Journal of Royal Science

Tracking number: JRS102Received: 29 April 2019Accepted: 02 May 2019Published: 07 May 2019

‡Corresponding Author

Haruo Sugi, Department of Physiology, Teikyo University Medical School, Tokyo, Japan. Email: sigi(at)kyf.biglobe.ne.jp

CitationSugi H (2019) Accumulating Evidence against the Current Dogmas of Muscle Contraction. J. Royal. Sci. Vol: 1, Issu: 1 (Pp 03-09).

Accumulating Evidence against the Current Dogmas of Muscle Contraction

AbstractThe framework of current dogma of muscle contraction can be stated as follows:

1. Muscle contraction results from cyclic attachment and detachment between myosin head (M) and actin filament (A), coupled with ATP hydrolysis;

2. In each cycle, M in the form of M-ADP-Pi passes through weak-to-strong binding transition with A to perform power stroke producing contraction;

3. At the end of power stroke, M forms rigor configuration A-M, until next ATP comes to bind it;

4. The power stroke of M is caused by active rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid and perpendicular to A.

We present the following evidence against the dogma stated above:

1. During muscle contraction, M may not pass through rigor A-M configuration;

2. ATP hydrolysis by A and M can take place without weak-to-strong A-M binding transition;

3. Both the lever arm domain and the subfragment-2 domain, connecting M to myosin filament backbone, regulate A-M binding strength;

4. At the end of power stroke, the catalytic domain in M is either perpendicular or oblique to A, depending on mechanical conditions. In conclusion, muscle contraction is still filled with mysteries.

Open Access

Haruo Sugi†‡,

Publisher Acknowledgement

IntroductionIn 1954, Huxley and Hanson found that muscle

contraction is caused by relative sliding between actin and myosin filaments, coupled with ATP hydrolysis [1]. More than 60 years have passed since their monumental discovery, and considerable progress has been made on the structure and function of actin and myosin filaments, especially with respect to cyclic attachment and detachment between myosin heads extending from myosin filaments and the corresponding myosin-binding sites in actin filaments. Figure 1 shows the structure of actin and myosin filaments, and their arrangement to form sarcomeres, i.e. structural and functional unit in muscle [2]. A myosin molecule consists of a long rod, called light meromyosin (LMM), and the rest of the molecule containing two pear-shaped heads, called heavy meromyosin (HMM). HMM can be further divided into two separate heads, known as myosin subfragment-1 (S-1), and a rod, known as myosin subfragment-2 (A). In myosin (or thick) filaments, LMM aggregates to form the filament backbone, while S-1 heads extend laterally from myosin filaments at an axial interval of 14.3nm (B). In this article, each S-1 head will be called myosin head. The arrangement of myosin molecules in a myosin filament is symmetrical with respect to the middle of the filament called the bare region, where myosin heads are absent (B). Meanwhile, actin (or thin) filament consists of two helical strands of globular actin monomers (G-actin) with a pitch of 35.5nm. The axial separation of G-actin is 5.46nm (C). Actin filaments also contain two regulatory proteins, tropomyosin and troponin, and extends in either direction from the Z-line to penetrate in between

myosin filaments, which are located centrally in each sarcomere, i.e. structures between adjacent Z-lines, so that each myosin head faces neighboring actin filaments (D).

Concerning the mechanism, with which actin and myosin filaments slide past each other, Huxley proposed a contraction model shown in Figure 2 [3]. Due to the difference in periodicity between actin and myosin filaments, the interaction between myosin heads and actin filaments takes place asynchronously. A myosin head (left), first attaches to adjacent actin filament (upper diagram), changes its configuration to produce unitary sliding (middle diagram), and then detaches from actin filament, while another myosin head (right) starts attaching to actin filament (lower diagram). This attachment-deformation-detachment cycle produces relative sliding between actin and myosin filaments, i.e. muscle contraction. The ingenious idea stimulated muscle investigators to prove the attachment-detachment cycle between myosin heads and actin filaments. Extensive studies have been made using a variety of techniques, and the dogmas on the mechanism of muscle contraction have been constructed. Although the dogmas have been described in many textbooks as if they are established facts. The purpose of this articles is to discuss shortcomings of the current dogmas, which retard progress of research work in this research field.

