Modulation of Striated Muscle Function is Reflected by Thick Filament Structure.

In mammalian skeletal and cardiac muscles, regulation of activity occurs when calcium binds to troponin on thin filaments, which ultimately results in exposure of myosin-binding sites on actin. However, modulation of contractile function, affecting such parameters as calcium sensitivity, the rate of rise of tension, the expression of maximum tension and/or the rate of onset of relaxation, is also calcium dependent. It is, in part, a property of the thick filament itself and its component myosin and/or accessory proteins. Among these are phosphorylation of myosin regulatory light chains or light chain 2 (RLCs; LC2) and in cardiac, but not skeletal fibers, phosphorylation of myosin-binding protein C (MyBP-C).Gentle methods of separating thick filaments from small tissue specimens, subjected to various experimental protocols designed to explore the functional parameters of such modulatory activities, allow examination of any accompanying structural changes.

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Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle

eLife ◽

10.7554/elife.24081 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Xuemeng Zhang ◽

Thomas Kampourakis ◽

Ziqian Yan ◽

Ivanka Sevrieva ◽

Malcolm Irving ◽

...

Keyword(s):

Heart Muscle ◽

Structural Changes ◽

Muscle Cells ◽

Thick Filament ◽

Calcium Sensitivity ◽

Cellular Level ◽

Thin Filaments ◽

Thick Filaments ◽

Filament Structure ◽

Heart Muscle Cells

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

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Structural changes that occur in scallop myosin filaments upon activation.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.830 ◽

1985 ◽

Vol 101 (3) ◽

pp. 830-837 ◽

Cited By ~ 39

Author(s):

P Vibert ◽

R Craig

Keyword(s):

Calcium Binding ◽

Structural Changes ◽

Striated Muscle ◽

Thick Filament ◽

Light Chains ◽

Free Calcium ◽

Thin Filaments ◽

Regulatory Light Chains ◽

Myosin Filaments ◽

Free Calcium Concentration

Myosin filaments isolated from scallop striated muscle have been activated by calcium-containing solutions, and their structure has been examined by electron microscopy after negative staining. The orderly helical arrangement of myosin projections characteristic of the relaxed state is largely lost upon activation. The oblique striping that arises from alignment of elongated projections along the long-pitched helical tracks is greatly weakened, although a 145 A axial periodicity is sometimes partially retained. The edges of the filaments become rough, and the myosin heads move outwards as their helical arrangement becomes disordered. Crossbridges at various angles appear to link thick and thin filaments after activation. The transition from order to disorder is reversible and occurs over a narrow range of free calcium concentration near pCa 5.7. Removal of nucleotide, as well as dissociation of regulatory light chains, also disrupts the ordered helical arrangement of projections. We suggest that the relaxed arrangement of the projections is probably maintained by intermolecular interactions between myosin molecules, which depend on the regulatory light chains. Calcium binding changes the interactions between light chains and the rest of the head, activating the myosin molecule. Intermolecular contacts between molecules may thus be altered and may propagate activation cooperatively throughout the thick filament.

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Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602776113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E3039-E3047 ◽

Cited By ~ 60

Author(s):

Thomas Kampourakis ◽

Yin-Biao Sun ◽

Malcolm Irving

Keyword(s):

Light Chain ◽

Heart Muscle ◽

Calcium Binding ◽

Structural Changes ◽

Thick Filament ◽

Calcium Sensitivity ◽

Active Force ◽

Thin Filaments ◽

Filament Axis ◽

Filament Structure

Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

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The Filament Lattice of Striated Muscle

Physiological Reviews ◽

10.1152/physrev.1998.78.2.359 ◽

1998 ◽

Vol 78 (2) ◽

pp. 359-391 ◽

Cited By ~ 142

Author(s):

BARRY M. MILLMAN

Keyword(s):

Muscle Contraction ◽

Structural Changes ◽

Striated Muscle ◽

Muscle Fibers ◽

Lattice Stability ◽

X Ray Diffraction ◽

Thin Filaments ◽

Intact Muscle ◽

Radial Forces ◽

Speed Of Shortening

Millman, Barry M. The Filament Lattice of Striated Muscle. Physiol. Rev. 78: 359–391, 1998. — The filament lattice of striated muscle is an overlapping hexagonal array of thick and thin filaments within which muscle contraction takes place. Its structure can be studied by electron microscopy or X-ray diffraction. With the latter technique, structural changes can be monitored during contraction and other physiological conditions. The lattice of intact muscle fibers can change size through osmotic swelling or shrinking or by changing the sarcomere length of the muscle. Similarly, muscle fibers that have been chemically or mechanically skinned can be compressed with bathing solutions containing very large inert polymeric molecules. The effects of lattice change on muscle contraction in vertebrate skeletal and cardiac muscle and in invertebrate striated muscle are reviewed. The force developed, the speed of shortening, and stiffness are compared with structural changes occurring within the lattice. Radial forces between the filaments in the lattice, which can include electrostatic, Van der Waals, entropic, structural, and cross bridge, are assessed for their contributions to lattice stability and to the contraction process.

