Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.

Abstract:The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cellloaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cellfree implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cellloaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.

The contraction of striated muscle (skeletal and cardiac muscle) is generated by ATP-dependent interactions between the molecular motor myosin II and the actin filament. The myosin motors are mechanically coupled along the thick filament in a geometry not achievable by single-molecule experiments. Here we show that a synthetic one-dimensional nanomachine, comprising fewer than ten myosin II dimers purified from rabbit psoas, performs isometric and isotonic contractions at 2 mM ATP, delivering a maximum power of 5 aW. The results are explained with a kinetic model fitted to the performance of mammalian skeletal muscle, showing that the condition for the motor coordination that maximises the efficiency in striated muscle is a minimum of 32 myosin heads sharing a common mechanical ground. The nanomachine offers a powerful tool for investigating muscle contractile-protein physiology, pathology and pharmacology without the potentially disturbing effects of the cytoskeletal—and regulatory—protein environment.

Muscle contraction is driven by a change in the structure of the head domain of myosin, the "working stroke" that pulls the actin filaments toward the midpoint of the myosin filaments. This movement of the myosin heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the myosin head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the head domain. The observed mass distribution matches that seen in electron micrographs of isolated myosin filaments in which the heads are folded back toward the filament midpoint. Following electrical stimulation, the heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the myosin filaments on activation, including the loss of helical and axial order of the myosin heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of myosin heads to actin in a conformation that is the same as that during steadystate isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.C ontraction of skeletal muscles is driven by a cyclical interaction between myosin and actin, fueled by the hydrolysis of ATP. The myosin and actin are polymerized into parallel thick and thin filaments, which themselves are organized into a hexagonal array in the muscle cell. The head domains of myosin lie on the surface of the thick filaments and bind to actin in the thin filaments. Filament sliding is driven by a change in conformation of the actin-bound myosin head: its working stroke (1-3). A detailed molecular model for the working stroke has been derived from biochemical and structural studies of isolated myosin head domains and their interaction with actin and ATP (3-6), and the quasi-crystalline organization of myosin and actin in muscle has allowed this model to be tested and elaborated by mechanical and structural studies on muscle cells (1, 2, 7-11).Many of these cell-based studies used rapid perturbations to synchronize the actions of the myosin heads in a muscle cell. Typically, the length of an active muscle fiber was rapidly decreased, displacing each set of myosin filaments by a few nanometers with respect to the opposing actin filaments (2). Such a shortening step produces an elastic force decrease during the step, followed in the next few milliseconds by rapid force regeneration driven by the working stroke in actin-attached myosin heads (2,7,8). This and related protocols have revealed fundamental properties of the working stroke, including its size, speed, and load dependence, and shown how ...

Muscle contraction is due to myosin motors that transiently attach with their globular head to an actin filament and generate force. After a sudden reduction of the load below the maximum isometric force (T0), the attached myosin heads execute an axial movement (the working stroke) that drives the sliding of the actin filament toward the center of the sarcomere by an amount that is larger at lower load and is 11 nm near zero load. Here, we show that an increase in temperature from 2 to 17°C, which increases the average isometric force per attached myosin head by 60%, does not affect the amount of filament sliding promoted by a reduction in force from T0 to 0.7T0, whereas it reduces the sliding under low load by 2.5 nm. These results exclude the possibility that the myosin working stroke is due to the release of the mechanical energy stored in the initial endothermic force-generating process and show that, at higher temperatures, the working stroke energy is greater because of higher force, although the stroke length is smaller at low load. We conclude the following: (i) the working stroke is made by a series of state transitions in the attached myosin head; (ii) the temperature increases the probability for the first transition, competent for isometric force generation; and (iii) the temperature-dependent rise in work at high load can be accounted for by the larger free energy drop that explains the rise in isometric force. muscle contraction ͉ muscle energetics ͉ myosin working stroke F orce and shortening in muscle are generated by cyclical interactions between the globular part of the myosin molecule (the myosin head) extending from the thick myosin filament and the thin actin filament. During each interaction, an interdomain structural change in the myosin head (the working stroke) produces a pull on the actin filament toward the center of the myosin filament, while one molecule of ATP is hydrolyzed (1, 2). The reduction of force from the steady isometric force T 0 to a load T induces a two-phase response in the attached myosin heads: first the instantaneous recoil of the elasticity in the myofilaments and in the myosin heads (phase 1) and then the rapid isotonic shortening due to the synchronous execution of the working stroke in the attached myosin heads (phase 2). Because phase 2 shortening occurs under isotonic conditions, it is not influenced by the myofilament compliance, and its amount (L T ) directly measures the size of the myosin working stroke under the load T (3, 4).A rise in temperature increases the isometric force developed on average by an attached myosin head (5), revealing the endothermic nature of isometric force generation (6-12). In contrast, shortening is known to be exothermic (13-16), but the temperature dependence of the steps involved in the execution of the working stroke itself is not known. Because the chemomechanical transduction process is unique, we could expect that the thermal energy captured in the reaction responsible for isometric force determines the energy released duri...

Between 1960 and 1991, 156 episodes of central nervous system (CNS) bleeding were documented in 106 patients from a total population of 1,410 hemophiliacs (7.5%). Ninety-one hemophilia A patients presented 131 bleeding episodes; 15 hemophilia B patients had 25 episodes. 32% of these episodes took place in patients less than 5 years of age. 46 % were age 10 or less, and 72% were age 20 or less. The mean age was 14.8 years in hemophilia A and 9 years in hemophilia B patients. A significant increase in the mean age of hemophilia A patients has been observed over the last 10 years; this may be related to HIV infection. A history of recent trauma was documented in 39.7% of the episodes. Spontaneous CNS bleeding was predominant in severe hemophilia (85.2%). One hundred and fifty-four CNS bleeding episodes were intracranial and 2 intraspinal. Of the intracranial episodes, 37.7% were subarach-noid, 29.8 subdural, and 22.7% intracerebral. Factor VIII or IX inhibitors were present in 11.3% of the patients; this figure is slightly lower than that observed in our total hemophilic population. Over 50% of the patients had psychoneurological sequelae; the most frequent were seizure disorders and motor impairment. The overall mortality rate was 29.2%. The mortality was more closely related to the CNS bleeding site than to the severity of hemophilia. Treatment should be based on prompt and prolonged replacement therapy to ensure hemostatic levels of antihemophilia factors.

Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4• C and 2.15 μm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T 0 ) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (S M3 and S M6 ) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere) −1 shortening at the velocity (V 0 ) that maintains zero force. Shortening at V 0 transiently reduces S M3 following the latent period and delays the subsequent increase in S M3 , but only delays the S M6 increase without a transient decrease. Shortening at V 0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (I M3 ), whereas the intensity of the M6 reflection (I M6 ) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.