The Lymn-Taylor schemeFirst, we shall describe the Lymn-Taylor

scheme, one of the current dogmas in the research

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Keywords: Muscle contraction, Lymn-Taylor Scheme, Swinging lever arm hypothesis, Myosin head power stroke.

Copyright: CC BY© 2019 Sugi H. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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field of muscle contraction mechanism. A muscle is regarded to be an engine converting chemical energy derived from ATP hydrolysis into mechanical work. In myosin molecules, both actin-binding and ATP-binding (ATPase) sites are localized in myosin heads. The extremely slow steady-state ATPase activity of myosin heads is enhanced more than 200 times in the presence of actin filament[4], and the enhanced ATPase activity is believed to correspond to that of muscle during contraction. Since the whole myosin molecule tends to form heterogeneous clumps with actin filament, biochemical studies on the actin-activated ATPase activity have been performed intensively using solutions containing HMM and actin filament (acto-HMM solutions). For convenience, the letters A and M will hereafter be used to denote actin filaments and myosin heads, respectively. At present, the ATPase reaction steps presented by Lymn and Taylor (the Lymn-Taylor scheme) [5] has generally been believed to take place in contracting muscle.

As shown in Figure 3, M is in the state of M-ADP-Pi in relaxed muscle, and on muscle activation binds with A weakly to form A-M-ADP-Pi (A). Then M binds with A strongly after releasing Pi to perform a power stroke, coupled with release of ADP (A to B). After completion of power stroke, M remains attached to A to form A-M, until next ATP comes to bind with it (B). Upon binding with next ATP, M detaches from A (B to C) to perform a recovery stroke, associated with hydrolysis of ATP to form M-ADP-Pi (C to D). Then M-ADP-Pi attaches to A to again perform a power stroke (D to A). It is well known that, in the absence of ATP, M forms rigor complex A-M with A, and it is implicitly believed that A-M in the Lymn-Taylor scheme is equal to the rigor complex A-M, so that M is expected to pass through rigor complex A-M during the attachment-detachment cycle producing muscle contraction.

The Swinging lever arm hypothesis Another powerful dogma in the research field of muscle contraction

mechanism is the swinging lever arm hypothesis put forward by muscle crystallographers. To obtain information about dynamic structural changes of myosin head during muscle contraction, Rayment et al. [6,7] intensively studied nucleotide-dependent changes in myosin head crystal structure. Because of difficulties in preparing crystal from rabbit skeletal muscle myosin head without some chemical modification, they used truncated myosin heads obtained from slime mold (Dictyostelium) myosin, from which lever arm domain (LD) was removed except for its remaining base. Rather surprisingly, the crystal structure of slime mold myosin head was almost identical with that of rabbit skeletal muscle myosin head, despite a considerable difference in phylum between amoeba-like single cell organism and mammalian animal. It seems possible that myosin heads would be closely packed to form crystals to result in deformation of functional regions, which differ depending on biological species.

Nevertheless, crystallographers intensively studied the nucleotide-dependent structural changes of slime mold myosin head crystal, using inorganic analogs of ADP and ATP, while the validity of the use of these analogs remained uncertain [8,9,10]. It was found that, depending on the kind of inorganic nucleotide analogs bound to truncated myosin head, the remaining base of LD rotated by 60o around the converter domain (COD). The rotation of the remaining LD base, depending on the kind of inorganic analogs, was regarded to represent pre- and post-power stroke states of myosin head. Thus, the swinging lever arm hypothesis was proposed as the mechanism producing myosin head power (and recovery) strokes, coupled with ATP hydrolysis. This hypothesis now appears in every textbook, as if it is an established fact, constituting a strong dogma in the research field of muscle contraction. Frankly speaking, the rotation of the LD base in truncated myosin head does not necessarily mean that the whole LD actually rotates by 60o around the COD. It seems unlikely that, in the myofilament-lattice structure of muscle, the small COD base structures can generate enough torque to swing the whole LD, which bears a large external load imposed on muscle. It reminds me of an idiom, “the tail wiggling the dog”. It may occur in water solutions, but never happens in muscle. In this

Figure 2: Proposed attachment-detachment cycle between myosin heads and corresponding myosin-binding sites in actin filament. From [3]. For further explanation, see text.

