The Comparative Structure and Function of Muscle

By Henry Huddart

The Comparative Structure and Function of Muscle is based upon a series of lectures given at the University of Lancaster over the last seven years, and it follows a natural division into structure, electrophysiology and excitation and mechanical activity. Within each section, an attempt is made to cover all muscle types in as wide a range of animals as the literature will allow. This book comprises 10 chapters, with the first one focusing on the fine structure of skeletal muscle. The following chapters then discuss the fine structure of cardiac and visceral muscle; the innervation of muscle; the ionic basis of the resting potential; the action potential and the activation of muscle; electrical activity and electrochemistry of invertebrate skeletal muscle; electrical activity of invertebrate and vertebrate cardiac muscle; the electrical activity and electrochemistry of visceral muscle; the mechanics of muscle; and excitation-contraction coupling and relaxation. This book will be of interest to practitioners in the fields of anatomy and the health sciences.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483280455

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The Comparative Structure and Function of Muscle

HENRY HUDDART

Department of Biological Sciences, University of Lancaster, England

Table of Contents

Cover image

Title page

Copyright

Introduction

SECTION 1: THE STRUCTURE OF MUSCLE

Introduction

The Fine Structure of Skeletal Muscle

Publisher Summary

The Contractile Component

The Non-contractile Component

Heterogeneity of Muscle Fibres

Chapter 2: The Fine Structure of Cardiac and Visceral Muscle

Publisher Summary

Cardiac Muscle

Visceral Muscle

Chapter 3: The Innervation of Muscle

Publisher Summary

The Vertebrate and Arthropod Pattern

SECTION 2: THE ELECTRICAL ACTIVITY OF MUSCLE

Introduction

Chapter 4: The Ionic Basis of the Resting Potential

Publisher Summary

The Ionic Hypothesis

The Experimental Testing of the Ionic Hypothesis

Membrane Structure and Permeability

Chapter 5: The Action Potential and the Activation of Muscle

Publisher Summary

The Sodium Hypothesis

Testing of the Sodium Hypothesis

Membrane Circuitry

Evocation of the Action Potential

Chapter 6: Electrical Activity and Electrochemistry of Invertebrate Skeletal Muscle

Publisher Summary

Introduction

Nematodes

Annelids

Arachnids

Crustaceans

Insects

Molluscs

Echinoderms

Chapter 7: Electrical Activity of Invertebrate and Vertebrate Cardiac Muscle

Publisher Summary

MYOGENIC HEARTS

Tunicates

Insects

Molluscs

The Vertebrates

Birds

Mammals

NEUROGENIC HEARTS

Arachnids

THE ELECTROCHEMISTRY OF CARDIAC MUSCLE

The Resting Potential

The Action Potential

Chapter 8: The Electrical Activity and Electrochemistry of Visceral Muscle

Publisher Summary

Introduction

The Resting Potential

The Action Potential

The Innervation, Neural Control and Pharmacology of Vertebrate Visceral Muscle

Innervation, Neural Control and Pharmacology of Invertebrate Visceral Muscle

The Electrochemistry of Visceral Muscle

SECTION 3: THE MECHANICAL ACTIVITY OF MUSCLE

Introduction

Chapter 9: The Mechanics of Muscle

Publisher Summary

Chapter 10: Excitation–Contraction Coupling and Relaxation

Publisher Summary

Disruption and Modification of EC Coupling

A Short Glossary of Fine Structural and Physiological Terminology

References

Author Index

Subject Index

OTHER TITLES IN THE ZOOLOGY DIVISION

Copyright

Pergamon Press Ltd., Headington Hill Hall, Oxford

Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, New York 10523

Pergamon of Canada Ltd., 207 Queen’s Quay West, Toronto 1

Pergamon Press (Aust.) Pty. Ltd., 19a Boundary Street, Rushcuttcrs Bay, N.S.W. 2011, Australia

Pergamon Press GmbH, Burgplatz 1, Braunschweig 3300 West Germany

Copyright © 1975 Pergamon Press Inc.

All Rights Reserved. No part of this publication may be reproduced, stored m a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior permission of Pergamon Press Limited

First edition 1975

Library of Congress Cataloging in Publication Data

Huddart, Henry.

The comparative structure and function of muscle.

(International series of monographs in pure and applied biology. Zoology Division, v. 53)

Bibliography: p.

1. Muscle. I. Title. [DNLM: 1. Anatomy, Comparative. 2. Muscles—Anatomy and histology. 3. Muscles—Physiology. 4. Physiology, Comparative WE500 H883c]

QP321.H8 1975 591.1′852 74–13870

ISBN 0 08 017845 6

Printed in Great Britain by Biddies Ltd, Guildford, Surrey

Introduction

MUSCLES are complex biological machines which convert chemical energy into mechanical energy for the support and maintenance of the body, permitting the body to act against the environment. The activity of the muscular system is part of the complex process of the reaction of animals to their environment, and it constitutes one of the major elements in homeostasis. No single text can hope to deal with muscular activity as part of the homeostatic process in any critical way, so the discussion here has been limited to the activity of muscle as a tissue, covering excitation, contraction and relaxation and the control mechanisms involved in these phenomena. These elements in muscular activity are considered from the point of view of the relationship between structure and function.

