Muscle Letters

Significance of human muscle fibers as research subject

muscletters — Thu, 20 Aug 2009 21:19:42 +0000

The relevance to the study of human performance, ageing, and disease;

Relatively slow kinetic properties make their study easier than that of muscle fibers obtained from smaller animal species;

As large fibers, they are stable in vitro after permeabilization, and maintain a good sarcomere pattern, even during repeated, long, maximum contractions;

He, ZH., ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition. Biophys J, 2000 Aug;79(2):945-61.

Hill Equation

muscletters — Thu, 20 Aug 2009 21:06:36 +0000

The Hill equation is an equation used in enzyme characterization. In biochemistry, the binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule (this is known as Cooperative binding).

The Hill Coefficient (n) describes the fraction of the enzyme saturated by ligand as a function of the ligand concentration; it is used in determining the degree of cooperativity of the enzyme.

Positively cooperative reaction (n > 1): Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules increases.
Negatively cooperative reaction (n < 1): Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules decreases.
Noncooperative reaction (n = 1): The affinity of the enzyme for a ligand molecule is not dependent on whether or not other ligand molecules are already bound.

Molecular mechanisms of muscle contraction (2)

muscletters — Sun, 05 Jul 2009 19:13:01 +0000

Troponin and tropomyosin bestow Ca2+ dependence on the productive interactions of actin and myosin: rapid ATPase activity, generation of force, and generation of movement.

In the absence of Ca2+, much of the myosin-binding site on actin is obscured by tropomyosin. Calcium binding to troponin causes tropomyosin to shift position, exposing much of the myosin-binding site. Strong actinmyosin binding requires a further repositioning of tropomyosin. … The shifting position of tropomyosin on actin is always a critical aspect of regulation.

The thin filament not only undergoes kinetic transitions, but also can have spatial transitions. Any local shift in tropomyosin position on the actin surface, for example caused by a lone, strongly bound cross-bridge, implies spatial transition(s) between actins with tropomyosin in one position, and actins with tropomyosin in another. Thus, the flexibility of tropomyosin on the actin filament is significant for full appreciation of its regulatory function.

Heller, M.J., et al., Cardiomyopathic tropomyosin mutations that increase thin filament Ca2+ sensitivity and tropomyosin N-domain flexibility. J Biol Chem, 2003. 278(43): p. 41742-8

Molecular mechanisms of muscle contraction (1)

muscletters — Sat, 04 Jul 2009 21:04:00 +0000

As a trimeric regulatory protein complex located on actin-tropomyosin filaments, troponin (Tn) together with tropomyosin (Tm), regulates cardiac muscle contraction in response to Ca²⁺.

Troponin I (TnI) is one of the subunits of Tn, which makes extensive contacts with the other subunits of the Tn trimer, troponin C (TnC) and troponin T (TnT), as well as actin-Tm.

In relaxed muscle at low intracellular Ca²⁺concentration, by virtue of its interaction with actin, TnI constrains Tm-Tn in a position on the actin filament that prevent myosin binding and subsequent crossbridge cycling.

Upon muscle stimulation, elevated intracellular Ca²⁺triggers muscle contraction by binding reversibly to TnC. Ca²⁺ binding causes a region of TnI to switch from its binding site on actin to a site on TnC. This conformational change removes the positional constraint of Tn-Tm on actin and permits the interaction of myosin with actin and the subsequent hydrolysis of ATP (actin-activated myosin ATPase activity) that provides energy for force production.

Foster, D.B., et al., C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning. Circ Res, 2003. 93(10): p. 917-24

Cap Disease & Nemaline Myopathy (2)

muscletters — Fri, 03 Jul 2009 11:56:48 +0000

The nemaline myopathy (NEM) nosography may encompass classical nemaline myopathy, cap disease, and actin filament aggregation myopathy, the latter with or without additional rods, as different stages under guidance or misguidance by different NEM-related genes during the development and maturational process of muscle fibre formation.

Goebel, H.H., Cap disease uncapped. Neuromuscul Disord, 2007. 17(6): p. 429-32

Cap Disease & Nemaline Myopathy (1)

muscletters — Fri, 03 Jul 2009 09:37:56 +0000

A missense mutation, Glu41Lys, in the β-tropomyosin gene (TPM2) was identified in both patients, with morphologic features of cap disease in the daughter but nemaline myopathy in the mother.

Four different mutations in TPM2 have so far been described and associated with 4 different phenotypes: nemaline myopathy, distal arthrogryposis type 1, myopathy without rods combined with distal arthrogryposis type 2B, and a congenital myopathy without rods. This variation indicates that the functional and structural consequences differ between the mutations. This may be due to interactions with other proteins, which are important for the function of TM.

It is possible that some of the myopathies with cap structures will turn out to be nemaline myopathy at a later stage because all the reported cases of cap disease were in patients younger than 32 years at the time of biopsy. The clinical expression along with the changes in the morphologic features suggest that cap disease may be a variant or early stage of nemaline myopathy.

Tajsharghi, H., et al., Congenital myopathy with nemaline rods and cap structures caused by a mutation in the beta-tropomyosin gene (TPM2). Arch Neurol, 2007. 64(9): p. 1334-8.