Figure 3: Putative diagrams of cyclic attachment and detachment between myosin head (M) extending from myosin filament and actin (A) in actin filament. For further explanations, see text. From [15].

Figure 1: Structure of myosin (thick) and actin (thin) filaments, and their arrangement within a sarcomere. (A) Structure of myosin molecule. (B) Ar-rangement of myosin molecules to form myosin filament. (C) Structure of actin (thin) filament. (D) Arrangement of actin and myosin filaments in a sarcomere. From [2].

Citation: Sugi H (2019) Accumulating Evidence against the Current Dogmas of Muscle Contraction. J. Royal. Sci. Vol: 1, Issu: 1 (Pp 03-09).

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article, we present accumulating evidence against the two current dogmas, i.e. the Lymn-Taylor-scheme and the swinging lever arm hypothesis.

Evidence against the Lymn-Taylor Scheme obtained from the Effect of Antibodies to Myosin head

We used three different antibodies to myosin head on both Ca2+-activated muscle fiber contraction and in vitro sliding of actin filaments on myosin molecules fixed on a glass surface [11]. Antibody 1 attaches to the junctional peptide between 50K and 20K segments of myosin heavy chain in myosin head catalytic domain (CAD)[12]; antibody 2 attaches to the reactive lysine residue in myosin head converter domain (COD) [12];

and antibody 3 attaches to two peptides of regulatory light chain in myosin head lever arm domain (LD)[13]. Figure 4 illustrates approximate regions of attachment of antibodies 1, 2 and 3 in myosin head by letters 1, 2 and 3 and 3’, respectively.

Although antibody 1 effectively position-mark myosin heads in living myosin filaments in our electron microscopic recording of ATP-induced myosin head movement, it has no effect on both Ca2+-activated permeabilized muscle fiber contraction and in vitro actin-myosin sliding. Figure 5 is a diagram showing primary structures of actin and myosin head (S-1) and their sites of interaction [17]; if massive antibody 1 molecule attaches to the junctional peptide between 50K and 20K fragments of myosin heavy chain, it completely covers actin-binding sites of myosin head to result in complete inhibition of actin-myosin binding. Consequently, the above finding can be taken to indicate that, during muscle contraction, myosin heads do not pass through rigor configuration A-M, obtained from extracted protein samples. To obtain further information about the possible configuration of myosin heads at the end of power stroke, we compared mechanical response of myosin head between high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states, and found distinct tension recovery following release in high-Ca rigor fibers, but not in low-Ca rigor fibers [18] (Figure 6). The tension recovery disappeared in the presence of EDTA, suggesting that at the end of power stroke, myosin heads in muscle fibers take the form of A-M-ADP, responsible for the tension recovery in high-Ca rigor fibers. Radcaj et al. [19] recorded X-ray diffraction pattern from skinned muscle fibers subjected to ramp-shaped releases, and also reached the conclusion that, at the end of power stroke, myosin heads do not take rigor configuration.

Evidence against the Swinging Lever Arm Hypothesis Based on the Effect of Antibodies to Myosin Head

In contrast with the ineffectiveness of antibody 1 on both muscle contraction and in vitro actin-myosin sliding, antibody 2, which also position-mark myosin heads effectively in our electron microscopic studies on the ATP-induced myosin head movement [13-15], exhibited marked inhibitory effect on in vitro actin-myosin sliding, while it had no appreciable effect on muscle contraction [16]. As shown in Figure 7, in vitro actin-myosin sliding was completely inhibited in the presence of antibody 2 < 0.15mg/ml, while the myosin head ATPase activity in the in vitro system did not change appreciably. On the other hand, the force-velocity relation of Ca2+-activated muscle fiber did not change appreciably in the presence of antibody up to 2mg/ml (Figure 8). It has been reported by Muhrad et al. that chemical modification of the RLR (trinitrophenylation) also inhibits in vitro actin-myosin sliding [20]. They explain the result as being due to mechanical collision of structures in the COD resulting from trinitrophenylation. A more or less similar explanation may apply to the marked inhibition of

Figure 4: Structure of myosin head. Catalytic domain (CAD) consists of 25K (green), 50K (red) and part of 20K (dark blue) fragments of heavy chain, while lever arm domain (LD) is composed of the rest of 20K fragments and essential (ELC, light blue) and regulatory (RLC, magenta) light chains. CAD and LD are connected with small converter domain (COD). Location of peptides around Lys83, and that of two peptides (Met58~Ala70 and Leu106~Phe120) in LD are colored yellow. Approximate regions of attachment of antibodies 1,2 and 3 are indicated by numbers 1, 2 and 3 and 3’, respectively. From [13].

