Muscle 2-Volume Set: Fundamental Biology and Mechanisms of Disease

A valuable study of the science behind the medicine, Muscle: Fundamental Biology and Mechanisms of Disease brings together key leaders in muscle biology. These experts provide state-of-the-art insights into the three forms of muscle--cardiac, skeletal, and smooth--from molecular anatomy, basic physiology, disease mechanisms, and targets of therapy. Commonalities and contrasts among these three tissue types are highlighted. This book focuses primarily on the biology of the myocyte.

Individuals active in muscle investigation--as well as those new to the field--will find this work useful, as will students of muscle biology. In the case of hte former, many wish to grasp issues at the margins of their own expertise (e.g. clinical matters at one end; molecular matters at the other), adn this book is designed to assist them. Students, postdoctoral fellows, course directors and other faculty will find this book of interest. Beyond this, many clinicians in training (e.g. cardiology fellows) will benefit.

• The only resource to focus on science before the clinical work and therapeutics
• Tiered approach to subject: discussion first of normal muscle function through pathological/disease state changes, and ending each section with therapeutic interventions
• Coverage of topics ranging from basic physiology to newly discovered molecular mechanisms of muscle diseases for all three muscle types: cardiac, skeletal, and smooth

• Language: English
• Publisher: Academic Press
• Release date: Aug 29, 2012
• ISBN: 9780123815118

Book preview

Olson

VOLUME 1

Chapter 1 An Introduction to Muscle

Chapter 2 A History of Muscle

Chapter 3 Cardiac Myocyte Specification and Differentiation

Chapter 4 Transcriptional Control of Cardiogenesis

Chapter 5 Cardiomyocyte Ultrastructure

Chapter 6 Overview of Cardiac Muscle Physiology

Chapter 7 Ionic Fluxes and Genesis of the Cardiac Action Potential

Chapter 8 G-Protein-Coupled Receptors in the Heart

Chapter 9 Receptor Tyrosine Kinases in Cardiac Muscle

Chapter 10 Communication in the Heart

Chapter 11 Calcium Fluxes and Homeostasis

Chapter 12 Excitation–Contraction Coupling in the Heart

Chapter 13 Role of Sarcomeres in Cellular Tension, Shortening, and Signaling in Cardiac Muscle

Chapter 14 Cardiovascular Mechanotransduction

Chapter 15 Cardiomyocyte Metabolism

Chapter 16 Transcriptional Control of Striated Muscle Mitochondrial Biogenesis and Function

Chapter 17 Mitochondrial Morphology and Function

Chapter 18 Genetics and Genomics in Cardiovascular Gene Discovery

Chapter 19 Cardiovascular Proteomics

Chapter 20 Adaption and Responses

Chapter 21 Regulation of Cardiac Systolic Function and Contractility

Chapter 22 Intracellular Signaling Pathways in Cardiac Remodeling

Chapter 23 Oxidative Stress and Cardiac Muscle

Chapter 24 Physiologic and Molecular Responses of the Heart to Chronic Exercise

Chapter 25 Epigenetics in Cardiovascular Biology

Chapter 26 Cardiac MicroRNAs

Chapter 27 Protein Quality Control in Cardiomyocytes

Chapter 28 Cardioprotection

Chapter 29 Cardiac Fibrosis

Chapter 30 Autophagy in Cardiac Physiology and Disease

Chapter 31 Programmed Cardiomyocyte Death in Heart Disease

Chapter 32 Wnt and Notch

Chapter 33 Congenital Cardiomyopathies

Chapter 34 Genetics of Congenital Heart Disease

Chapter 35 Mechanisms of Stress-Induced Cardiac Hypertrophy

Chapter 36 Ischemic Heart Disease

Chapter 37 The Pathophysiology of Heart Failure

Chapter 38 The Right Ventricle

Chapter 39 Mammalian Myocardial Regeneration

Chapter 40 The Structural Basis of Arrhythmia

Chapter 41 Molecular and Cellular Mechanisms of Cardiac Arrhythmias

Chapter 42 Genetic Mechanisms of Arrhythmia

Chapter 43 Infiltrative and Protein Misfolding Myocardial Diseases

Chapter 44 Cardiac Aging

Chapter 45 Adrenergic Receptor Polymorphisms in Heart Failure

Chapter 46 Cardiac Gene Therapy

Chapter 47 Protein Kinases in the Heart

Chapter 48 Cell Therapy for Cardiac Disease

Chapter 49 Chemical Genetics of Cardiac Regeneration

Chapter 50 Device Therapy for Systolic Ventricular Failure

Chapter 51 Novel Therapeutic Targets and Strategies against Myocardial Diseases

Part I: Introduction

Chapter 1

An Introduction to Muscle

Joseph A. Hill¹,² and Eric N. Olson²

¹Departments of Internal Medicine (Cardiology)

²Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX

Movement through space is fundamental to life: movement of the entire organism, such as walking; movement of a part of the organism, such as an arm; movement of materials within the organism, such as food in the stomach; movement of offspring into the external world, as in birth. Each is a mechanism indispensable to virtually all members of the animal kingdom. In the end, it is muscle that accomplishes movement. As a consequence, muscle is the most abundant tissue in most animals and accounts for much of the energy-consuming cellular work in an active animal.

Movement occurs in a wide range of contexts and accomplishes a multitude of tasks. To meet this need, a variety of highly specialized forms of muscle have evolved. In this volume, we will explore the multifaceted variety of muscle types, focusing on the origin, function, control, and disease mechanisms in each. Importantly, although muscle is tremendously complex, there are numerous common features across muscle types. All types of muscle harbor a common molecular mechanism of contraction: the sliding of actin and myosin filaments past one another. In all muscle types, electrical activation triggers a rise in intracellular Ca²+, the primary, proximal events that elicit contraction. Finally, diseases that affect one muscle type often touch others.

The many different types of muscle found in the animal kingdom are broadly categorized as striated or smooth. The former term derives from the striped or striated appearance of the muscle cell (myocyte) under the microscope, a consequence of overlapping filaments arranged within the cell. There are two types of striated muscle, skeletal and cardiac, and skeletal muscle is characterized by two general phenotypes, fast twitch and slow twitch.

