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Graduate Physiology 503 Muscle Physiology Hurley

MUSCLE PHYSIOLOGY

Part 1: Ultrastructure and cross-bridge cycling Learning Objectives:

 Describe a sarcomere, and the characteristics of the major contractile proteins associated with this structure.

 Describe the sliding filament theory and its relationship to force generation.  Describe the cross‐bridge cycle for muscle contraction and the role of ATP, ADP,

and Pi in the cycle.  Describe how calcium initiates contraction of striated muscle.

A. OVERVIEW

The essential feature of muscle is the ability to convert energy derived from the hydrolysis of ATP into mechanical work. Mechanical work is expressed as a contractile event in which the muscle develops force and, if not constrained, shortens. Each muscle fiber in a whole muscle performs its function in a coordinated way that is controlled by the nervous system, so that purposeful motor activity can be produced. Nevertheless, muscle cells possess important intrinsic properties or mechanisms that also serve to regulate function. These will be discussed in the following lectures, starting with the functional organization of muscle, and tracing events that occur from the moment the muscle is activated to relaxation and the subsequent metabolic recovery processes that replenish the ATP consumed during contraction.

Three Muscle Types: Skeletal, Cardiac, and Smooth Muscle makes up approximately 50% of the body weight of a healthy individual. A variety of bodily functions involve muscle contraction: locomotion, blood circulation, and gastrointestinal motility. Muscle represents a major consumer of metabolic energy, ranging from approximately 30% of basal metabolism to upwards of 90% during vigorous exercise and is the major source of heat to maintain body temperature. Muscle can be classified in several different ways. Perhaps the most general one is in terms of structure, i.e. striated vs. non‐striated or smooth muscle. When examined via light microscopy, both skeletal and cardiac muscle appear striated,

Graduate Physiology 503 Muscle Physiology Hurley

while smooth muscle is non‐striated. Most skeletal muscle is under voluntary control while cardiac and smooth muscle are principally under involuntary control. From a structural and biochemical standpoint, all striated muscle is similar. Most of our knowledge and therefore most of what will be described in these lectures comes from studies of skeletal muscle. Cardiac muscle is similar to skeletal muscle in many regards. Their differences will also be highlighted at various times in these lectures. Smooth muscle has a number of unique properties and will be discussed in a separate lecture.

B. FUNCTIONAL ORGANIZATION OF SKELETAL MUSCLE: THE

CONTRACTILE MACHINERY

Whole Muscle Substructure Sarcomere: the smallest mechanical functional unit of muscle, extends from Z‐line to Z‐line (~2.3 μm, see Figure 1 and 2). A‐band ‐ under the light microscope appears dark or Anisotropic; region of thick filament (~1.6 μm). I‐band ‐ appears clear or Isotropic, region on either side of A‐band; thin filaments are in the I‐ band and extend into the A‐band where thick and thin filaments overlap.

M‐line ‐ center of A‐band and the H‐zone and marks center of sarcomere. Lateral organizer for thick filaments. Z‐line ‐ defines end of

sarcomere. H‐zone ‐ area in the center of sarcomere between opposing thin filaments. Titin molecules ‐ run from Z‐line to M‐line, connects Z‐lines to thick filaments; provides lateral stability, major source of parallel elasticity and passive force of cell. Nebulin molecules – each thin filament in skeletal muscle has one nebulin

Figure 1

Graduate Physiology 503 Muscle Physiology Hurley

molecule believed to set thin filament length at 1.05 μm. Also believed to be important in anchoring of capping proteins. Tropomodulin – actin capping protein on end of thin filament, protects actin filaments from depolymerization.

CapZ – actin capping protein on other end of thin filament, protects actin filaments from depolymerizing and is probably involved in anchoring thin filament to Z‐line proteins such as alpha‐actinin. Thick filaments ‐ 1.6 μm long, composed primarily of myosin; one thick filament contains approximately 320 myosin molecules.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 2

Graduate Physiology 503 Muscle Physiology Hurley

Figure 3

Myosin Molecule (Figure 3 and 4) The myosin superfamily comprises at least 18 different classes of myosins across plant and animal kingdoms with tremendous diversity in function. Muscle myosin II is the best studied of all the myosins. Muscle myosin II molecules are non- processive molecular motors; i.e. they are released from actin after each catalytic cycle (see cross-bridge cycling below). Muscle myosin is a hexamer consisting of two so-called heavy chains and two pairs of light chains. The COOH-terminal ends of the heavy chains form a coiled coil of two α-helices that aggregate in the cell to create the backbone of the thick filaments. The remainder of the myosin molecule projects outward from the thick filament, forming the cross-bridge portion of the molecule to which the essential and regulatory light chains are noncovalently bound. Mild proteolytic treatment of thick filaments results in the generation of the light (LMM) and heavy meromyosin (HMM) fragments, and further proteolysis of the HMM cross-bridge fragment gives rise to the subfragments S1 and S2. The myosin S1, also referred to as the “head” of the cross-bridge, retains all of the motor functions of the molecule, i.e., the ability to produce movement and force. The globular NH2-terminal “catalytic domain” contains both the actin-binding site and the active site for ATP hydrolysis. The long α-helical “neck domain” extends toward the tail of the mole-cule and appears to be stabilized by its interaction with the two light chains. Finally, between the cata-lytic and neck domains is the “converter domain” (Figure 4). A conformational change within S1 while tightly bound to actin appears to provide the “power stroke” that underlies force production and movement.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 4

