To define the mechanics and energetics of the myosin motor action in muscles, it is mandatory to know fundamental parameters such as the stiffness and the force of the single myosin motor, and the ...fraction of motors attached during contraction. These parameters can be defined in situ using sarcomere-level mechanics in single muscle fibers under the assumption that the stiffness of a myosin dimer with both motors attached (as occurs in rigor, when all motors are attached) is twice that of a single motor (as occurs in the isometric contraction). We use a mechanical/structural model to identify the constraints that underpin the stiffness of the myosin dimer with both motors attached to actin. By comparing the results of the model with the data in the literature, we conclude that the two-fold axial stiffness of the dimers with both motors attached is justified by a stiffness of the myosin motor that is anisotropic and higher along the axis of the myofilaments. A lower azimuthal stiffness of the motor plays an important role in the complex architecture of the sarcomere by allowing the motors to attach to actin filaments at different azimuthal angles relative to the thick filament.
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 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.
Key points
Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load.
Accordingly, the unloaded shortening velocity (V0) is ...already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state.
Here the number of actin‐attached motors per half‐myosin filament (n) during V0 shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half‐sarcomere compliance and force during the force redevelopment after shortening.
The value of n decreases progressively with shortening and, during V0 shortening starting at the end of LR, is 1–4.
Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening.
The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V0) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca2+‐induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin‐attached motors per half‐thick filament during V0 shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half‐sarcomere (hs) compliance and force during the force development following V0 shortening. When V0 shortening is superimposed on the maximum isometric force T0, n decreases progressively with the increase of shortening (range 30–80 nm per hs) and, when V0 shortening is imposed at the end of LR, n can be as low as 1–4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V0 shortening.
Key points
Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load.
Accordingly, the unloaded shortening velocity (V0) is already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state.
Here the number of actin‐attached motors per half‐myosin filament (n) during V0 shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half‐sarcomere compliance and force during the force redevelopment after shortening.
The value of n decreases progressively with shortening and, during V0 shortening starting at the end of LR, is 1–4.
Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening.
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.
Background
Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene ...mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.
Methods
Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.
Results
MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.
Conclusions
Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
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