Cardiac Muscle

Vertebrate cardiac muscle is found in only one place, namely the heart, forming the contractile walls of the organ. Whereas cardiac muscle shares a striated appearance with skeletal muscle, a number of important structural differences exist between these two muscle types. For one, cardiac myocytes are branched, in contrast with the long, multinucleated myocytes in skeletal muscle (Figure 1.1). Second, the points of apposition between cardiac muscle cells contain specialized regions called intercalated discs, where gap junctions provide for regulated transport of molecules and electrical signals from myocyte to myocyte. Thus, an action potential generated in one part of the heart will spread spontaneously to neighboring myocytes via gap junctions. Very rapidly, the electrical signal traverses the entire myocardium, eliciting a coordinated contraction event termed systole. Indeed, cardiac myocytes are connected in a network that functions as a syncytium, which is critical to the coordinated and efficient function of the myocardium during each heartbeat. Skeletal muscle cells, in contrast, will not contract unless triggered to do so by input from a controlling motor neuron; electrical activation of a neighboring fiber will not provoke a contraction.

Figure 1.1 Cardiac muscle: Heart muscle is striated, harboring contractile proteins aligned in a regimented fashion to yield alternating light–dark patterns on light microscopic examination. Cardiac muscle fibers branch and are interconnect via intercalated discs, facilitating a synchronized heartbeat. Skeletal muscle: Skeletal muscle, also striated, consists of bundles of long, multinucleated cells called fibers. Each fiber, in turn, is a bundle of strands termed myofibrils. As with cardiac muscle, the striations arise due to the arrangement of contractile units (sarcomeres) along the length of the fiber. Smooth muscle: Smooth muscle consists of spindle-shaped cells lacking striations.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

Cardiac myocytes can generate action potentials on their own, without input from the nervous system. The spontaneous electrical activation of cardiac muscle is accomplished by specific features of the electrical properties of the cardiac myocyte plasma membrane, including ion channels and transporters with intrinsic pacemaker properties. Indeed, cardiac muscle isolated and placed in tissue culture manifests rhythmic electrical depolarizations and coordinated contractions.

Contraction of the heart muscle would be highly inefficient if all the myocytes were competing for electrical control of the tissue. Nature has solved this problem with two highly specialized features of cardiac tissue. In the wall of the right atrium, near the attachment site of the superior vena cava, lies the sino-atrial (SA) node, a small region of tissue with the fastest rate of spontaneous depolarization; under normal conditions, the SA node functions as the over-riding pacemaker of the heart. Second, rapid spread of electrical activity through the ventricles is accomplished via a network of specialized myocytes, termed Purkinje cells, lying on the endocardial surface of each of the ventricles. These cells do not contribute in a significant way to muscular contraction; rather, they are endowed with electrical cable properties such that electrical signals travel very rapidly through them, substantially faster than through the contractile myocytes themselves.

The cardiac action potential lasts up to 20 times longer than the skeletal myocyte action potential, a critical feature that allows for complete ejection of blood from each cardiac chamber during systole. By contrast, the much shorter skeletal muscle action potential serves only as a trigger for contraction and does not control the duration of the contraction.

Skeletal Muscle

Attached to the bones by tendons, skeletal muscle is responsible for voluntary movements of the body. Adults have a fixed number of muscle cells; weight lifting and other methods of building muscle do not increase the number of cells but simply enlarge those already present. Like cardiac muscle, skeletal muscle has a striated appearance on microscopic examination, owing to the regimented alignment of contractile proteins within each cell.

A skeletal muscle consists of a bundle of long fibers running the length of the muscle (Figure 1.2). Each fiber is actually a single cell with many nuclei, reflecting its formation by the fusion of many embryonic progenitor cells. Each fiber is itself a bundle of smaller myofibrils arranged longitudinally. The myofibrils, in turn, are composed of two kinds of myofilaments. Thin filaments consist of two strands of actin and one strand of regulatory proteins coiled around one another, while thick filaments contain staggered arrays of myosin molecules.

Figure 1.2 Structure of skeletal muscle. Skeletal muscle consists of bundles of long, multinucleated cells called fibers. Each fiber, in turn, is a bundle of strands called myofibrils. As with cardiac muscle, the striations arise due to the arrangement of contractile units (sarcomeres) along the length of the fiber. Sarcomeres, in turn, comprise thick and thin protein filaments which slide past each other in an energy-dependent fashion, thereby effecting contraction.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

As noted, skeletal muscle comprises two types of fibers. Fast muscle fibers are used for short, rapid, powerful contractions. Slow muscle fibers, in contrast, are often found in muscles that maintain posture and are capable of sustaining long contractions. A slow fiber has a less extensive network of sarcoplasmic reticulum and slower calcium pumps than a fast fiber, so calcium remains elevated in the cytoplasm longer. As a result, a twitch in a slow fiber can last approximately five times longer than in a fast fiber. Slow fibers are also specialized to utilize a steady supply of energy; they have a rich blood supply, many mitochondria, and an abundance of the oxygen-storing protein myoglobin.

The cells of skeletal muscles, which we use for voluntary movements such as walking or lifting, are long fibers containing many nuclei within a single plasma membrane. They contain high concentrations of proteins specific to muscle tissue, such as muscle-specific versions of the contractile proteins myosin and actin, and membrane receptor proteins that detect and transduce signals from nerve cells.

A fundamental feature of skeletal muscle action, distinct from cardiac muscle, is the fact that it is graded; we can voluntarily alter the extent and strength of contraction. This critical feature of skeletal muscle occurs despite the fact that at the cellular level, any stimulus that depolarizes the plasma membrane of a single muscle fiber triggers an all-or-none response. A number of mechanisms working in concert contribute to allow for finely tuned muscle contraction, even to the point of playing a Mozart concerto or wielding a scalpel in surgery. First, the rapidity of action potential arrival from a motor neuron can vary. A single action potential will produce an increase in muscle tension lasting about 100 milliseconds or less, termed a twitch (Figure 1.3). If the second action potential arrives before the response to the first is over, the tension will sum and produce a greater response. If a muscle receives an overlapping series of action potentials, further summation will occur. Thus, the level of tension that develops depends on the rate of motor neuron stimulation. Ultimately, if the rate of stimulation is fast enough, the twitches will blur into one smooth and sustained contraction termed tetanus.