Figure 5

Thin Filament (Figure 5)

1.05 μm long Composed primarily of actin One thin filament contains approximately 360 42kDa globular F‐actin molecules Thin filament is a double stranded helix of actin monomers Thin filaments insert into Z‐lines Thin filaments contain the regulatory proteins tropomyosin and troponin Tropomyosin is an elongated protein that winds along the actin helix. Each tropomyosin extends over seven actin monomers.

Graduate Physiology 503 Muscle Physiology Hurley

Troponin is a globular protein bound to tropomyosin and actin. It is composed of 3 subunits called troponin C (TnC, binds calcium); troponin T (TnT, binds tropomyosin), and troponin I (TnI, binds to actin and prevents myosin/actin interactions.

C. MOLECULAR BASIS OF MUSCLE CONTRACTION

Sliding filament mechanism of contraction Critical Concept: All muscles, both striated and smooth, contract by means of the sliding and the interdigitating of the thick and thin filaments with NO change in the length of the filaments themselves. In striated muscle, this is readily seen as a decrease in sarcomere length; the Z‐lines come closer together, the I‐band narrows.

Figure 6. Detail of the interaction of thin and thick filaments. Three myosin heads are shown depicting progressively three stages, from left to right, of the power stroke itself. All stages shown occur after calcium binds to troponin C permitting myosin to bind following the release of inorganic phosphate (middle head) and activation of the myosin head.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 7

The sliding of filaments is due to the action of cross‐bridges (the S‐1 head) that link myosin with actin. Once activated, the action of the cross‐bridge is to PULL in the direction of the M‐line (the center of the sarcomere) and away from the Z‐line on itsʹ half of the sarcomere. Cross‐bridges attach to actin (see Figure 6 and 7), swivel to a new angle, thereby generating force and moving the thin filaments towards the center of the sarcomere (work or power stroke). The cross‐bridge then detaches, swivels back to its original, or relaxed orientation and can again bind to actin.

Graduate Physiology 503 Muscle Physiology Hurley

The attachment, movement and detachment of the cross‐bridges constitutes the CROSS‐ BRIDGE CYCLE. The energy for this process is derived from the hydrolysis of ATP on the globular head of the myosin molecule. Active cross‐bridges function asynchronously during muscle contraction, cross‐bridges are distributed among the various stages of their cycle allowing for a smooth and continuous contraction.

The working distance of the cross‐bridge is 10 nm. If all cross‐bridges went through the cycle once, the muscle would shorten by only 1% of its overall length. Therefore, repeated cycling of cross‐bridges is required to account for the amount of shortening a muscle can undergo. If the muscle is free to shorten, the cross‐bridges cycle and upon reattachment, bind to another actin site further along the thin filament as the filament slides and interdigitates farther. This occurs during an isotonic (same force) contraction. However, if the muscle is held so that it cannot shorten, the cross‐bridges cycle on and off the thin filament at or near the same actin site; the working stroke is curtailed, so there is minimal sliding of filaments, but force is produced. This occurs during an isometric (same length) contraction.

Figure 8

Cross‐bridge cycle – detailed description:

In normal resting muscle the crossbridges are in the myosin.ADP.Pi state due to the high cellular ATP concentration and the inhibition of actin‐myosin interactions by troponin. Therefore, the myosin crossbridges are NOT attached to actin and are arranged perpendicular to the filament backbone. In resting muscle the myosin

Graduate Physiology 503 Muscle Physiology Hurley

ATPase has a very low basal activity where ATP is rapidly hydrolyzed to ADP plus Pi (termed the products), followed by the very slow release of ADP and Pi, and immediate rebinding of ATP to the myosin ATP binding site. Therefore: M + ATP → M.ATP → M.ADP.Pi → M + ADP + Pi → M + ATP …. and so on.

Graduate Physiology 503 Muscle Physiology Hurley

If the muscle is stimulated and myosin is capable of binding actin (regulation to be discussed later), then following hydrolysis of ATP, myosin.ADP.Pi binds to actin, much faster release of products, myosin power stroke which “slides” the actin filament past the myosin filament, rebinding of ATP, and dissociation of myosin from the thin filament and the cycle continues.