Since muscle is so exceedingly varied both in structure and function, the methods which biologists have employed to study it have of necessity drawn upon the experimental expertise of many varied disciplines. Biophysical techniques of X-ray diffraction and nuclear magnetic resonance have provided much information about the components of muscle and their interactions at the molecular level. Biochemical investigations have revealed much about subcellular control systems within muscle cells, and traditional physiological studies have told us much about electrochemistry and muscle mechanics. With the addition of fine structural information from electron microscopists, a comprehensive picture is emerging about the total behaviour of muscle which is a synthesis of the above data. This text attempts to provide an overall picture of just what happens in muscle during excitation, contraction and relaxation and how the structures present are involved in these processes.

Over the past decade, a considerable literature has been produced concerned with invertebrate muscle. While this area is not as well documented and the data not as well disseminated as that of vertebrate muscle, it is now possible to examine muscle function in a comparative manner as the literature permits. Since invertebrate muscle is exceedingly varied in structure and function, this diversity serves to remind us of the strong relationship between structure and physiology, a relationship which should always be kept in mind.

The literature on muscle is so immense, and its production rate accelerates each year by such a degree, that it is now impossible for one investigator to be in touch with the newer developments in all fields. It is also very doubtful whether the mastery of technical detail needed to evaluate information from so many disparate disciplines lies within the compass of one person. This means that much information contributing to our understanding of muscle has to be examined at second hand, with some inevitable loss of critical understanding.

This text is based upon a series of lectures given at the University of Lancaster over the last seven years, and it follows a natural division into structure, electrophysiology and excitation and mechanical activity. Within each section, an attempt is made to cover all muscle types in as wide a range of animals as the literature will allow.

Clearly, a book of this type cannot be brought to fruition without the help and advice of many people. I am particularly indebted to my friend and colleague Dr. Stephen Hunt for much helpful discussion and the loan of many of his unpublished micrographs of visceral muscle. I also wish to thank my research students for their time and effort in reading parts of the text, suggesting clarifications and additions and for the loan of their unpublished work and electron micrographs. Without the help of my technician, Mr. G. R. Abram, the preparation of much of the illustrative material would have been a most burdensome task. Lastly I wish to thank Mr. David Inglis and the production team of Pergamon Press for their sheer expertise in translating my manuscript and illustrations into book form.

SECTION 1

THE STRUCTURE OF MUSCLE

Introduction

THE widespread application of electron miscroscopic techniques to muscle tissue during the last twenty years has revealed just how diverse in detail but uniform in fundamental characteristics muscle is. Fine structural studies, of course, are not an end in themselves. Of great importance, however, is the attempt to correlate differences in fine structure with differences in function. In this approach, the electron microscope is simply used as another analytical tool, no less important than the oscilloscope or the intracellular electrode.

Fortunately, muscle has attracted many microscopists/physiologists who have used this structure/function approach, and as a result, much is now known about how variations in structures such as myofibrils, the T system and the sarcoplasmic reticulum are correlated with variations in contractility, speed of response and excitation–contraction coupling mechanisms.

Fine structural studies have shown that the convenient physiological classification of muscle into skeletal (or striated), cardiac and visceral (or smooth) has some basis in structural reality, and so this division of muscle has been retained for both vertebrates and invertebrates throughout this book. In a work of this nature it would be somewhat pointless to engage in a detailed description of the fine structure of all muscle types since only the basic concepts need be known to clarify the analysis of muscle functioning. Much more detail of muscle fine structure will be found in specialist works such as Bourne (1960, second edition 1972–) and Huxley (1960) and in volumes on certain animal groups such as molluscs (Hoyle, 1964) and insects (Hoyle, 1965). Many of the recent excellent ultrastructural atlases also deal with muscle and are well worth examining (Fawcett, 1966; Toner and Carr, 1968; Smith, 1968; Sandborn, 1970).

CHAPTER 1

The Fine Structure of Skeletal Muscle

Publisher Summary

The morphology of skeletal muscle varies enormously in different animals and at different positions in the same animal body, dependent upon the function the muscle executes. Not only is the obvious feature of size variation seen, but also variations are seen in the arrangement of the individual fibers within the muscle. Even among a relatively uniform group of animals, such as the vertebrates, muscles may be either long-fibred and strap-like or short-fibred and highly divided, the latter being often called multipennate muscles indicating multiple tendon insertions. Among the invertebrates, skeletal muscle is even more varied in its gross morphology. It may be tubular, sheet-like or highly spongy, and diffuses in animals relying upon a hydrostatic skeleton, but it is usually more orientated in the discrete muscle systems associated with the external skeletons of arthropods. Even within the arthropods, however, the arrangement of the individual fibers within a muscle can be variable. Muscles may be basically strap-like in fiber orientation as in many coxal, flight, and intersegmental muscles or pinnate in form as in many femoral muscles, but all of them show the advantages of first-order lever systems.