Figure 5: Schematic diagram of myosin head-actin molecule complex (acto-S1 complex), illustrating their likely regions of interaction. From [17]

Figure 6: Tension response of a single high-Ca rigor muscle fiber to repeated application of ramp-shaped releases (0.5% of fiber slack length; duration, 5ms). The fiber was first activated with contracting solution (pCa, 4; downward arrow), and after the maximum tension was reached, transferred into high-Ca rigor solution (pCa, 4; upward arrow). Then, a series of release-restretch cycles were applied to the fiber at appropriate intervals (downward and upward arrowheads). Note that the drop in rigor tension coincident with applied release is followed by tension recovery. From [18].

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actin-myosin sliding by antibody 2. In the in vitro system, myosin heads are randomly oriented on a glass surface, and should have flexibility to catch and interact with actin filaments moving in random directions; if antibody 2 binds with the RLR, the myosin head flexibility is markedly reduced to result in inhibition of actin-myosin sliding. On the other hand, in muscle fibers, each myosin filament is surrounded by six actin filaments at distances appropriate to interact with actin. Consequently, myosin heads, with antibody 2 attached to their RLR in the COD may still interact with actin to produce contraction. The ineffectiveness of antibody 2 on muscle contraction strongly suggests that the swinging lever arm hypothesis, another dogma of muscle contraction, may be invalid. As already mentioned, myosin heads in living hydrated myosin filaments mounted in the EC can be position-marked by antibody 1,2 or 3, and are shown to move in response to applied ATP [13-15] (Figure 9). All the results described in this section indicate that the swinging lever arm mechanism may not be involved in muscle contraction.

Evidence against the Swinging Lever Arm Hypothesis Based on the Eeect of Antibodies to Myosin Head Lever Arm Domain and Myosin Subfragment-2

In 1992, I was asked by Harrington to examine the effect of antibody to myosin subfragment -2 (anti-S-2 antibody), prepared in his laboratory, on the contraction characteristics of muscle fibers. The anti-S-2 antibody had no effect on in vitro actin-myosin sliding, but showed striking effect

on Ca2+-activated contraction of permeabilized muscle fibers. As shown in Figure 10, however, the anti-S-2 antibody (1.5mg/ml) reduced Ca2+-activated isometric tension in single permeabilized muscle fiber to zero in a time dependent manner, while its MgATPase activity remained unchanged. Muscle fiber stiffness, measured by applying small sinusoidal length changes was observed to decrease in parallel with isometric force [21] (Figure 11). These results indicate that, despite its ineffectiveness on in vitro actin-myosin sliding, anti-S-2 antibody inhibits isometric tension and reduces muscle fiber stiffness in muscle fibers without affecting their MgATPase activity. We further studied the effect of anti-S-2 antibody on the force-velocity relation of muscle fibers. As shown in Figure 12, the maximum unloaded shortening velocity Vmax did not change despite marked reduction of isometric tension (A); if the values of isometric tension were normalized with respect to the maximum, the force-velocity curves were found to be identical (B).

We also examined the effect of antibody 3, attaching to myosin head LD domain, and similar results were obtained concerning the reduction of isometric tension without changing MgATPase activity (Figure 13), and without changing the maximum unloaded shortening velocity Vmax [16] (Figure 14). Therefore, we conclude that, if antibodies attach to myosin S-2 region or myosin head LD region, with which myosin head CAD is connected to myosin filament backbone, the strength of actin-myosin binding at the actin-myosin interface at the distal region of myosin head CAD is markedly reduced, so that the weakened actin-myosin binding may be readily broken by small sinusoidal length changes and by small relative sliding between actin and myosin filaments. If this explanation is correct, the ATP hydrolysis during the attachment-detachment cycle between myosin head and actin (Figure 3) may not necessarily be coupled with the weak-to-strong transition of A-M linkage, coupled with reaction, A-M-ADP-Pi → A-M-ADP + Pi.

