© 1998 American Heart Association, Inc.
Editorial |
How Actin-Myosin Interactions Differ With Different Isoforms of Myosin
Correspondence to Saul Winegrad, MD, Department of Physiology, School of Medicine, University of Pennsylvania, 37th and Hamilton Walk, Philadelphia, PA 19104-6085.
Key Words: myosin • myosin heavy chain • in vitro motility • unitary force • unitary displacement • actin-myosin interaction
The development of in vitro techniques for assaying the mechanical properties of individual actin-myosin interactions has provided investigators with a powerful tool to address questions about fundamental properties of the force-generating reactions that produce movement of cells or organelles within cells. These techniques are capable of measuring the force and/or displacement produced by the interaction of a single force generator with a single actin filament. The load on the force generator can be varied in a controlled manner to allow the sampling of force and velocity under a variety of conditions. Progress is being made toward measuring the amount of ATP split during these reactions. There are, however, limitations to the existing in vitro motility assays, such as the inability to control the orientation of the molecules rigorously and the quantitative effects of brownian motion on the force and displacement transients that are produced by the interaction between myosin and actin. One consequence of these problems and the different approaches of the several laboratories using in vitro assays is the existence of a range of values for the force and displacement produced by the unitary force–generating set of reactions. Most results indicate forces between 2 and 10 pN and displacements from 5 to 15 nm with means of about 5 pN and 5 nm, respectively. The latter is about the periodicity of actin in the thin filament. In spite of these problems, which are slowly responding to the ingenuity of investigators using the techniques, much useful information has been produced. (For a more detailed discussion of the application of in vitro motility techniques to the study of the mechanics of the interaction of actin with myosin, see References 1 and 21 2 .)
In intact cardiac tissue and cells, unloaded shortening velocity in
muscles homogeneous for ß-myosin heavy chain
(V3) is less than 40% of that in muscles
homogeneous for 
-myosin heavy chain
(V1),3 4 and the rate of
hydrolysis of ATP by myosin is similarly slower when the heavy chain is
the beta isoform. The mechanical basis for these differences has not
been shown. In addition, there has been controversy about whether the
individual V3 force generators produce more force
per cycle and greater time-averaged force than V1
ones. An understanding of the mechanism for the different rates of
shortening and ATP hydrolysis has broader implications than just
details of the function of V1 and
V3. Comprehension of the basis for this
difference should lead to further insights into the mechanism and
reactions that produce force.
In an important application of the in vitro motility assay to the study
of cardiac muscle, Sugiura et al5 have addressed
the question of the basis for the differences in the unloaded
shortening velocities and ATPase activities in cardiac muscle
containing - or ß-myosin heavy chain. In their in vitro motility
assay, a single myosin molecule interacts with an individual actin
filament attached to a bead, which in turn is suspended by "optical
tweezers" that can control the stiffness of the preparation. With
stiffness set at a high level, the bead is held stable, and isometric
contractile force can be measured. When the stiffness is low,
displacement of the actin filament occurs, as in a lightly loaded
isotonic contraction.
Sugiura et al5 found that V1 and V3 force generators produce the same force and the same displacement. The difference between the force and displacement transients of the two is in the duration of the individual events. Both the force and the displacement transients are approximately 40% longer with V3 than with V1. These results provide the strongest evidence to date in favor of a difference in duration without a difference in generation of force.
The results of Sugiura et al5 lead to some new
and interesting questions. Although there is remarkable homology in
amino acid sequence between - and ß-myosin heavy chains, there are
differences at several crucial locations in the molecule, including the
rod, the hinge, the light chain domain, the actin binding site, and the
ATP binding site.6 The difference in the
durations of the force and displacement transients of
V1 and V3—about 40% —is
significantly smaller than the 150% difference in unloaded shortening
velocity found in intact cardiac cells and in velocity measured when
multiple actomyosin interactions occur
simultaneously.3 The experiments
should detect functional differences due to amino acid differences in
the myosin head but probably not in the rod. The discrepancy in the
size of the effect of isoform on velocity of shortening could be due to
difference in the conditions under which myosin and actin interact in
the in vitro assay or the existence of another factor contributing to
the slower cycling of V3 myosin. For instance, a
difference in lateral interactions between rod portions of myosin in
the intact filament could alter compliance in thick filaments and
modify force and displacement.
The velocity of shortening for V1 that was measured in the in vitro assay, approximately 45 nm/s, is considerably slower by more than an order of magnitude than that measured in intact sarcomeres, but this can be attributed to the low concentration of ATP used in the in vitro assay. In order to be able to resolve individual contractile events, 0.5 µmol/L ATP was used. At this concentration of ATP, there is a marked limitation in the velocity of shortening in intact cardiac cells and in the rate of ATP hydrolysis in actomyosin in solution.
An additional provocative result obtained by Sugiura et al5 is the difference in the duration of the force and displacement transients regardless of the isoform of myosin heavy chain. The force transient is about 65% longer than the displacement transient with each isoform. This suggests that the rate of detachment of myosin from actin, which is consistent with first-order kinetics in their data, is either dependent on strain or inversely related to load. Intuitively, the latter seems less likely than the former. Huxley7 first proposed a relation between strain and detachment rate in his presentation of a model for crossbridge cycling, and evidence to support it has been generated subsequently. If it is feasible to analyze the shape of the transient, either by achieving greater time resolution, time-averaging multiple transients, or using various analogues of ATP, it should be possible to learn more about the factors that influence the rate of detachment of myosin from actin and gain additional information about the mechanical correlates of different chemical events within the crossbridge cycle. This is an exciting prospect.
With this study, Sugiura et al5 have provided answers to some questions about the force-generating reactions between actin and myosin and posed others that have broad implications for all molecular motors.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editor or of the American Heart Association.
References
1.
Cooke R. Actomyosin interaction in striated muscle.
Physiol Rev. 1997;77:671–697.
2. Spudich JA. How molecular motors work. Nature. 1994;372:515–518.[Medline] [Order article via Infotrieve]
3. Harris DE, Work SS, Wright RK, Alpert NR, Warshaw DM. Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro. J Muscle Res Cell Motil. 1994;15:11–19.[Medline] [Order article via Infotrieve]
4.
Pagani ED, Julian FJ. Rabbit papillary muscle myosin isozymes
and the velocity of muscle shortening. Circ Res. 1984;54:586–594.
5.
Sugiura S, Kobayakawa N, Fujita H, Yamashita H, Momomura S,
Chaen S, Omata M, Sugi H. Comparison of unitary displacements and
forces between 2 cardiac myosin isoforms by the optical trap technique:
molecular basis for cardiac adaptation. Circ Res. 1998;82:1029–1034.
6. McNally EM, Kraft R, Bravo-Zehnder M, Taylor DA, Leinwand LA. Full-length rat alpha and beta cardiac myosin heavy chain sequences. J Mol Biol. 1989;210:665–671.[Medline] [Order article via Infotrieve]
7. Huxley AF. Muscle structure and theories of contraction. Prog Biophys Biophys Chem. 1957;7:255–317.[Medline] [Order article via Infotrieve]
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