Figure 1.3 Individual twitches sum to enhance the force of contraction. Tension develops in a muscle in response to a single action potential triggered by the innervating motor neuron. When two or more action potentials fire in succession, the force of contraction increases.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

A second mechanism that produces graded contraction of a whole muscle derives from organization of muscle cells into motor units (Figure 1.4). In a vertebrate muscle, each muscle cell is innervated by only one motor neuron; however, motor neurons can branch, making synaptic contacts with many muscle cells. In some instances, there may be hundreds of motor neurons controlling an individual muscle, each with its own pool of muscle fibers scattered throughout the muscle. When a motor neuron fires, all the muscle cells to which it connects (the motor unit) contract as a group. The strength of the resulting contraction will therefore depend on how many muscle fibers the motor neuron controls, a number that varies widely.

Figure 1.4 Motor units in skeletal muscle. Each muscle fiber (cell) has a single neuromuscular junction, or synaptic connection, with a motor neuron. A given motor neuron, however, typically branches and controls several – even many – muscle fibers. A motor neuron and all the fibers it controls constitute a motor unit.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

Thus, the nervous system can regulate the strength of skeletal muscle contraction by both regulating how many motor units are activated at a given instant and selecting whether large or small motor units are activated. Tension in a muscle can be progressively increased by activating more and more motor neurons that control the muscle, a process termed recruitment.

Finally, a third mechanism whereby the nervous system governs the graded activation of skeletal muscle relates to alternating activation of different motor units. For example, muscles that control posture must remain partially contracted for extended periods of time, which would lead to fatigue of any single muscle fiber. To obviate that, motor units making up the muscle are activated alternately so that they take turns maintaining the prolonged contraction.

Smooth Muscle

Smooth muscle lacks the striations of skeletal and cardiac muscle because the actin and myosin filaments are not regularly arrayed along the length of the cell. Instead, the arrangement of filaments is less regimented and occurs as a spiral. Smooth muscle cells are spindle-shaped, and they contain less myosin than striated muscle. Further, the myosin is not associated with specific actin strands. Smooth muscle has neither a transverse (T)-tubule system, plasma membrane infoldings characteristic of striated muscle, nor a well-developed sarcoplasmic reticulum. Calcium ions must enter the cytoplasm via the plasma membrane during an action potential, and the amount actually reaching the filaments is relatively small.

Smooth muscle contractions are relatively slow with an extensive range of control, exceeding that of striated muscle. Also, smooth muscle can contract over a much greater range of lengths than striated muscle. Whereas they contract more slowly than skeletal muscle, smooth myocytes can remain contracted for longer periods of time.

Smooth muscle is found mainly in the walls of hollow organs such has blood vessels, digestive tract organs, urinary bladder, and uterus. Controlled by different kinds of nerves than those controlling skeletal muscles, smooth muscles are responsible for involuntary body activities, such as churning of the stomach or constriction of arteries.

Common Molecular Mechanisms

As noted, striated muscle is so termed because the highly regular arrangement of the myofilaments creates a repeated pattern of light and dark bands (Figure 1.5). Each repeated unit is a sarcomere, the fundamental contractile unit. The borders of the sarcomere, the so-called Z-lines, are lined up in adjacent myofibrils to contribute to the striations visible under the light microscope. The thin filaments are attached to the Z-lines and project toward the center of the sarcomere, while the thick filaments are centered in the sarcomere. At rest, the thick and thin filaments do not overlap completely in the area near the edge of the sarcomere; rather, only thin filaments are found in this so-called I-band. The A-band is the region that corresponds to the length of the thick filaments.

Figure 1.5 Sliding-filament model of muscle contraction. As contraction occurs, the lengths of the thick (myosin) filaments (black) and the thin (actin) filaments (red) remain the same as contraction occurs (transmission electron micrographic image above, schematic below). In relaxed muscle, the length of each sarcomere is greater than in a contracted muscle. When the muscle is fully contracted, the sarcomere is markedly shortened, the thin filaments overlap, and there is little space between the ends of the thick filaments and the Z-lines.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

Thousands of actin filaments are arranged in parallel to one another, interdigitated with thicker filaments made of myosin. Myosin acts as a motor molecule by means of projections (arms) that walk along the actin filaments. In this so-called sliding-filament model, contraction of the muscle cell results from the actin and myosin filaments sliding past one another, shortening the sarcomere and ultimately the entire cell. In other words, when muscle contracts, the length of each sarcomere is reduced; that is, the distance between Z-lines diminishes. Neither the thin filaments nor the thick filaments change in length; rather, the filaments slide past each other longitudinally, such that the degree of overlap of thin and thick filaments increases.

When muscle is at rest, the myosin-binding sites on the actin molecules are blocked by the regulatory protein tropomyosin (Figure 1.6). This, in turn, is controlled by another set of regulatory proteins, the troponin complex, which controls the positioning of tropomyosin on the thin filament. For a muscle cell to contract, the myosin-binding sites on actin must be exposed by a displacement of the troponin and tropomyosin elements. This critical step occurs when calcium ions bind to troponin, altering its interaction with tropomyosin and uncovering the myosin-binding sites on actin. In the presence of calcium, the sliding of thin and thick filaments can occur, and muscle contraction proceeds. When calcium concentrations in the cytosol fall, the binding sites on actin are covered, and contraction stops.