The regulation of actin-myosin binding strength by myosin S-2 and by myosin head LD described in this section constitutes strong evidence against the swinging lever arm hypothesis, since the crystallographers completely ignore the role of these regions. Figure 15 illustrates diagrammatically the change in configuration of myosin head and S-2 during power and recovery strokes in muscle contraction. It is clear that, during the power and recovery

Figure 7: Marked inhibitory effect of anti-RLR antibody on in vitro actin-myosin sliding. The mean sliding velocities of actin filaments on myosin fixed to a glass slide are plotted against antibody concentration with vertical bars indicating SD. Each data point was obtained from 80―120 measurements. From [16].

Figure 8: Force-velocity relation of single Ca2+-activated muscle fiber in the absence of antibody 2 (slid line) and in the presence of antibody 2 (2mg/ml)(broken line). From [16].

Figure 9: (A―C) Histograms showing amplitude distribution of ATP-induced myosin head movement, position-marked with antibody 1 (A), antibody 2 (B), and antibody 3 (C). (D and E), Diagrams illustrating that the amplitude of movement is the same between the distal and the proximal regions of myosin head, where antibodies 1 and 2 attach, respectively. Note that myosin heads move in response to ATP, even when antibody 2 attaches to the reactive lysine residue (RLR) in the COD (B). From [13].

Citation: Sugi H (2019) Accumulating Evidence against the Current Dogmas of Muscle Contraction. J. Royal. Sci. Vol: 1, Issu: 1 (Pp 03-09).

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Figure 10: Simultaneous recordings of MgATPase activity (upper traces), measured by decrease of NADH fluorescence, and Ca2+-acivated isometric tension (lower traces) in permeabilized muscle fibers before (A) and at 100min (B) and 150min (C) after application of anti-S-2 antibody (1.5mg/ml).Note that, even after complete disappearance of isometric tension, muscle MgATPase activity remains unchanged. From [21].

strokes, the change in angle takes place in both the CAD-LD junction and the LD-S-2 junction. Our experimental results using antibodies indicate that the active change in angle at the LD-S-2 junction play an essential role in producing power and recovery strokes, while the change in angle at the CAD-LD junction may be largely passive in character.

Meanwhile, the swinging lever arm hypothesis predicts that, during each attachment-detachment between myosin head and actin filament, myosin head CAD is rigid and perpendicular to actin filament (Figure 3). Using the actin-myosin filament mixture mounted in the EC, we could record ATP-induced myosin head movement, i.e. myosin head power

Figure 11: Relation between muscle fiber stiffness, measured by applying small sinusoidal length changes (peak-to-peak amplitude, 0.1% of slack length; frequency, 1kHz), and isometric tension, during the development of Ca2+-activated isometric force before (curve A), and at 30min (curve B), 60min (curve C), and 90min (curve D) after application of anti-S-2 antibody (1.5mg/ml). Stiffness versus tension curves A,B,C and D were obtained from records a,b,c and d, respectively. From [21].

Figure 12: Effect of anti-S-2 antibody on the force-velocity relation of Ca2+-activated muscle fibers. (A) Force-velocity curves obtained at various magnitudes of steady isometric force in the presence of antibody. (B) Force-velocity curves normalized with respect to the maximum steady isometric tension. Note that the value of Vmax does not change despite reduction of isometric tension. From [16].

Figure 13: Simultaneous recordings of MgATPase activity of muscle fibers, measured by NADH fluorescence (upper traces), and Ca2+-activated isometric force (lower traces). Note that MgATPase activity of the fibers remains unchanged despite marked reduction of isometric force. From [16].