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The myosin interacting-heads motif present in live tarantula muscle explains tetanic and posttetanic phosphorylation mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921312117 ◽

2020 ◽

Vol 117 (22) ◽

pp. 11865-11874 ◽

Cited By ~ 5

Author(s):

Raúl Padrón ◽

Weikang Ma ◽

Sebastian Duno-Miranda ◽

Natalia Koubassova ◽

Kyoung Hwan Lee ◽

...

Keyword(s):

Striated Muscle ◽

Thick Filament ◽

X Ray Diffraction ◽

Thin Filaments ◽

Time Resolved ◽

Vertebrate Muscle ◽

Before And After ◽

Filament Activation ◽

Interacting Heads Motif ◽

Filament Surface

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing—proposed in vertebrate muscle—is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.

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LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

The Journal of Cell Biology ◽

10.1083/jcb.37.1.105 ◽

1968 ◽

Vol 37 (1) ◽

pp. 105-116 ◽

Cited By ~ 72

Author(s):

Robert E. Kelly ◽

Robert V. Rice

Keyword(s):

Smooth Muscle ◽

Cross Section ◽

Striated Muscle ◽

Thick Filament ◽

Longitudinal Axis ◽

Thin Filaments ◽

Chicken Gizzard ◽

Thick Filaments ◽

Myosin Filaments ◽

Muscle Myosin

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.

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Changes in thick filament length in Limulus striated muscle.

The Journal of Cell Biology ◽

10.1083/jcb.75.2.366 ◽

1977 ◽

Vol 75 (2) ◽

pp. 366-380 ◽

Cited By ~ 21

Author(s):

M M Dewey ◽

B Walcott ◽

D E Colflesh ◽

H Terry ◽

R J Levine

Keyword(s):

Horseshoe Crab ◽

Striated Muscle ◽

Thick Filament ◽

Filament Length ◽

Thin Filaments ◽

Thick Filaments ◽

Wide Range ◽

The Difference

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.

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Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522236113 ◽

2016 ◽

Vol 113 (12) ◽

pp. 3239-3244 ◽

Cited By ~ 38

Author(s):

Michael J. Previs ◽

Ji Young Mun ◽

Arthur J. Michalek ◽

Samantha Beck Previs ◽

James Gulick ◽

...

Keyword(s):

Binding Protein ◽

Cardiac Contractility ◽

Thick Filament ◽

Structural Effects ◽

Thin Filaments ◽

Intrinsically Disordered ◽

Myosin Binding Protein ◽

Myosin Binding ◽

And Function ◽

The Impact

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C’s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain’s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C’s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C’s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C’s phosphorylation state.

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Models of contractile units and their assembly in smooth muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y05-052 ◽

2005 ◽

Vol 83 (10) ◽

pp. 825-831 ◽

Cited By ~ 11

Author(s):

Farah Ali ◽

Peter D Paré ◽

Chun Y Seow

Keyword(s):

Smooth Muscle ◽

Striated Muscle ◽

Dense Body ◽

Unit Length ◽

Muscle Length ◽

Thick Filament ◽

Contractile Apparatus ◽

Thin Filaments ◽

Dense Bodies ◽

In Series

It is believed that the contractile filaments in smooth muscle are organized into arrays of contractile units (similar to the sarcomeric structure in striated muscle), and that such an organization is crucial for transforming the mechanical activities of actomyosin interaction into cell shortening and force generation. Details of the filament organization, however, are still poorly understood. Several models of contractile filament architecture are discussed here. To account for the linear relationship observed between the force generated by a smooth muscle and the muscle length at the plateau of an isotonic contraction, a model of contractile unit is proposed. The model consists of 2 dense bodies with actin (thin) filaments attached, and a myosin (thick) filament lying between the parallel thin filaments. In addition, the thick filament is assumed to span the whole contractile unit length, from dense body to dense body, so that when the contractile unit shortens, the amount of overlap between the thick and thin filaments (i.e., the distance between the dense bodies) decreases in exact proportion to the amount of shortening. Assembly of the contractile units into functional contractile apparatus is assumed to involve a group of cells that form a mechanical syncytium. The contractile apparatus is assumed malleable in that the number of contractile units in series and in parallel can be altered to accommodate strains on the muscle and to maintain the muscle's optimal mechanical function.Key words: contraction model, ultrastructure, length adaptation, plasticity.

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