Figure 1.6 Molecular control of muscle contraction. The thin filament consists of two strands of actin twisted into a helix. (a) When a muscle is at rest, the long, rod-like tropomyosin molecule blocks the myosin-binding sites required for formation of cross-bridges. (b) When another protein complex, troponin, binds calcium ions, conformational changes lead to uncovering of the binding sites on actin. As a result, cross-bridges with myosin form, and the muscle contracts.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

The molecular architecture of myosin is notable for a long tail region and a globular head region protruding off to the side. The tail domain is where the individual myosin molecules align to form the thick filaments. The myosin head is the site of the bioenergetic reactions that power muscle contractions. The myosin head binds ATP and hydrolyzes it into ADP, inorganic phosphate, and free energy. Some of the energy released by ATP hydrolysis is transferred to the myosin head itself, which undergoes an allosteric change in conformation to a high-energy configuration. This energized myosin binds to a specific site on actin, forming a cross-bridge. The stored energy is then released, and the myosin head relaxes to a low-energy configuration. This relaxation event alters the angle of attachment between the myosin head and tail. Thus, as myosin turns inward on itself, it exerts tension on the thin filament to which it is bound, pulling the thin filament toward the center of the sarcomere. Finally, the bond between actin and myosin in the low-energy state is broken when a new molecule of ATP binds to the myosin head. In a repeating cycle, the free head cleaves the new ATP to revert to the high-energy configuration and attach to a new binding site on another actin molecule site farther along the thin filament. Each of the approximately 350 heads of a thick filament forms and reforms about five cross-bridges per second.

Contracting muscle requires a great deal of energy. However, a muscle cell typically stores only enough ATP for a few contractions. Muscle cells also store glycogen between the myofibrils. However, most of the energy needed for repetitive muscle contractions is stored in substances called phosphagens. Creatine phosphate, the phosphagen of vertebrates, can restore ATP levels by providing a phosphate group to ADP to generate ATP.

Calcium concentrations in the myocyte cytoplasm are regulated by the sarcoplasmic reticulum, a specialized endoplasmic reticulum, and by transporters that pump calcium out of the cell (Figure 1.7). The membrane of the sarcoplasmic reticulum actively sequesters high concentrations of calcium within its interior. Calcium-selective molecular pumps fill the sarcoplasmic reticulum, and specialized ion channels release the calcium back out. In the end, the sarcoplasmic reticulum is a major site of intracellular calcium homeostasis and storage, available for triggered release to launch a contraction.

Figure 1.7 Cellular architecture of the myofibril, sarcoplasmic reticulum, and neuromuscular junction. Action potentials, triggered by release of acetylcholine from the motor neuron at the neuromuscular junction, sweep across a muscle fiber and deep into the T-tubules. Calcium enters the cytoplasm from the extracellular space. In addition, Ca ²+ is released into the cytoplasm from the sarcoplasmic reticulum. The increased Ca ²+ levels in the cytoplasm elicit contraction by triggering the binding of myosin to actin.

Campbell, Neil A.; Reece, Jane B., Biology, 6th Edition, © 2002. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.

The activity of all muscle is governed, at least in part, by the nervous system. In the case of cardiac muscle, sympathetic and parasympathetic nerves impinge on the tissue at multiple sites, eliciting events which are largely antagonistic. Sympathetic drive accelerates heart rate (chronotropy), facilitates relaxation (lusitropy), enhances contractility (inotropy), and accelerates the spread of electrical depolarization. Parasympathetic drive, mediated by the vagus nerve, antagonizes these actions.

In the case of skeletal muscle, a motor neuron that makes a synaptic connection with the muscle cell triggers a myocyte action potential. The synaptic terminal of the motor neuron releases a neurotransmitter at the neuromuscular junction which depolarizes the post-synaptic zone of the muscle cell, triggering an action potential. That action potential, the signal for contraction, spreads deep into the interior of the myocyte along T-tubules. The spreading membrane depolarization transmits a signal to the sarcoplasmic reticulum, provoking release of calcium from that intracellular store. The signal from plasma membrane to sarcoplasmic reticulum differs in key ways among muscles, being largely dependent on calcium influx in the case of heart and on depolarization-induced changes in protein conformation in the case of skeletal muscle. Either way, calcium in the cytoplasm – deriving from both extracellular and intracellular stores – binds troponin, exposing the myosin head to actin, and allowing the muscle to contract. Muscle contraction stops when the sarcoplasmic reticulum resequesters the calcium, lowering cytoplasmic concentrations, and releasing the tropomyosin–troponin complex to once again block the myosin-binding sites on actin.

Summary

A vast array of muscle types exists in nature, contributing to a multitude of physiological processes. Muscle is characterized by highly specialized structure and function in order to accomplish both powerful and delicate tasks. At the same time, many molecular and cellular mechanisms are shared in common across all muscle types. Muscle, under the governance of a sophisticated nervous system, contributes to some of the grandest accomplishments of humankind – from athletics to music and dance – and yet it is the site of horrific diseases with profound individual and societal consequences. Whereas much is known about the fascinating biology of muscle, a great deal remains unknown. It is our sincere hope that this book will engender in our readers enthusiasm and fascination for the astonishing biology of muscle.

Chapter 2

A History of Muscle

Arnold M. Katz

University of Connecticut School of Medicine, Farmington, CT; Dartmouth Medical School, Hanover, NH; Harvard Medical School, Boston, MA

Movement, in its various forms, is considered one of the most typical vital phenomena. Existing at all levels of evolutionary development, it reaches its highest specialization and perfection in the striated muscle of arthropods and vertebrates.

W.F.H.M. Mommaerts, 1950 (1)

Introduction

Motility is essential for life. Movements of animals are obvious, and even plants with fixed roots can have motile gametes. Single-celled organisms move to search for food and escape predators, and many cellular functions require energy-dependent transport of intracellular organelles. Biological motility can occur when kinesins or dyneins travel along tubulin filaments, and myosin moves along actin filaments. All participate in motile functions that include cytokinesis, endocytic trafficking, chemotaxis, and cytoplasmic streaming. Beating of cilia and flagellae depends on microtubule-based kinesins and dyneins, while myosin–actin interactions are responsible for muscular contraction.

Advances in our understanding of muscle have paralleled the evolution of science (Table 2.1). Reviewing this fascinating history not only tells us how science has progressed, but can also help predict how future knowledge might emerge. As noted by Winston Churchill: The longer you look back, the farther you can look forward. This is not a philosophical or political argument – any occulist can tell you it is true (2).