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stroke [15]. In our experimental condition, the amount of ATP applied to myosin heads was limited (<10µM), so that only a small proportion of myosin heads could be activated while majority of myosin heads were in rigor state. In such a condition, the activated myosin head can not cause gross myofilament sliding, and can only stretch adjacent elastic structures. This condition resembles contraction of muscle with both ends fixed in position, i.e. nominally isometric condition. In this condition, myosin heads exhibit two different modes of power stroke depending on experimental conditions as shown in Figure 16; in the standard ionic strength, myosin heads are oblique to actin filaments at the end of owe stroke (B), while at low ionic strength, which is known to enhance isometric tension in muscle fibers twofold [22], myosin heads are perpendicular to actin filaments (C). This finding also constitutes evidence against the swinging lever arm

Figure 14: Effect of antibody 3 (anti-LD antibody) on the force-velocity relation of permeabilized muscle fibers. (A) Force-velocity curves in the absence (solid line) and in the presence (broken line) of antibody (1.5mg/ml). Note that the maximum unloaded shortening velocity Vmax remains unchanged while Ca2+-activated isometric force is markedly reduced. (B) Force-velocity curves normalized with respect to the maximum isometric force. From [16].

Figure 15: Diagram showing change in myosin head configuration before (solid line) and after (broken line) power stroke. Black circles in myosin head COD indicate approximate position, around which myosin head LD rotates according to the swinging lever arm hypothesis. Shaded area indicates boundary between myosin head LD and myosin S-2. Note that the angle between LD and S-2 changes during myosin head power stroke.

hypothesis, which predicts that myosin head CAD is always perpendicular to actin filaments.

ConclusionIn this article, we discussed the validity of the two current dogmas

of muscle contraction mechanism, the Lymn-Taylor Scheme and the Swinging lever arm hypothesis, which appear in every textbook as if they are established facts, with the following conclusion. (1) In the Lymn-Taylor Scheme, it is implicitly believed that, during cyclic attachment and detachment between myosin head and actin filament, myosin head pass through rigor A-M configuration, determined from extracted protein samples. However, antibody 1, attaching to the junctional peptide between

Figure 16: Diagrams illustrating two different modes of myosin head power stroke, based on our EC experiments on the ATP-induced power stroke of myosin head at the distal (antibody 1 attached) and the proximal (antibody 2 attached) regions of myosin head CAD. (A) Structure of myosin head showing approximate regions of attachment of antibodies 1 and 2 by numbers 1 and 2, respectively. (B) The mode of myosin head power stroke in the nearly isometric condition (external KCl, 120mM), in which gross myofilament sliding does not occur due to small proportion of ATP-activated myosin heads. Note that the amplitude of power stroke is larger at the CAD distal region than at the CAD proximal region. (C) the mode of myosin head power stroke at low ionic strength (externa KCl, 20mM). Note that the amplitude of power stroke is similar at both the distal and the proximal regions of myosin head CAD. From [15].

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mechanism of muscle contraction. Adv. Protein Chem. 71: 161-193.

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Author infomation†

Haruo Sugi {Sugi H}

‡Corresponding Author

Haruo Sugi, Department of Physiology, Teikyo University Medical School, Tokyo, Japan. Email: sigi(at)kyf.biglobe.ne.jp

50K and 20K fragments of myosin heavy chain in myosin head to completely cover actin-binding sites in myosin head CAD, has no effect on both muscle contraction and in vitro actin-myosin sliding, indicating that, during muscle contraction, myosin heads pass through some unknown configuration. (2) Antibody 2, attaching to the reactive lysine residue (RLR) in myosin head COD, shows no effect on muscle contraction, while it inhibits in vitro actin-myosin sliding. Since antibody 2 bound to the RLR is expected to impair active rotation of myosin head LD around COD, the ineffectiveness of antibody 2 on muscle contraction indicates that the swinging lever

arm mechanism does not work in producing muscle contraction. (3) Both myosin head LD and myosin S-2 are shown to regulate actin-myosin binding strength at the distal region of myosin head CAD, indicating their essential role in muscle contraction, although these structures are completely ignored in constructing the swinging lever arm mechanism.

Finally, we emphasize that, to reach full understanding of muscle contraction at the molecular and submolecular level, muscle investigators should continue research work intensively without being affected by the current dogmas, which are no longer useful as discussed in this article.

Department of Physiology, Teikyo University Medical School, Tokyo, Japan