THE gross morphology of skeletal muscle varies enormously in different animals, and at different positions in the same animal body, dependent upon the function the muscle executes. Not only is the obvious feature of size variation seen, but, more significantly, variations are seen in the arrangement of the individual fibres within the muscle. Even among a relatively uniform group of animals such as the vertebrates, muscles may be either long-fibred and strap-like (such as the sartorius and the gastrocnemius), or short-fibred and highly divided (such as the deltoid), the latter being often called multipennate muscles, indicating multiple tendon insertions. A very readable account of the gross morphology of skeletal muscle and its relation to lever factors and the skeleton can be found in Young

Among the invertebrates, skeletal muscle is even more varied in its gross morphology. It may be tubular, sheet-like or highly spongy and diffuse in animals relying upon a hydrostatic skeleton (such as annelids and molluscs), but it is usually more orientated in the discrete muscle systems associated with the external skeletons of arthropods. Even within the arthropods, however, the arrangement of the individual fibres within a muscle can be very variable. Muscles may be basically strap-like in fibre orientation, as in many coxal, flight and intersegmental muscles or pinnate in form (with radiating fibres) as in many femoral muscles, but all seem to show the advantages of first order lever systems (see Fig. 1.1). Chapter 9 gives a fuller treatment of this topic.

FIG. 1.1 The structure and arrangement of some insect skeletal muscles. (a) Main femoral musculature of a typical insect. The apodemes are inserted above and below the tibial articulation, which thus restricts movement into the vertical plane in relation to the femur. (b) The femur/tibia dicondylic joint. The cross-hatched area is flexible cuticle known as the articular corium. (c) Isolated single unit insect muscle (e.g. a coxal muscle). (d) Multi-unit muscle with separate muscle units (e.g. stick insect flexor tibialis). (c) Undivided multi-unit muscle (e.g. flexor tibialis of cockroach and Lcpidoptcra. From Huddart (1971b).

No matter how varied the fibre arrangement within muscles, each individual fibre has a basically similar ultrastructure. In physiological terms, the ideal physical model of a muscle cell consists of a contractile component and a non-contractile component. This physiological description is mirrored in the cell’s fine structure, where the contractile component is seen to consist of a series of rod-like elements orientated in the longitudinal axis of the cell, the myofibrils, and the non-contractile component consists of ground sarcoplasm containing nuclei, mitochondria, glycogen deposits and the longitudinal tubules of the sarcoplasmic reticulum. The relative balance between these two major cellular components varies in different muscle fibres and this has important consequences in terms of fibre power output. Variation in the myofibril fraction of the cell is strongly correlated with variations both in the speed of contraction and tension exerted. Since the non-contractile fraction of the cell represents a considerable scries and parallel elastic component, causing great viscous damping and drag on the myofibrils, it will be obvious that cells with a large non-contractile component will be inherently inefficient. In terms of maximizing the tension output per unit cross-sectional area, the greater the myofibril proportion of cell volume, the greater the contractile efficiency of the cell. The sketch in Fig. 1.2 shows just how variable the myofibril density can be in various skeletal muscle fibres.

FIG. 1.2 The distribution of myofibrils in some skeletal muscle fibres. (a) Anodonta (Mollusca); (b) Octopus (Mollusca); (c) hirudinean (Annelida); (d) insect leg muscle and vertebrate skeletal muscle; (e) cockroach flight muscle; (f) Thyone (Echinodermata). (a), (b), (c) and (f) redrawn from Hoyle (1957) and Florey (1966), (d) and (c) drawn from fresh material (frozen sections examined with phase contrast).

The greatest overall myofibril density seems to be that in insect flight muscles (Fig. 1.3) where the myofibrils may account for anything from 70 to 80% of fibre cross-sectional area. In these fibres the ground sarcoplasm is reduced to an absolute minimum, the only major non-contractile inclusions being the mitochondria and the rows of sarcoplasmic reticular tubules (Fig. 1.4). This situation is very different from that seen in many skeletal muscles such as the stick insect leg muscles (Huddart and Oates, 1970a; Fig. 1.5), crustacean phasic and tonic fibres (Jahromi and Atwood, 1967), and in visceral muscle (see Chapter 2), where a considerable ground sarcoplasm is present and where contraction speed and mechanical output are considerably lower than that seen in flight muscle.

FIG. 1.3 Low power survey electron photomicrograph of cockroach flight muscle. Portions of three muscle fibres are visible in the field and a single axon near its terminal with various tracheal profiles. Note that there is little wastage of fibre area on non-contractile inclusions, and the periphery of the fibre has little sarcoplasm. Obvious structures visible arc columnar myofibrils (M), darker-staining dense mitochondria (Mi) and sarcoplasmic reticulum (S). The axon contains many mitochondria and abundant transmitter vesicles. Original plate courtesy of Mr. M. Greenwood. Print magnification:× 12,000.