Table 2.1. Major Areas in the Study of Muscle

Early Observations

Animal movement was obvious to Stone Age humans, who viewed muscle as food (Figure 2.1). Differences in the flavors of muscles from different animals and different parts of the same animal must also have been apparent to our early ancestors: for example, that the breast muscles of birds that fly long distances, like geese and ducks, are darker in color and have a richer taste than the whiter muscles of birds like chickens that flap their wings during brief bursts of activity. The ability of cooking to modify muscle flavor can be viewed as the first discovery in the history of muscle, but it was only after the advent of modern chemistry that these observations could be explained in terms of heat-induced reactions between lipids, aldehydes, alcohols, ketones, and heterocyclic phenolic and sulfur-containing molecules (3).

Figure 2.1 Portion of a hunting scene painted in the Lascaux Caves approximately 15,000 years ago.

Animal Spirits and the Vital Force

The emergence of natural science from mythology can be traced to the Ionian Greek philosophers Thales, Anaximander, and Anaximenes, who in the 6th century BCE sought to explain natural phenomena in terms of interactions between natural forces, rather than battles among gods (4). The central role of conflict, which was to dominate Western science for almost 2,000 years, led health to be viewed as a balance between four humors. Within this paradigm, muscular contraction was thought to be initiated when the animal spirit (cold humor) passes from the brain to the muscles through channels in nerves. As late as 1671, Thomas Willis (5) concluded that contraction occurs when:

animal spirits being brought from the head by the passage of the nerves to every muscle … which as they are naturally nimble and elastick … are permitted, expanding themselves, [to] leap into the fleshy fibres [of the muscle]. (6)

William Harvey’s discovery of the circulation in 1628, along with van Leeuwenhoek’s description of innumerable small vesicles and globules within muscles, which had been circulated among members of the Royal Society in London, led Croone to suggest in 1664 (7) that nutrients which reach the muscles via arteries and nerves increase the volume of tiny bladders:

from the artery of each particular muscle the nourishing juice of the muscle was thrown out and extravasated to run at large among the carious fibres, and insinuating itself by the constant pulse of the heart was driven on [to meet] the fibres in the muscles … And likewise, that from each ramification of the nerve within the muscle, that second sort of matter much more fluid and active than the former is extravasated, and these mixed together … enter into each little bladder, and by their constant agitation, ebullition, or effervescence … makes that which we call the very life of each part … (6)

Croone’s postulate that contraction and the increased stiffness of contracted muscle are caused by increased volume of little bladders provides an early example of an explanation based on a physical mechanism (see below).

Electrical Activation

One of the major discoveries in biology, that electrical stimulation causes muscles to contract, was made in 1756 when Caldani found that the discharge from a Leyden jar could excite an isolated nerve-muscle preparation (8). Thirty-five years later, Galvani demonstrated that electrical currents generated by friction machines and batteries made of dissimilar metals also stimulate nerve-muscle preparations (9). He also discovered that muscles themselves generate animal electricity when, after placing the nerve of one nerve-muscle preparation over a second muscle, he observed that stimulating the latter caused the first muscle to contract (10). In a spectacular series of demonstrations, Galvani’s nephew Aldini caused convulsive contractions by applying electrical arcs at different points on recently deceased human cadavers, some of which had been beheaded (11) (Figure 2.2). These experiments, which were discussed widely throughout Europe, led Mary Shelley to describe the use of electricity to bring the monster to life in her Frankenstein, which was first published in 1818.

Figure 2.2 Depictions of the ability of electric arcs applied at different points on the head and body of cadavers to cause muscular contractions.

(From Aldini, 1804 (11).)

Energy Utilization: Work and Heat

The view that non-quantifiable vital forces control biological function – and life itself – dominated physiology until the beginning of the 19th century, when Claude Bernard, Herrmann Helmholtz, and others showed that biological responses can be explained in terms of quantifiable physical and chemical laws. According to Bernard:

It matters little whether or not we admit that [the force which governs life] differs essentially from the forces presiding over the phenomena of inorganic bodies, the vital phenomena which it governs must still be determinable; for the force would otherwise be blind and lawless, and that is impossible …

Biology must borrow the experimental method of physico-chemical sciences, but keep its special phenomena and its own laws [which are] immutable, and the phenomena governed by these laws are bound to the conditions on which they exist, by a necessary and absolute determinism. (12)

Helmholtz (13), whose participation in the emerging science of thermodynamics was to shape muscle physiology for more than a century (14), wrote:

the phenomena of nature are to be referred back to the motions of material particles possessing unchangeable moving force … Motion, as a matter of experience, can only appear as a change in the relative positions of at least two material bodies. Force, which originates motion … is therefore to be defined as the endeavour of two masses to alter their relative positions. Finally … we discover the problem of physical natural science to be to refer natural phenomena back to unchangeable attractive and repulsive forces … (6)

Applications of thermodynamics to active muscle had its roots in the discovery of the equivalence of work and heat at the end of the 18th century, when Benjamin Thompson found that heat liberation was proportional to the work expended during the boring of cannons. Recognition of the First Law of Thermodynamics, that the sum of the energies in an isolated system is constant, stimulated efforts to quantify total energy expenditure by muscle. Although muscular work had been measured since the 17th century, it was not until 1848 that Helmholtz clearly demonstrated that working muscles also generate heat (15).

In the early 20th century, muscles were thought to shorten and generate tension by assuming stiffer spring-like properties. According to this New Elastic Body theory, the transition from rest to activity occurs when chemical energy provided to the muscle during activation forms new elastic bonds within the contractile machinery (Figure 2.3a). Because this theory predicts that a fixed amount of energy is provided during activation to establish the new bonds, the total energy released as work plus heat by an active muscle should be independent of load (Figure 2.3b). In 1923, however, Wallace Fenn, working with A.V. Hill, showed that total energy release increases when more work is performed (Figure 2.3c) (16). This finding, called the Fenn effect, demolished the new elastic body theory by demonstrating that the total energy liberated by a contracting muscle is not constant, but instead depends on load. It is a historical curiosity that the Fenn effect had been documented in cardiac muscle almost a decade earlier, when Evans and Matsuoka found that cardiac oxygen consumption increases when the heart does more work (17).

