rabbit skeletal muscle). The former is a crystal structure of an isolated myosin head at the PPS state (orange), whereas the latter is a cryo-EM structure of the actin (gray)–myosin (red) complex at the rigor state. Five Rosetta models are displayed for the PPS ensemble, after aligning the complex to the upper actin molecule (myosin motor in orange; actin in gray).A clear atomistic-level picture for the allosteric regulation encoded in the motor domain is crucial to understanding how small-molecule drugs impact myosin function and to help design and optimize molecules targeting allosteric sites based on key intermediate states. Recently, a machine learning-based method, AlphaFold2, has successfully demonstrated the ability in predicting protein folds (25) and multimeric interfaces (26) given a query sequence. However, this approach is limited in studying the actomyosin cycle, due to its inability to handle ligand and mutation effects as well as multiple conformational states and transitions (27, 28). Developed over decades, all-atom molecular dynamics (MD) simulations have become powerful in studying the mechanisms of biomolecular machines (29–31). By combining Rosetta comparative modeling and Gaussian accelerated molecular dynamics (GaMD) (32), we develop a computational approach that characterizes the conformational ensembles of actomyosin at different ligand states. Our results reveal the coupling between actin-binding cleft closure and structural rearrangement at the active site. The population distribution of the PPS state along the conformational coordinates of the motor domain is shifted to that of the rigor state, as a consequence of hydrolysis product release. The predicted actin–myosin interactions agree with the existing rigor cryo-EM structures and mutagenesis studies. Our work highlights the principles underlying force-producing mechanisms of myosin–actin systems.     

Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step.

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-1) with actin must dissociate into S-1.ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state ATPase rate. We find that the rate of the initial Pi burst markedly increases at high actin concentration although the Lymn-Taylor model predicts the rate should remain nearly constant or decrease. In addition, at high actin concentration, the magnitude of the initial Pi burst is much larger than is predicted by the Lymn-Taylor model. Finally, at 360 microM actin, at which more than 90% of the S-1.ATP is bound to actin, there is no inhibition of the steady-state ATPase activity although the Lymn-Taylor model predicts that 70% inhibition should occur. We conclude that the acto-S-1 complex is not dissociated by ATP during each cycle of ATP hydrolysis; in fact, the rate of the initial Pi burst appears to be even faster when S-1.ATP is bound to actin than when it is dissociated.     

On the origin and transmission of force in actomyosin subfragment 1.

A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstruction of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crosslinkings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site--i.e., as indications of how energy is transduced in this system.     

Estimate of cellular force generation in an arterial smooth muscle with a high actin: myosin ratio

An in vitro preparation from the media of the pig carotid artery develops somewhat higher force/cell cross-sectional area with one-fifth the myosin content of skeletal muscle cells. The following results suggest that this performance reflects cellular properties rather than the arrangement of cells within the tissue: (1) force development at the peak of the length-force curve is independent of the length of the tissue segment in a strip of constant cross-section, and (2) average cell length is directly proportional to tissue length. We conclude that the contractile system of arterial smooth muscle cells is specialized for force generation and that the mechanical properties of the pig carotid media preparation provide valid estimates of cellular function.     

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

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

Altered cations and muscle membrane ATPase activity in deoxycorticosterone acetate-salt spontaneously hypertensive rats.

The role of ions and cell membrane function in the pathogenesis of benign and malignant hypertension was investigated in spontaneously hypertensive rats (SHR). Ten-week-old male SHR (n = 50) and SHR treated with deoxycorticosterone acetate (DOCA; n = 70) and 1% NaCl drinking water were studied weekly for 14 weeks. Malignant hypertension developed only in DOCA-salt SHR and was characterised by severe hypertension, failure to thrive and renal fibrinoid necrosis. Fourteen DOCA-salt SHR and one SHR died. Extracellular (serum) and intracellular (erythrocyte and muscle) Na+, K+, Mg2+, Ca2+ and muscle membrane Na+,K(+)-adenosine triphosphatase (ATPase), Ca(2+)-ATPase and Mg(2+)-ATPase were measured at various stages in the development of malignant hypertension. Three developmental phases were defined: benign, premalignant and malignant. DOCA-salt SHR showed persistent hypokalaemia. In the benign phase, there were no differences in Na+, Mg2+ and Ca2+ between SHR and DOCA-salt SHR. In the premalignant phase, serum and erythrocyte Mg2+ and ATPase activity were significantly lower in DOCA-salt SHR compared with SHR. During the late premalignant and malignant phases, intracellular Ca2+ and Na+ were significantly higher in the DOCA-salt SHR compared with SHR. In view of these findings, the abnormalities in DOCA-salt SHR during the early phases of blood pressure elevation could be contributory factors to the development of malignant hypertension.     

Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1).ATP complex.

In our model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1.ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca2+, S-1.ATP should not turn on the regulated actin filament. In the present study, we tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM X S-1) so that functionally it acts like S-1.ATP, although it does not hydrolyze ATP. We found that, in the absence of Ca2+, neither S-1.ATP nor pPDM X S-1.ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto X S-1). In contrast, in the presence of Ca2+, pPDM X S-1.ATP binding almost completely turns on the regulated acto.S-1 ATPase activity. These results can be explained by our original cooperativity model, with pPDM X S-1.ATP binding only approximately equal to 2-fold more strongly to the turned-on form than to the turned-off form of regulated actin. However, our results are not consistent with our alternative model, which predicts that if pPDM X S-1.ATP binds to actin in the absence of Ca2+ but does not turn on the ATPase activity, then it should also not turn on the ATPase activity in the presence of Ca2+.     

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.