FIG. 1.4 Cockroach flight muscle fibre showing five myofibrils and parts of three mitochondria separated by rows of sarcoplasmic reticular tubules. Note the great density of the mitochondria, and the multiplication of SR tubules. Print magnification: ×60,000. H. Huddart, previously unpublished.

FIG. 1.5 Longitudinal section of stick insect flexor tibialis muscle fibre cut at the fibre periphery. Notice the complexity of the ground sarcoplasm and its involvement with the basement membrane material of the tracheoles (T). Collagen fibrils (C), mitochondria (M) and glycogen deposits arc also present. Print magnification: ×20,000. From Huddart and Oates (1970a).

The Contractile Component

The myofibrils

Perhaps the most characteristic feature of skeletal muscle is its regular pattern of light and dark bands, their presence being responsible for the name ‘striated’ which is often given to this type of muscle. This banding pattern is clearly visible in individual muscle cells examined by light microscopy (Fig. 1.6A) and the regular banding is even more apparent when individual myofibrils are examined in the electron microscope (Fig. 1.6B). It can be clearly seen that the rod-like myofibril is divided into a series of many hundreds of identical repeated units, the deep-staining Z discs forming the limits of the units. The unit distance between two adjacent Z discs is called a sarcomere, each sarcomere representing one of the major periods in the banding pattern of the whole cell. The sarcomere is thus the basic building brick of the contractile machinery of the cell. What gives skeletal muscle its characteristic striated appearance is the arrangement of parallel bundles of myofibrils with their sarcomeres ‘in register’, that is, with their Z discs in almost perfect alignment. This condition can be seen in Fig. 1.6B.

FIG. 1.6 A, light micrograph of crustacean skeletal muscle showing clear myofibrillar banding patterns. Print magnification: ×3,000. B, electron photomicrograph of crustacean skeletal muscle showing a group of myofibrils exhibiting a clear banding pattern. The basic myofibrillar unit is the sarcomere, delimited by two adjacent Z discs (Z). The clear appearance of banding of the whole cell in the light microscope is due to the ‘in register’ alignment of Z discs of parallel fibrils, a characteristic of striated muscle. M lines are clearly visible (see text). Print magnification: ×18,000. (courtesy of Mr. K. Oates); Courtesy of Mr. S.J. Bradbury.

The impression is often given in elementary accounts that the myofibrils are relatively static structures within the muscle cell, but this is far from the case. During the post-natal growth of mouse skeletal muscle, Goldspink (1970) showed that the number of myofibrils within a fibre progressively increased, in some cases from 75 to as many as 1200. The bimodal distribution of myofibrils within the normal 0.4 to 1.2 μm range is suggestive of an increase by longitudinal splitting, a phenomenon Goldspink often observed in his sectioned material. Increase in myofibril number by longitudinal splitting is also seen in insect and crustacean skeletal muscle, where it would appear that even in the fully adult stage splitting occurs involving some considerable turnover of myofibrillar material (Fig. 1.8). The myofibrillar system of a skeletal muscle cell must thus be considered a dynamic, highly plastic, and constantly evolving structure.

FIG. 1.8 Myofibril splitting in crustacean skeletal muscle. In both cases, myofibrils show proliferation by the development of longitudinal splits (at arrows). M lines are again clearly visible. Magnifications in both A and B are × 32,000. Huddart, previously unpublished.

Table 1.1 is an attempt to summarize some of the more important myofibrillar characteristics of representative vertebrate skeletal muscles. It must be stressed that great caution has to be exercised in comparing measurements from muscles fixed under different conditions by different experimenters since some variation will occur in the degree of shortening of the sarcomere. The data in this and the following table have been taken from published micrographs showing least obvious distortion of sarcomere structure. However, it must be borne in mind that sarcomere ‘length’ is very labile and whether a true resting length exists at all is open to question. This is particularly true of supercontracting muscle fibres in, for example, barnacles (Hoyle et al.,, 1965) and insects (Osborne, 1967).

TABLE 1.1

PHYSICAL CHARACTERISTICS OF THE MYOFIBRILS OF SOME TYPICAL VERTEBRATE SKELETAL MUSCLES

What stands out in Table 1.1 is that despite the variability of fibre diameter and arrangement of fibres within muscles, the basic myofibrillar machinery is almost unbelievably uniform, particularly in the case of the lengths of the contractile sub-components. Only sarcomere length and diameter seem to be subject to significant size variation, which is understandable in the light of what is known about longitudinal splitting. Corresponding data from some typical representative invertebrate skeletal muscle myofibrils are shown in Table 1.2. It can be seen that while major myofibrillar characteristics are somewhat more variable than in vertebrates, they are still surprisingly consistent in such vastly different muscles in such very different animal phyla.