Figure 2.3 Load dependence of energy released during muscle contraction. (a) The New Elastic Body Theory, which until 1923 was generally believed to explain muscular contraction, postulated that a fixed amount of energy is delivered to a relaxed muscle during activation. This energy is then used to create new, stiffer, spring-like properties within the contractile machinery. (b) Load dependence of the energy released by a contracting muscle as work (blue) and heat (green) as predicted by the New Elastic Body Theory. Because a constant amount of energy is assumed to be added during activation, to maintain total energy release constant heat production should decrease at intermediate loads, where work is maximal. (c) Actual load dependence of energy release as work and heat observed by Fenn (16) . The increased total energy released by a muscle contracting at intermediate loads, where more work is done, is the Fenn effect.

(Modified from Katz (67), 2011.)

In seeking to understand how increasing work increases total energy liberation by a muscle, A.V. Hill developed a series of equations that were to dominate thinking about muscle contraction until the end of the 20th century (18). Hill found that the rate of extra energy liberation as work plus heat decreases with increasing load according to the equation:

(Eq. 2.1)

where P is the load on a muscle, P0 is the maximal load the muscle can lift, v is the velocity of muscle shortening at load P, a is the amount of heat liberated per cm of shortening, and b is a constant. Eq. 2.1 can be rearranged so that P and v, the two variables, are on the left, and a, b, and P0 – all of which are constants for a given muscle – are on the right:

(Eq. 2.2)

The significance of Eq. 2.2, often called the Hill Equation, is that because it plots the product of P times v versus a constant, graphs of the dependence of shortening velocity (v) on developed force (P) describe a hyperbola (x times y=constant) (Figure 2.4a). Hill found that the curves based on his measurements of heat and work were the same as the hyperbolic force–velocity relationships measured directly by Fenn and Marsh more than a decade earlier (19). Because an ordinary stretched spring follows Hooke’s Law, where shortening velocity increases in a linear manner as load decreases (Figure 2.4a), the hyperbolic relationship between load (force) and velocity indicates that a contracting muscle is much more complex than a new elastic body.

Figure 2.4 Dependence of shortening velocity and the rate of extra energy liberation on load (force). (a) Force velocity curves showing the linear relationship expected if the contractile machinery has spring-like characteristics that obey Hooke’s Law, and the hyperbolic curve measured by Fenn and Marsh (19) and predicted by the Hill equations (18) . (b) Dependence of the rate of extra energy liberation as work plus heat on load (force) measured by Hill (18) .

(Modified from Katz (67), 2011.)

Hill also found that the rate of total energy liberation as work plus heat is a linear function of the load on a muscle (Figure 2.4b). The significance of this linear relationship was eloquently stated by Hill:

The control exercised by the tension P existing in the muscle at any moment, on the rate of its energy expenditure at that moment, may be due to some such mechanisms as the following. Imagine that the chemical transformations associated with the state of activity in muscle occur by combination at, or by the catalytic effect of, or perhaps by passage through, certain active points in the molecular machinery, the number of which is determined by the tension existing in the muscle at the moment. We can imagine that when the force in the muscle is high the affinities of more of these points are being satisfied by the attractions they exert on one another, and that fewer of them are available to take part in chemical transformation. When the tension is low the affinities of less of these points are being satisfied by mutual attraction, and more of them are exposed to chemical reaction. The rate at which chemical transformation would occur, and, therefore, at which energy would be liberated, would be directly proportional to the number of exposed affinities or catalytic groups, and so would be a linear function of the force exerted by the muscle, increasing as the force diminished. (18)

Hill’s statement, written when virtually nothing was known of contractile protein interactions and a year before myosin was discovered to be an ATPase enzyme (see below), explains muscle energetics in terms of interactions between hypothetical active points within the muscle that can exist in either of two states. In one, the active points are attached and maintain tension; in the other, the active points are free to liberate chemical energy and so cycle at their maximal velocity. Today we recognize these active points as interactions between myosin cross-bridges and actin (see below).

The Hill equations were, for many years, a touchstone for theories of muscle contraction. For example, adherence to predictions from these equations provided support for a model of the contractile process that was based on hypothetical macromolecular phase transitions (20). However, these and other efforts to explain the contractile process in terms of changes in the folding of macromolecules ended with the sliding-filament hypothesis (see below).

Energy Production: Metabolism

The first clue as to how muscles generate energy emerged in the late 18th century when Lavoisier, Priestly, and others observed that oxygen is essential for animal life. Berzelius, in the early 19th century, found that lactic acid, which had been found in sour milk, appeared in the muscles of a stag that had been exhausted during a long hunt (21). In 1907, Fletcher and Hopkins (22) discovered that lactic acid generated during activity disappears when a muscle is allowed to recover in the presence of oxygen. Myerhof’s subsequent finding that lactic acid production is proportional to the work done when a muscle contracts in the absence of oxygen, where lactate cannot be oxidized (23), suggested that energy released by anaerobic glycolysis is directly coupled to muscle contraction:

The hypothesis that muscles use energy derived directly from glycolysis to perform work, although it explained a large body of experimental data, collapsed in 1930 when Lundsgaard discovered that muscles still contract after glycolysis is blocked with iodoacetic acid (24). The subsequent observation that working muscles hydrolyze phosphocreatine, a labile compound composed of creatine and phosphoric acid, and the demonstration by Eggleton and Eggleton that the amount of work performed during muscle contraction is proportional to a decrease in phosphocreatine content (25), led to a new hypothesis, that the breakdown of phosphocreatine to creatine and inorganic phosphate (Pi) provides energy directly to contracting muscles:

According to the new hypothesis, glycolysis supplies energy to form phosphocreatine from creatine and Pi instead of delivering energy directly to the contractile machinery.