Relative force measured by mixtures assay

muscletters — Thu, 02 Jul 2009 15:15:51 +0000

In an in vitro motility assay, multiple myosin molecules pull simultaneously but asynchronously on actin filaments, generating continuous motion at a velocity characteristic of the isoform and the experimental conditions. When the assay is performed with two different populations of myosin, the different myosin molecules pulling on the same actin filament are essentially in a “tug-of-war.”

If the two myosins propel actin at different velocities, then the velocity of an actin filament will depend on the relative propotions of the two myosins and the relative pulling strengths or kinetics of the two myosins; in a mixture, filaments will tend to run dispropotionately cliser to the velocity of the stronger myosin. Myosin mixtures assays can therefore be used to estimate the relative difference in force or detachment kinetics of two different populations of myosin.

Snook, J.H., et al., Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility. Free Radic Biol Med, 2008. 44(1): p. 14-23

In Vitro Motility Assay (2)

muscletters — Thu, 02 Jul 2009 07:06:32 +0000

A flow cell (i.e., an open system that allows solution exchange during the experiment) is assembled from a glass slide and a nitrocellulose coated coverslip using silicon grease along the edges of the coverslip as spacer between the two surfaces.

A myosin solution is then allowed to flow into the cell. Within seconds, the myosin molecules bind to the coverslip. The nitrocellulose prevents myosin denaturation on the highly charged glass surface.

After a washing step to remove excess myosin, free binding sites in the flow cell are blocked to prevent actin filaments from sticking to the glass surface. Fluorescent derivatives of phalloidin (e.g., rhodamin-phalloidin) – a mushroom toxin that tightly binds to actin filaments – are used to fluorescently label actin filaments indirectly.

When added to the flow cell, these fluorescent actin filaments bind to the myosin molecules, and the motility assay system is ready for use. Adding “fuel” in the form of ATP produces a sliding movement of the actin filaments powered by the myosin motors attached to the coverslip.

http://www.mih.unibas.ch/Booklet/Booklet96/Chapter2/Chapter2.html

In Vitro Motility Assay (1) — Classical Paper

muscletters — Wed, 01 Jul 2009 19:41:28 +0000

The first quantitative measurement of rates of movement of purified myosin along actin in vitro were made by using the Nitella-based movement assay. That depends on the biochemically undefined actin cables of the Nitella cell, which are stabilized by unknown factors and may be contaminated by components of the Nitella cytoplasm.

The first approach to establish a purified movement system was use an array of polar, aligned actin filaments bound to the substrated by the Dictyostelium protein severin. These experiments provided quantitative in vitro measurement of movement of purified actin and myosin. However, this system has been difficult to develop into a practical myosin movement assay. Many beads attach to the substratum without moving, and those that move do so for relatively short distances.

Yanagida et al. observed single fluorescent actin filaments in solution by using a video light microscope. They found that the amplitude and frequency of bending of the filaments increased in the presence of soluble myosin fragments and ATP. We considered that we might observe linear movement of single fluorescent actin filaments along myosin filaments by inverting our movement system, immobilizing the myosin on the substrate, and allowing single actin filaments to attach to the bound myosin.

In this study, we report that, in the presence of ATP, myosin filaments attached to glass are indeed capable of supporting movement of single actin filaments labeled with rhodamine phalloidin. The rates of movement of these single actin filaments are consistent with those measured in our previous assays and depend on the concentration of ATP, the buffer pH, and the type of myosin.

The most important feature of this assay is that it gives rapid, quantitative, and reproducible myosin movement data from small samples of purified proteins. And this assay offers the opportunity to examine the direct effects of modifications of either actin or myosin or of added factors on the movement process.

Kron, S.J. and J.A. Spudich, Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A, 1986. 83(17): p. 6272-6.

Models for muscle contraction

muscletters — Wed, 01 Jul 2009 12:32:58 +0000

Huxley’s model in 1957 —- The sliding movement is driven by thermal fluctuation of the position of a myosin head and the directionality of the movement is ascribed to asymmetry of kinetic parameters for actin and myosin interaction; that is, in the forward position a myosin favorably attaches to an actin filament, and detaches from an actin filament in the backward position. The ATP hydrolysis is supposed to supply the energy for the cyclical attachment/detachment process.

Crossbridge swinging model —- The myosin head rigidly binds to actin at 45◦ before ATP binds (strong binding state). Upon binding of ATP, the myosin rapidly dissociates from actin, and then ATP molecule is hydrolyzed to ADP and Pi on the myosin head. In the complex with the products myosin repeats attachment at 90◦ and detachment with actin rapidly (weakly binding state). After the release of Pi and ADP, the myosin head returns to the strong binding state.(Huxley, 1969; Lymn and Taylor, 1971)

Lever-arm swinging model —- Myosin neck domain next to a motor domain rotates depending on the chemical states of nucleotide (Houdusse et al., 1999). A small conformational change in a ATP binding domain is transmitted to the neck domain. Thus the neck domain acts as a lever-arm that can cause a large displacement (Rayment et al., 1993). This model predicts that the displacement accompanied by a single ATPase cycle would be constant 5–10 nm for the muscle myosin.

The lever-arm swinging model and the crossbridge-swinging model has been a paradigm in the muscle research.

Esaki, S., et al., Cooperative actions between myosin heads bring effective functions. Biosystems, 2007. 88(3): p. 293-300