TABLE 1.2

PHYSICAL CHARACTERISTICS OF MYOFIBRILS OF SOME REPRESENTATIVE INVERTEBRATE SKELETAL MUSCLES

Composition of the sarcomere

The banding pattern of the myofibril is of significance only in that it mirrors the relative density and nature of the contractile proteins which constitute the sarcomere. The banding of a typical sarcomere is shown in diagrammatic form in Fig. 1.7 and the actual appearance of a sarcomere in the electron microscope is shown below it. It can be seen that the bands represent the relative distribution of two sets of fine filaments (the myofilaments) which are more or less constant in diameter and length in any one muscle fibre. The banding seen in the light microscope shows the sarcomere to consist of two outer light-staining zones, a central medium-staining zone surrounded by lateral dark-staining zones. The outer limits of these dark zones constitute what is called the A band, the two outer light zones constitute (along with their partners from the adjacent sarcomeres) the I bands. The very deep staining line in the centre of the I bands (used to delimit the sarcomere) is called the Z disc while the dark line (or group of lines) often seen in the centre of the A band is called the M line and the clearer regions on either side of the M line constitute the H zone.

FIG. 1.7 A, diagrammatic representation of the sarcomere structure in a typical striated muscle myofibril. B and C, actual sarcomeres from crayfish and copepod muscle respectively, with interpretations of the banding shown at the side. See text for details. B from Huddart and Oates (1970b), C courtesy of Mr. K. Oates.

The two types of myofilaments constituting the contractile machinery of the sarcomere are polymers of the proteins myosin and actin. A glance at Fig. 1.8 shows that the myofilaments are of two types, thick and thin, the thick filaments consisting of longitudinally orientated molecules of myosin, these being responsible for the deep-staining appearance of the A band in electron microscopic sections. The thin filaments are composed of double-stranded chains of the globular protein actin, these being responsible for the less dense I band region. The actin polymer passes through the Z disc (see later in text) and in each half sarcomere in vertebrate muscle there are 400 molecules of actin (200 in each strand) in the filament, the whole filament having 800 of these globular units. The results of X-ray diffraction studies and the molar and weight ratios of actin to myosin suggests the presence of about 360 molecules of myosin in each thick filament of vertebrate skeletal muscle.

Myosin and actin substructure

The more important data from electron microscopy, X-ray diffraction and protein chemistry studies concerning contractile protein structure have been reviewed by Bendall (1969) and Huxley (1971).

Although the myosin molecules in the thick filaments are all longitudinally orientated, the molecules in one-half of the filament have an orientation which is the reverse of that shown by the molecules in the other half of the filament. The individual myosin molecules, resembling a somewhat elongate tadpole, have a head and tail structure, the tails forming the centre rod or backbone of the thick filament, the tails always pointing towards the centre of the A band. This is shown in diagrammatic form in Fig. 1.9a. This reversal of molecular orientation means that the force developed at each interacting site (the head end of the molecule) will act to pull the actin filaments into the A band from both sides of the H zone, thus reinforcing the efficiency of shortening of the sarcomere (Fig. 1.9b).

FIG. 1.9 (a) The arrangement of the individual myosin molecules in the thick filament of skeletal muscle. The central ‘backbone’ of the filament is formed by the long tails of the molecules, and the reversal of polarity in the centre of the filament is clearly visible. (b) Diagram showing how the structural polarity of the myosin cross-bridges and the polarity of the actin molecules in the thin filaments act to slide the actin filaments towards the centre of the A band. (c) Diagram showing the arrangement of cross-bridges on a 6/2 helix (i.e. six bridges arranged in pairs) around the central axis. The helix repeat is about 42.9 nm, and the linear bridge distance is 14.3 nm. All redrawn from Huxley (1971).

The diagram in Fig. 1.9a may give the impression that the myosin molecules are arranged in the filament in a two-dimensional manner, but that this is not the case is shown by X-ray diffraction studies (Huxley and Brown, 1967) which indicate that the myosin molecules are arranged around the longitudinal axis of the filament backbone in a spiral manner. This is shown in diagrammatic form in Fig. 1.9c where the spirally placed pegs represent the molecular heads. The helical repeat distance (that is, the distance along the backbone for one complete revolution of the heads on the filament) is 429 A, and the distance between adjacent heads is about 143 Å linear distance along the filament. For data from insect muscle see Reedy (1967).

The individual myosin molecules consist of long rods about 1500 Å long with globular regions containing both the ATPase activity and the actin-binding sites at one end. The rod is, in fact, a double α-helix chain (Fig. 1.10), and the total molecular weight is about 500,000. Of interest to biophysicists in determining the molecule’s orientation and reactivity is the observation that the molecule has two obvious points of weakness at which it can be attacked by the proteolytic enzymes trypsin or papain and broken up into sub-units. Trypsin attack breaks the molecule initially into two fractions, light and heavy meromyosin (LMM and HMM respectively). LMM is the major part of the tail region of the molecule, having a length of about 930 Å and a molecular weight of about 150,000. This part of the molecule possesses no ATPase activity and its tendency to aggregate suggests that it is bound in the spine of the filament. The HMM fraction has a length of about 600 Å and a molecular weight of about 340,000. This heavy but short fraction contains the head region and it possesses the ATPase activity and actin-combining property of the whole parent molecule.

FIG. 1.10 Diagrammatic representation of the structure of the individual myosin molecule. The total molecular weight of about 500,000 is roughly distributed as LMM 150,000 and HMM 350,000. For further details see the text. Redrawn and based on data in Lowey et al., (1969), Bendall (1969) and Huxley (1971).