After a few years, however, the new hypothesis also collapsed when adenosine triphosphate (ATP) was found to be essential for phosphocreatine breakdown during muscle contraction, and phosphocreatine was shown to form ATP when high-energy phosphate (~P) is transferred from phosphocreatine to ADP. The demonstration that ~P supplies chemical energy to a number of energy-consuming reactions led to yet another hypothesis, that the role of phosphocreatine is to form ATP by transferring ~P to ADP according to the reactions:

and

Although ATP hydrolysis was almost universally believed to be coupled directly to muscle contraction, it was not until 1962 that discovery of a specific creatine phosphokinase inhibitor that prevents ~P transfer from phosphocreatine to regenerate ATP from ADP made it possible to demonstrate that ATP concentration decreases during contraction (26).

The final chapter in this story was written when studies of the reactions between actin, myosin, and ATP elucidated the mechanism by which chemical energy released by ATP hydrolysis is converted to mechanical energy during muscular contraction.

Contractile Proteins

In 1864, Kühne (27) obtained a viscous protein that he called myosin when he extracted skeletal muscle minces with solutions containing high concentrations of neutral salts. An important clue regarding the energetics of contraction was provided in 1939 when Engelhardt and Ljubimova demonstrated that myosin is an ATPase enzyme (28).

All myosin preparations were initially believed to consist of a single protein until 1941, when Banga and Szent-Györgyi (29) found that myosin B, which was obtained after prolonged extraction of the muscle mince, had different properties than myosin A, which was obtained by short extraction. The following year, Straub found that myosin B contained an additional protein that he called actin because it activates myosin ATPase activity (30–31). A key discovery was made in 1943, when Albert Szent-Györgyi demonstrated that ATP can cause threads made of actomyosin (the new name for myosin A) to shorten (32) (Figure 2.5). Commenting on this observation, Mommaerts (1) cited Oliver Heaviside’s statement: The best of all proofs is to set out the fact descriptively so that it can be seen to be a fact.

Figure 2.5 Fibers made by extruding actomyosin solubilized at high ionic strength through a fine needle into a solution where salt concentration is low. Addition of ATP has caused the lower fiber to contract.

(Modified from a photograph given to the author by W.F.H.M. Mommaerts in 1962.)

In 1962 Drabikowski and Gergely (33) demonstrated that actin preparations often contain tropomyosin, a third protein that had been discovered by Bailey 15 years earlier (34), and Katz (35) showed in 1964 that tropomyosin regulates the interactions between actin and myosin. In 1963, after several groups had demonstrated that calcium is able to activate some, but not all actomyosin preparations in vitro, Weber and Winicur (36) reported that the calcium-sensitivity of actomyosins reconstituted from purified myosin and actin depended on the actin preparations, and Ebashi (37) found that a protein that resembled tropomyosin could confer calcium-sensitivity to actomyosins. In 1965, Ebashi and Kodama identified a protein factor that modifies the aggregation of tropomyosin (38) and, the following year, found that this factor, which they had named troponin, sensitizes reconstituted actomyosins to Ca²+ (39); at the same time Katz (40) isolated a similar Ca²+-sensitizing protein complex from actin. The final chapter of this story was added in 1972, when troponin was found to include three distinct proteins (41).

The Sliding-Filament Hypothesis

Until the middle of the 20th century, contraction had been attributed to conformational changes in macromolecules within muscle fibers (see above). However, in 1951 Hugh Huxley provided X-ray diffraction data which suggested that stretching a muscle causes elongated structures to pull away from one another, rather than changing their internal molecular conformations (42), and two years later Huxley published an electronmicroscopic study which showed that sarcomeres are made up of arrays of elongated filaments whose organization accounts for the striations in skeletal muscle (43). Evidence that muscles contract when thin actin filaments slide by thick myosin filaments, both of whose lengths remain constant, was published in 1954 by Hugh Huxley and Hanson (44), and by A.F. Huxley and Niedergerke (45). However, several years were to pass before the sliding-filament hypothesis was universally accepted as part of the foundation of our understanding of muscle contraction (46).

A link between muscle energetics and contractile protein interactions was suggested in 1957, when A.F. Huxley postulated that shortening velocity is determined by the rate of myosin cross-bridge movement along the thin filaments, and developed force by the number of active interactions between the cross-bridges and actin (47). Ten years later, when Bárány (48) demonstrated a direct relationship between muscle shortening velocity and myosin ATPase activity, it became clear that Hill’s active points (see above) are interactions between myosin cross-bridges and actin.

Calcium, Excitation–Contraction Coupling, Relaxation

The first clue that muscle contraction is activated by calcium was published in 1883, when Sydney Ringer observed that hearts cease to beat when placed in a calcium-free solution, and that calcium salts restore their ability to contract (49). In the 1930s and early 1940s, several investigators obtained evidence that direct application of calcium to muscle fibers could cause contraction (50–53). However, a problem arose in 1949, when Hill recognized that diffusion of an activator from the cell surface is too slow to initiate contraction in frog sartorius muscle:

It is quite … impossible to explain the rapid development of full activity in a [skeletal muscle] twitch by assuming that it is set up by the arrival at any point of some substance diffusing from the surface: diffusion is far too slow. Either we must suppose that [the muscle is stimulated by] excitation (natural or artificial) throughout the interior, not merely at the surface: or we must look for some physical or physico-chemical process which is released by excitation at the surface and then propagated inwards. (54)

Hill’s concerns were resolved when it was realized that calcium derived from an internal membrane system, the sarcoplasmic reticulum, mediates excitation–contraction coupling, and that relaxation occurs when calcium is sequestered within these membranes. The initial observation, made in the early 1950s, was that addition of the supernatants obtained after muscle minces were fractionated by the highest speed centrifugation then available causes actomyosin preparations to relax in vitro (55–56). This effect, which requires the presence of ATP and can be abolished by calcium, was initially believed to be caused by a soluble relaxing factor that is inactivated by calcium (57). In the early 1960s, however, the relaxing effect was recognized not to be caused by a soluble substance, but instead occurs when tiny membrane vesicles, called microsomes, pump calcium into their interior after they are energized by ATP. This made it clear that what had appeared to be soluble fractions of muscle homogenates actually contained sealed membrane vesicles derived from the sarcoplasmic reticulum, and that relaxation occurs when an ATP-dependent calcium-pump in these membranes removes calcium from the cytosol (58–60).

