A dual regulation of contraction operates in both skeletal and cardiac muscles. The first mechanism, based on Ca
-dependent structural changes of the regulatory proteins in the thin filament, makes ...the actin sites available for binding of the myosin motors. The second recruits the myosin heads from the OFF state, in which they are unable to split ATP and bind to actin, in relation to the force during contraction. Comparison of the relevant X-ray diffraction signals marking the state of the thick filament demonstrates that the force feedback that controls the regulatory state of the thick filament works in the same way in skeletal as in cardiac muscles: even if in an isometric tetanus of skeletal muscle force is under the control of the firing frequency of the motor unit, while in a heartbeat force is controlled by the afterload, the stress-sensor switching the motors ON plays the same role in adapting the energetic cost of the contraction to the force. A new aspect of the Frank-Starling law of the heart emerges: independent of the diastolic filling of the ventricle, the number of myosin motors switched ON during systole, and thus the energetic cost of contraction, are tuned to the arterial pressure. Deterioration of the thick-filament regulation mechanism may explain the hyper-contractility related to hypertrophic cardiomyopathy, an inherited heart disease that in 40% of cases is due to mutations in cardiac myosin.
The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between ...mechanical and biochemical steps in the myosin-actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin-actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.
The stiffness of the single myosin motor (
ɛ) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the ...half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (
T
0) modulated by changing either Ca
2+ or temperature. The hs compliance is 43.0
±
0.8
nm
MPa
−1 in isometric contraction at saturating Ca
2+, whereas in rigor it is 28.2
±
1.1
nm
MPa
−1. The equivalent compliance of myofilaments is 21.0
±
3.3
nm
MPa
−1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5
±
1.7
kPa
nm
−1 in isometric contraction at saturating Ca
2+ (
e
0), and in rigor (
e
r) it rises to 138.9
±
21.2
kPa
nm
−1.
ɛ, calculated from
e
r and the lattice molecular dimensions, is 1.21
±
0.18
pN
nm
−1.
ɛ estimated, using a thermodynamic approach, from the relation of
T
0 at saturating Ca
2+ versus the reciprocal of absolute temperature is 1.25
±
0.14
pN
nm
−1, similar to that estimated for fibers in rigor. Consequently, the ratio
e
0/
e
r (0.33
±
0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating Ca
2+. If the osmotic agent dextran T-500 (4
g/100
ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both
e
0 and
e
r increase by ∼40%.
ɛ becomes ∼1.7
pN
nm
−1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.
Abstract
Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro ...it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca
2+
-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca
2+
. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever Pi increases with heart-beat frequency.
A conventional five-step chemo-mechanical cycle of the myosin-actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding ...of a new ATP molecule, is able to fit the Pi dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in Pi much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with Pi. The large dissociation constant of the product release in the branched pathway allows the isometric myosin-actin reaction to fit the equilibrium constant of the ATPase.
In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of ...the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.
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.
The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from ...the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force T
P to a value T (0.8–0.2 T
P). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s−1 at high load to ∼8 nm·hs−1 and 6,000 s−1 at low load. Increases in sarcomere length (1.9–2.2 μm) and external Ca2+ₒ (1–2.5 mM), which produce an increase of T
P, do not affect the dependence on T, normalized for T
P, of the size and speed of the working stroke. Thus, length- and Ca2+-dependent increase of T
P and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.
Contraction of striated muscle is regulated by a dual mechanism involving both thin, actin-containing filament and thick, myosin-containing filament. Thin filament is activated by Ca
binding to ...troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors, extending from the neighbouring stress-activated thick filaments. Motor attachment to actin contributes to spreading activation along the thin filament, through a cooperative mechanism, still unclear, that determines the slope of the sigmoidal relation between isometric force and pCa (-logCa
), estimated by Hill coefficient n
. We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca
at different temperatures (12-35 °C) to show that n
depends on the motor force at constant number of attached motors. The definition of the role of motor force provides fundamental constraints for modelling the dynamics of thin filament activation and defining the action of small molecules as possible therapeutic tools.
Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and ...shortening by cyclic ATP-driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform-dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors and the rate of transition through the attachment-detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.
PDF