The LMM fraction consists of two identical polypeptide chains in an α-helical conformation and the aggregation of the LMM chains of the spirally arranged myosin molecules is responsible for the deep-staining bulk of the A band thick filament which we see in electron microscopic sections. The HMM fraction consists of the head and part of the tail of the molecule (Fig. 1.10) and further enzymatic attack breaks HMM into subfragments 1 and 2 (HMM S1 and HMM S2 respectively). HMM S1 possesses all the ATPase and actin-combining activity of the parent molecule. In the electron microscope, the individual molecular heads appear to be about 100 Å long and 200 Å wide, and their molecular weight is about 120,000 to 180,000 (Perry, 1967). It is clear that two such globular head-like units constitute the HMM S1 fraction proper (Fig. 1.10).

The HMM S2 fraction, length about 370 Å and molecular weight about 60,000, is a double helix like the LMM fraction, but unlike the latter it does not form aggregates either with itself or with LMM. This evidence suggests that the HMM S2 fraction is not bound into the backbone of the thick filament along with the LMM fraction. This deduction has given rise to the view that the head part of the myosin molecule may be hinged out of the thick filament at the junction between HMM S2 and the LMM fraction. The interactive arc thus formed will have the radius of the HMM S2 length (about 370 Å) (see Lowey, 1967). This hinging–out hypothesis is of fundamental importance since it postulates how attachment of cross-bridges to specific actin loci can take place over a wide range of interfilament spacings (Fig. 1.11).

FIG. 1.11 Diagrammatic representation of the ‘hinging out’ hypothesis. This explains how the head subunits of the thick filaments can attach to cross-bridges on the thin filaments over a wide range of interfilament spacings, yet always maintaining the same attachment angle. This hypothesis assumes flexible linkages at either end of the S2 part of the chain. Redrawn from Huxley (1971).

The actin monomers are about 55 Å in diameter and they polymerize into a double-stranded super helix (Fig. 1.12). The actin monomer is globular (G actin) and its transformation into the thin filament (the fibrous form, or F actin) takes place with dephosphorylation of ATP in the following manner:

FIG. 1.12 Diagrammatic representation of the structural polarity of the actin molecules in the thin filaments. Note that in any one F actin filament and on any one side of the Z disc, all the actin monomers have the same relative polarity, but this polarity is reversed on the other side of the Z disc. This reversal allows matching of the actins with the polarity of the myosin cross-bridges on either side of the Z disc (see Fig. 1.9b). Redrawn from Huxley (1971).

The double helices of F actin have a regular cross-over point of about 360 Å (Huxley and Brown, 1967). Within the super helix, the actin molecules are arranged in strict structural polarity. All of the actin molecules on one side of the Z disc are orientated in the same direction and this orientation is reversed on the other side of the Z disc (Fig. 1.12). Since the myosin heads also show orientation reversal in the two sides of the A band, it is clear that the actin and myosin molecules always have the same relative orientation to each other (Fig. 1.9b).

Other structural proteins of the sarcomere

Actin and myosin are by no means the only proteins associated with the contractile apparatus. At least three other well-defined molecules are present—tropomyosin, troponin and α-actinin. The tropomyosin molecule is a long straight double helix, but unlike myosin it lacks the head region and is thus shorter and possesses no ATPase activity. Tropomyosin, along with troponin, is present in the thin filament between the actin super helix formations and it is also present, along with α-actinin, in the Z disc. There is evidence that tropomyosin and troponin act as a core for the F actin filaments and are in some way responsible for the control of filament length (Barany et al.,, 1962; Bendall, 1964). It now seems clear that what in the past has passed for tropomyosin is in reality a mixture of tropomyosin and troponin. The ATPase activity of purified actin and myosin becomes extremely sensitive to calcium ions in the presence of tropomyosin, but it is now known that highly purified tropomyosin lacks this calcium-sensitizing action, the latter property appears to be conferred on the system by troponin (Briggs and Fuchs, 1967). A likely role for troponin is to bind the tropomyosin double helices together within the actin super helices, and antibody staining has shown troponin at intervals of about 400 Å along the thin filament (Ebashi and Kodama, 1966). Unlike tropomyosin, troponin is a globular protein with molecular weight about 80,000 (Ebashi et al.,, 1967).

Recent studies have shown that troponin is not a single protein, and it has been fractionated into a number of components by a variety of investigators. Schaub et al., (1967) and Schaub and Perry (1969) have separated troponin into A and B components, Ebashi et al., (1971) have resolved on three components, troponins I and II and fraction III, while Greaser and Gergely (1971) have identified four components, fractions 1 to 4. There does seem to be agreement that troponin has two major components, one producing inhibition of ATPase activity in the presence or absence of calcium, the other conferring calcium-sensitivity, but there is still much doubt as to whether the components are pure proteins or not.