The mechanism by which activation at the cell surface signals contraction deep within myocytes came into focus when Hugh Huxley demonstrated that transverse tubular structures, called T-tubules, are plasma membrane extensions whose lumens open into the extracellular space (61). Evidence that action potentials are propagated down the T-tubules was provided by A.F. Huxley and Taylor (62), who found that small electrical currents applied through a microelectrode placed near the mouth of a T-tubule induce contractions that are limited to the sarcomeres adjacent to the point of stimulation. Further evidence that T-tubules propagate action potentials into the interior of the muscle cells was obtained when disruption of the connections between the T-tubules and the plasma membrane was found to make it impossible for electrical stimuli to activate contraction (63). These and other findings completed the answer to Hill’s question by demonstrating that transmission of a wave of depolarization down the T-tubules into the cell interior, which is more rapid than diffusion of an activator substance, plays a critical role in excitation–contraction coupling.

Skeletal, Cardiac, and Smooth Muscle

The heart has been known to be a muscle for at least 2,500 years. The Heart, a book of the Hippocratic corpus that is probably of Hellenistic origin, states: The heart is a very strong muscle … because of the firmness of its flesh (64). The heart’s function as a reciprocal pump, in which phases of filling alternate with phases of ejection, was understood by Erasistratus, an Alexandrian physiologist active in the 3rd century BCE, and by Galen, who was a leading physician in the Roman empire in the 2nd century CE. The latter recognized that fiber orientation differs in cardiac and skeletal muscle, noting that unlike skeletal muscles which have their fibres going only in one direction … the heart has both length-wise and cross fibres, as well as a third kind running diagonally, inclined at an angle (65). The distinction between smooth and striated muscle was made much later, in 1672, when van Leeuwenhoek described cross-striations (21).

Most of the seminal work on muscle took advantage of availability and relative simplicity of fast white skeletal muscle. It is not an accident that the pioneering studies of muscle energetics used the frog sartorius, which has parallel fibers, an active state that can be stabilized in tetanic contractions, and few intrinsic regulatory mechanisms. Similarly, most landmark studies of muscle biochemistry used preparations from rabbit white skeletal muscle, which has relatively few intrinsic regulatory mechanisms because the number of contracting motor units is controlled and integrated by the central nervous system. In 1970, I noted that the heart’s contractile proteins are harder to purify and more labile than the homologous proteins of skeletal muscle, and suggested that it would be wise to become familiar with the latter before attempting to characterize the more difficult cardiac systems (66). This is also true of the cardiac sarcoplasmic reticulum, which is more difficult to purify and less stable than the sarcoplasmic reticulum from rabbit white skeletal muscle. The value of characterizing simpler tissues before attempting to understand more complex systems is also apparent in electrophysiology, where studies of the relatively simple squid giant axon provided tools and guidelines that were invaluable for subsequent investigations of the more complex electrophysiology of heart muscle.

A number of homologies in key regulatory systems attest to the common evolutionary ancestry of different muscle types. These homologies also provide fascinating insights into how members of a single protein family can carry out similar functions, but in different ways (Figure 2.6). For example, contraction can be activated by members of the E-F hand family of calcium-binding proteins when calcium binds to troponin C in the thin filaments (Figure 2.6a), a myosin light chain in the thick filaments (Figure 2.6b), and calmodulin, a soluble calcium-binding protein that stimulates a kinase which phosphorylates a myosin light chain that is another E-F hand protein (Figure 2.6c). Another homology is the ability of different L-type calcium channel proteins to use different mechanisms to initiate calcium release from the sarcoplasmic reticulum. In skeletal muscles, calcium release occurs when plasma membrane depolarization removes a plug that occludes an intracellular calcium release channel (Figure 2.6d), whereas in the heart, membrane depolarization opens L-type calcium channels that admit a small amount of calcium that activates the intracellular calcium release channels (Figure 2.6e).

Figure 2.6 Homologous regulatory mechanisms in muscle. Top: Three mechanisms by which contraction is activated when calcium binds to an E-F hand protein (italic labels within boxes). (a) Troponin-linked regulation, seen in most skeletal and cardiac muscles, where the calcium-binding E-F hand protein is incorporated into the thin filament. (b) Myosin light chain-linked regulation, an uncommon mechanism seen in a few striated muscles, where contraction is activated when calcium binds to an E-F hand myosin light chain in the myosin cross-bridge. (c) Calmodulin-linked regulation, seen in many smooth muscles, where calcium binding to calmodulin, a soluble E-F hand protein, forms a complex that activates a protein kinase called myosin light chain kinase (MLCK). The latter then phosphorylates a myosin light chain, another E-F hand protein which, although it has lost its ability to bind calcium, still participates in calcium-mediated activation of contraction. Bottom: Two mechanisms by which an action potential propagated across the cell surface opens sarcoplasmic reticulum calcium release channels. (d) Mechanical coupling in skeletal muscle occurs when plasma membrane depolarization changes the conformation of a membrane protein, called a dihydropyridine receptor, that is related to the L-type calcium channels. By removing a plug that occludes the pore of the sarcoplasmic reticulum calcium release channel, this voltage-dependent conformational change activates contraction. (e) Calcium-triggered calcium release in cardiac muscle occurs when plasma membrane depolarization allows a small amount of calcium to enter the cytosol through an L-type calcium channel. This calcium then serves as a trigger that opens intracellular calcium release channels which activate contraction by releasing a much larger amount of calcium from within the sarcoplasmic reticulum.

(Modified from Katz (67), 2011.)

Conclusion

The history reviewed in this chapter represents the foundation for much of our current knowledge of the molecular entities that participate in and regulate muscle contraction and relaxation. In addition, the observations and interpretations described in the present chapter hold considerable promise in adding to our understanding of – and ability to treat – human disease.

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