D. THE MOLECULAR BASIS OF THE LENGTH‐TENSION RELATIONSHIP

(Figure 8)

Critical Concept: The length of a muscle prior to stimulation (pre‐load) is a fundamental determinant of the amount of force that it can develop upon stimulation, the active force. The amount of active force can be traced to the number of sites of mechanical interaction between the thick and thin filaments of the sarcomere, through the number of cross‐ bridges. This relationship holds for all muscle, both striated and smooth muscles. The sliding filament mechanism of contraction predicts that the degree of actin and myosin overlap will determine the maximal amount of active force that can be developed upon stimulation. In other words, because the cross‐bridges are the sites of interaction between actin and myosin filaments, one would predict that the contractile force should vary with the number of interacting cross‐bridges. The force should range from zero, at long sarcomere lengths at which no overlap of the filaments occurs, to maximal force at the sarcomere length at which maximal overlap of the filaments occur – that is where ALL of the crossbridges can bind to actin molecules. The study from which Figure 8 was derived was performed in isolated single skeletal muscle fibers. The fibers were activated at each of a series of sarcomere lengths and isometric force was measured. The maximum force produced was plotted as function of the sarcomere length. As predicted, the results show a dependence of tension on sarcomere length. There is an optimum length for force production: this was designated as Lo or Lmax.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 9

For simplicity of explanation, the length‐tension relationship is usually discussed from long to short sarcomere lengths; that is from right to left. However, the “limbs” of the curve are labeled by convention from left to right. Please do not be confused by the terms ascending and descending limbs as they describe the graph from left to right, NOT from longer to shorter sarcomere lengths. Length tension curve: Detailed description At long lengths with no overlap of filaments ‐ force equals zero. As the sarcomere is shortened, filament overlap increases, the number of crossbridge interactions increase, force increases linearly, this is termed the descending limb (remember terminology is left to right but we are discussing the graph right to left). Maximal force occurs where maximum actin‐myosin interactions occur ‐ this is defined as the length of optimal force or Lo. Due to the bare zone, where no myosin heads are present (refer to Figure 3), force remains constant as the sarcomere is shortened the length of this zone. As the sarcomere is shortened from the length of maximal active force to a point where the two thin filaments meet and in fact overlap (approximately 1.67 μm

Graduate Physiology 503 Muscle Physiology Hurley

sarcomeric length) the decrease in force is linear and the slope equal to that of the descending limb. The decrease in force is due solely to a decrease in number of actin and myosin interactions. As the sarcomere is shortened below about 1.67 μm, force decreases again but the fall in force is steeper than in the descending limb. This is due to the fact that the thin filaments are overlapping and produce a steric hindrance for crossbridge binding and then at approximately 1.5 μm, the myosin containing thick filaments abut the Z lines. At these stages of filament overlap, the interaction of cross‐bridges is impaired and as a consequence, active force production is reduced.

E. CONTROL OF ACTIN‐MYOSIN INTERACTION: REGULATORY

PROTEINS

In striated muscles, a combination of pure actin and myosin, in the presence of ATP, would result in the interaction of actin and myosin and an increased rate of ATP hydrolysis (actin‐activated myosin ATPase). In living muscle, the resting, non‐contracting state, is due to the inhibition of the actin‐ myosin interaction by the regulatory proteins troponin and tropomyosin, which form a complex on the thin filament. Once stimulated, control of actin‐myosin interaction

Graduate Physiology 503 Muscle Physiology Hurley

(release of inhibition) is achieved by the binding of calcium to one of the subunits of troponin.

Tropomyosin (Tm). This is a long, rod‐shaped molecule, lying in the groove formed by the helix of the actin filament. Each tropomyosin molecule extends over 7 actin monomers, forming a continuous flexible structure. Troponin (Tn). This is a globular molecule that ʺsitsʺ astride one end of the tropomyosin molecule. Troponin is comprised of three subunits: TnT: site of attachment of troponin to tropomyosin. TnI: inhibitory site of troponin, inhibits actin activation of myosin ATPase. TnC: Ca2+ binding site. The Troponin‐Tropomyosin Complex (Tn‐Tm). consists of 1 troponin and 1 tropomyosin molecule. This complex, then, exerts control over 7 actin monomers.

F. CALCIUM AND THE REGULATORY PROTEINS OF STRIATED MUSCLE.

In all muscle, the degree of interaction of actin and myosin is regulated by calcium, and in striated muscle this calcium regulation is expressed by the troponin‐tropomyosin complex (Figure 9).

Resting Muscle. Free Ca2+ concentration is about 10‐8 to 10‐7 M. There is not enough Ca2+ bound to TnC to unblock the interaction of actin and myosin. Activated Muscle. Free Ca2+ concentration increases 100‐fold. Ca2+ binds to TnC, initiating a series of conformational changes in the thin filament regulatory proteins that lead to a shift in the position of tropomyosin in the grooves formed by the actin helices, exposing sites on actin to which myosin can bind (ʺsteric hindranceʺ mechanism of thin‐filament regulation). Actin‐activated myosin ATPase activity increases, with the transduction of the chemical energy from ATP hydrolysis into mechanical output. Critical Concept: The regulatory proteins inhibit the interaction of actin and myosin in the absence of calcium. Calcium initiates contraction by reversing this inhibitory effect. Striated muscle is regulated by a disinhibition system. Myosin is always turned‐on but inhibited from interacting with actin. Removal of this inhibition (thus disinhibition) allows contraction to occur.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 10