Troponins A and B possess differing sedimentation constants and differing solubility characteristics. Troponin B binds to tropomyosin B in a weight ratio of 1 : 1, and it produces calcium-independent inhibition of ATPase activity. Troponins A and B form a complex soluble at low ionic strength, and troponin A will bind calcium, but troponin A plus tropomyosin has practically no effect on ATPase activity.

The three fractions of Ebashi and co-workers differ in molecular weight and many other characteristics. Troponin I (4×10⁴ molecular weight) interacts with tropomyosin, is insoluble at low ionic strength, and does not bind calcium. Troponin II (2.2 × 10⁴ molecular weight) binds about two calcium ions per mole, and also gives rapid superprecipitation of actomyosin only slightly affected by calcium. Troponins I and II plus tropomyosin induce rapid actomyosin superprecipitation in the absence, but not the presence of calcium. Fraction III (1.7 × 10⁴ molecular weight) is present in variable amounts, but is not actually essential for the reconstitution of the relaxing system. Although it binds calcium to the extent of one mole per mole, it docs not confer calcium-sensitivity on Troponin I.

The four fractions of Greaser and Gergely (1971) also differ both in molecular weight and calcium-activity characteristics. Fraction I (molecular weight 1.4×10⁴) gives calcium-independent inhibition of ATPase activity, as does fraction 2 (molecular weight 2.4×10⁴), although the former is not indispensible in the reconstructed troponin system. Fraction 3 (molecular weight 3.5×10⁴) gives slight inhibition of ATPase activity, while fraction 4 (molecular weight 2.1×10⁴) binds calcium. Fractions 2 plus 4 showed some calcium-sensitive inhibitory activity and fractions 2, 3 and 4 possess full activity. Hence fraction 4 appears to be similar to troponin A (and troponin II) and fractions 2 and 3 resemble troponin B (troponin I).

The weight ratio of troponins I and II is in the range of 1.1—1.3 to 1, and it seems likely that the troponin complex consists of one molecule of troponin I and two molecules of troponin II. The molecular weight of this complex would then be about 8×10⁴, within the range of reported values. If this assumption is correct, then troponin would bind three to four moles of calcium per mole, rather than the two supposed until recently. For a more detailed treatment of troponin, and its relationship with other proteins of the contractile system, see Taylor (1972).

There is now conclusive evidence for the existence of a third filament in skeletal muscle additional to actin and myosin. The simple sliding filament theory involving actin and myosin alone is not able to explain many of the contractile properties of muscle, such as passive elasticity and the ability of overstretched muscle to develop tension. If muscle is so stretched that actins and myosins no longer overlap, not only does the muscle hold together and the sarcomere retain its integrity, but it is able to contract on KCl depolarization. This evidence suggests the presence of a third filament which is not only elastic but also contractile, and present in both the A and I band. McNeill and Hoyle (1967) have now produced electron-microscopic evidence which can no longer be ignored for the existence of this third filament, called the superthin or T filament in skeletal muscles of barnacle, Podophthalmus (Malocostraca), Doropygus (Copepoda), garter snake and rabbit.

The T filament is about 25 Å in diameter and passes from Z disc to Z disc through all the zones of the sarcomere, and it can be seen bridging the gap between the actin and myosin filaments in highly stretched muscle. Walcott and Ridge way (1967) have produced further evidence for the existence of the T filament in rabbit psoas muscle and in skeletal muscles of Homarus and Benacus (Coleoptera) subjected to myosin extraction. In these myosin extracted muscles, the T filament networks are clearly visible in the A band passing right through the sarcomere. It seems likely that the T filament may be composed of the protein myofibrillin which can be extracted from muscle residues which have already been actomyosin extracted (Guba and Harsanyi, 1966).

The sliding filament hypothesis

The evidence is now conclusive that tension in muscle fibres is developed when interactions occur between actin and myosin filaments resulting in a transient or sustained shortening of the sarcomere. Only one hypothesis explaining how this may come about has so far been able to withstand the tests of both rigorous biophysical analysis and critical electron microscopy. This is known as the sliding filament hypothesis and it proposes that cross-bridges occur between myosin heads and neighbouring actin molecules in the actin super helix causing a progressive, ratchet-like interdigitation of actin within the myosin (A band) zone of the sarcomere. On this hypothesis, contraction results in the sarcomere telescoping into the A band. The most comprehensive statement of this hypothesis can be found in Huxley and Hanson (1960).

