Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb−) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb− fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb− fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k−4) are larger in Neb+ fibers than in Neb− fibers. The rates of the force generation step (k4) and the reversal detachment step (k−2) change in the opposite direction. These effects can be explained by Le Chatelier’s principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

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Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties

AJP Cell Physiology ◽

10.1152/ajpcell.00503.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1123-C1132 ◽

Cited By ~ 43

Author(s):

David S. Gokhin ◽

Marie-Louise Bang ◽

Jianlin Zhang ◽

Ju Chen ◽

Richard L. Lieber

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Expression Patterns ◽

Nemaline Myopathy ◽

Myosin Heavy Chain Isoforms ◽

Filament Length ◽

Force Transmission ◽

Thin Filaments ◽

Tension Properties ◽

Tension Curves

Nebulin (NEB) is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy.

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Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.120.2.411 ◽

1993 ◽

Vol 120 (2) ◽

pp. 411-420 ◽

Cited By ~ 98

Author(s):

V M Fowler ◽

M A Sussmann ◽

P G Miller ◽

B E Flucher ◽

M P Daniels

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Spatial Organization ◽

Membrane Skeleton ◽

Filament Length ◽

Human Erythrocyte Membrane ◽

Thin Filaments ◽

Muscle Tropomyosin ◽

Immunofluorescence Labeling ◽

Pointed End

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.

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Titin Isoform Size is not Correlated with Thin Filament Length in Rat Skeletal Muscle

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2951 ◽

2010 ◽

Vol 98 (3) ◽

pp. 544a

Author(s):

Jonathan M. Pleitner ◽

Marion L. Greaser

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Filament Length ◽

Rat Skeletal Muscle

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Thin Filament Length in Mouse Skeletal Muscle and its Relationship to Differential Splicing of Nebulin

Biophysical Journal ◽

10.1016/j.bpj.2010.12.3388 ◽

2011 ◽

Vol 100 (3) ◽

pp. 587a ◽

Cited By ~ 1

Author(s):

Danielle Buck ◽

Paola Tonino ◽

Adam Hoying ◽

Henk Granzier

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Filament Length ◽

Differential Splicing ◽

Mouse Skeletal Muscle

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Thin filaments are not of uniform length in rat skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.96.1.100 ◽

1983 ◽

Vol 96 (1) ◽

pp. 100-103 ◽

Cited By ~ 15

Author(s):

L Traeger ◽

M A Goldstein

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Fast Muscle ◽

Filament Length ◽

Rat Skeletal Muscle ◽

Adult Rats ◽

Thin Filaments ◽

Filament Assembly ◽

Old Rats ◽

Two Stages

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3-, 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9-d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.

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Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

AJP Cell Physiology ◽

10.1152/ajpcell.00491.2009 ◽

2010 ◽

Vol 299 (5) ◽

pp. C1091-C1099 ◽

Cited By ~ 7

Author(s):

Ryan S. Lee ◽

Svetlana B. Tikunova ◽

Kristopher P. Kline ◽

Henry G. Zot ◽

Javier E. Hasbun ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Muscle Force ◽

Force Production ◽

Dissociation Rate ◽

Wild Type ◽

Binding Properties ◽

Cross Bridge ◽

Skeletal Muscle Contraction ◽

Filament Activation

To investigate effects of altering troponin (Tn)C Ca2+ binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca2+ sensitivities. Ca2+ binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys99 residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca2+ sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca2+ dissociation rate ( koff), respectively. On the basis of Kd and koff, these results suggest that the Ca2+ association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca2+ dependence of force development and rate of force redevelopment ( ktr) at 15°C, resulting in sensitization of both force and ktr to Ca2+ for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and ktr to Ca2+, I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (Fmax) and maximal ktr. Although V43QTnC and I60QTnC had drastically different effects on Ca2+ binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated ktr at force levels <30%Fmax vs. wild-type TnC. However, at matched force levels >30%Fmax ktr was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected ktr through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal ktr for I60QTnC at low Ca2+ concentration. Thus TnC mutants with altered Ca2+ binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

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New Insights into the Structural Roles of Nebulin in Skeletal Muscle

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/968139 ◽

2010 ◽

Vol 2010 ◽

pp. 1-6 ◽

Cited By ~ 15

Author(s):

Coen A. C. Ottenheijm ◽

Henk Granzier

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Force Production ◽

Nemaline Myopathy ◽

Structure And Function ◽

Filament Length ◽

Thin Filaments ◽

Muscle Structure ◽

And Function

One important feature of muscle structure and function that has remained relatively obscure is the mechanism that regulates thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of nebulin KO models, reveal that nebulin plays an important role in the regulation of thin filament length. Another structural feature of skeletal muscle that is not well understood is the mechanism involved in maintaining the regular lateral alignment of adjacent sarcomeres, that is, myofibrillar connectivity. Recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils. Thus, novel structural roles of nebulin in skeletal muscle involve the regulation of thin filament length and maintaining myofibrillar connectivity. When these functions of nebulin are absent, muscle weakness ensues, as is the case in patients with nemaline myopathy with mutations in nebulin. Here we review these new insights in the role of nebulin in skeletal muscle structure.

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