The primary detective work which led to this hypothesis was the observation firstly, that during the contraction-relaxation cycle the A band did not significantly change in length but the I band did; and secondly, during contraction, as the I band became narrower, the H zone disappeared, this latter zone being significantly larger than normal in stretched muscle. Coupling these observations with electron microscopic evidence of the distribution of the two types of contractile proteins within the sarcomere, we arrive at the sliding filament hypothesis which can be diagrammatically summarized as shown in Fig. 1.13. The electron microscopic evidence for this hypothesis is overwhelming, and clearly shows actin/myosin interdigitation, but what is often forgotten in this simple picture is the immense stress that the whole filament lattice must sustain during contraction. The ability of the actomyosin lattice to withstand such stresses is largely due to extra strengthening interlinks between the myofibrillar complement of myosin filaments and also between the actin filaments. The myosin-myosin interlinking occurs in the M band region (the region of tail-to-tail abutment of the molecules) and these interlinks (which probably involve other proteins such as tropomyosin) are responsible for the line (or group of lines) clearly resolvable in many sections (Knappeis and Carlsen, 1968). The actin-actin interlinks occur in the Z disc, probably involving tropomyosin. A reasonable analogy is the actin filaments ‘wired’ to the disc lattice by their central tropomyosin cores. The actin filament is thus a sort of sleeve fitted over a perforated disc lattice with projecting tropomyosin pegs. A much more detailed analysis of the composition and structure of the Z disc and its relation to actin anchorage can be found in Huxley (1963), Armstrong and Porter (1964), Stromer et al., (1967), Armstrong (1970a) and Rowel (1971).

FIG. 1.13 Diagram of the sarcomeric banding changes associated with contraction in skeletal muscle (upper row) and the interpretation of these changes in terms of the relative sliding together of the two contractile filaments (lower row). The left-hand figures show stretched muscle in which the actin/myosin overlap is small and hence the moderately-dense H zone is large. In relaxed muscle (centre pairs), overlap of actin and myosin is extensive resulting in a narrow H zone, while in contracted muscle (right-hand pairs), actin/myosin overlap is total and the H zone disappears. In all conditions, the A zone (myosin area) remains the same, the I band being reduced as contraction proceeds.

No matter how perfectly the electron microscopic evidence supports observed gross banding pattern changes during contraction, it in no way provides any clues as to the molecular nature of the actin/myosin cross-links. This latter problem has been elucidated in a most elegant manner by Davies (1963, 1964), and his views have now withstood much critical analysis (see Bendall, 1969).

In essence, the Davies theory proposes that the active actin-binding region of the myosin molecule head consists of a long polypeptide chain terminated by an ATP molecule, and that this chain has two alternative configurations. In the resting state, the polypeptide chain is extended since the residual negative charge on the terminal ATP is repelled by the fixed negative charge believed to reside near the ATPase site on the myosin head (Fig. 1.14). Activation commences when a calcium ion attaches to the terminal ATP of the myosin chain and forms a link between it and the bound ADP of one of the actin molecules of the thin filament. The calcium linkage neutralizes the residual negativity on the myosin ATP and the polypeptide chain contracts to an α-helix configuration due to hydrogen bond formation (Fig. 1.14). This shortening of the polypeptide chain to an α-helix pulls the actin filament one cross-bridge distance towards the centre of the A band in relation to the myosin filament, and brings the cross-bridge ATP into the activity zone of the myosin ATPase site. As a result of this, the ATP is split by the myosin ATPase and the chain relaxes as the ADP is rephosphorylated due once again to electrostatic repulsion of the negative charges. This breakage and relaxation of the cross-bridge allows rebinding to take place at a new actin site in the continued presence of calcium ions when a new myosin–ATP–calcium–ADP–actin link is established. The result is that a series of miniature contractions and relaxations of the cross-bridges occurs and each contraction pulls the actin filament one interbinding site distance into the A band. Each microcontraction is thought to pull the actin approximately 100 Å into the A band (Davies, 1963). This process will continue until the sarcoplasmic calcium is reduced to a low level by the calcium-pumping action of the sarcoplasmic reticulum. It is thought that a calcium level of about 10−7M is low enough to reverse active cross-bridge formation when the actin filaments assume a position (the resting state) related to the resting level of calcium in the cell. The cycle of these processes is represented in diagrammatic form in Fig. 1.15.

FIG. 1.14 The postulated resting (upper) and contracted (lower) state of the myosin molecule head. In the extended resting form, ATP is bound to the reactive site, but after the formation of the calcium link with the actin ADP site, the bridge contracts to the α-helical form. Redrawn from Davies (1963)

FIG. 1.15 Diagrammatic representation of the cycle of events leading to a single miniature contraction cycle, involving one myosin head pulling an attached actin a distance of 100 Å into the A band. Redrawn from Davies (1963).

As was pointed out earlier in this chapter, both the electron microscopic and X-ray analysis evidence supports the hypothesis that the myosin heads (i.e. the cross-bridge sites) are organized with six sites per pitch or revolution of the filament. The X-ray and electron microscopic evidence only differs in that the latter suggests that the six cross-bridges are arranged in a simple spiral, whereas the former indicates the presence of three pairs of cross-bridges per revolution of the filament.

In view of the six-fold cross-bridge configuration it would be reasonable to expect each myosin filament to be surrounded by six actin filaments for the purpose of cross-linking (Fig. 1.16). This is, in fact, the actual orbital actin configuration seen in many vertebrate skeletal muscles, and particularly in insect flight muscle (Fig. 1.17), but it is by no means the only actin orbit configuration seen in either vertebrate or invertebrate skeletal muscle (Fig. 1.17). Table 1.3 is a compilation of actin : myosin orbital arrangements from a range of skeletal muscles and it can be seen that great deviation occurs from the simple