Regulation of Skeletal Muscle Contraction

The main feature of muscle contraction is the interaction of actin, myosin and ATP. This fundamental process of contraction is regulated by the tropomyosin-troponin-Ca2+ system. According to the current theory, in the resting muscle TM is positioned in the groove of the actin double helix in a way that it sterically blocks the combination of myosin with actin. This is illustrated in Fig. RE1.

Fig. RE1. A scheme for the steric hindrance model. (From Vander, 1980).

In the absence of Ca2+, TM blocks the cross-bridge binding sites on actin. Binding of Ca2+ to TN moves the TM to one side exposing the binding sites and allowing cross bridges to bind to the thin filament. The binding of Ca2+ to TN-C is the starting point in the chain of events; this initiates the TM movement through TN-T. In addition, the TN-C-Ca2+ complex removes TN-I from its inhibitory position on actin; thus the combination of the myosin head with actin can proceed to full extent. Since in the thin filament there is only one TN and one TM molecule per seven G-actin molecules, one has to assume that cooperative interactions play a major role in the regulation of contraction.

Lehman and collaborators (1994) provided evidence for a TM based steric mechanism in Limulus thin filaments (Figs. RE2 and RE3). Further experiments with frog skeletal muscle confirmed the steric-model for activation of muscle thin filaments (Vibert et al., 1997). Recently, by using cryoelectron microscopy and helical image reconsruction the location of tropomyosin in troponin regulated thin filaments has been resolved under both relaxing and activating conditions (Xu et al., 1999).

Fig. RE2. Ca2+-induced tropomyosin movements in Limulus thin filaments revealed by three-dimensional reconstruction of electron micrographs. (From Lehman et al., 1994).


Fig. RE3. Density maps of the strands, shown in Fig. RE2.

Picture on Fig. RE2 shows surface views of reconstructed densities of thin filaments left in the presence of EGTA (a strong Ca2+ complexing agent, thus in the presence of EGTA there is practically no free Ca2+) right in the presence of Ca2+. The helically wound strands on the surface of actin have a diameter of about 20 Å (that is the width of TM). The inner (Ai) and outer (Ao) domains of one actin monomer are marked. The actin monomer shapes from both the EGTA- and Ca2+-treated filaments are very similar, but the elongated strands of density originating from TM are located in different positions on the actin filaments. Fig. RE3 shows helical projections formed by projecting the map densities of RE2 onto a plane perpendicular to the helix axis, a) thin filaments in EGTA, b) thin filaments in Ca2+. The TM strand, which makes contact with actin monomers at A0 and Ai in a) and b), respectively, is indicated by arrows. The difference map in c) was calculated by subtracting densities in the Ca2+ map from those in the EGTA map. The regions of the maps that are significantly different are indicated by white (positive differences) and by black (negative differences). The pairs of positive and negative peaks are located at the respective strand positions and demonstrate strand movement.

The role of Ca2+ in Regulation of Skeletal Muscle Contraction

Historical experiments: In 1883, Ringer observed that contraction of isolated frog heart ceases when CaCl2 was omitted from the bathing solution. This was reversible.

Fifty years later, Heilbrunn and Wiercinsky showed that injection of CaCl2 directly into skeletal muscle fibers causes contraction and no other cation duplicated this effect. They concluded that "Calcium might be an activator of muscle."

A.V. Hill, the founder of muscle physiology, predicted that the activator of muscle must come from an internal source, since membrane depolarization is quickly followed by mechanical response, e.g., a skeletal muscle can be fully activated within a few milliseconds. Diffusion of an activator from the surface to the interior of a fiber would take a much longer time.

In the 1960s it was shown that sarcoplasmic reticulum (SR) preparations exhibit Ca2+-ATPase activity. That is, the SR membrane contains Ca2+ pump that transports Ca2+ from the sarcoplasm into the SR lumen at the expense of ATP. Furthermore, it was established that the SR contains large amounts of calcium salts.

The experiments of Huxley and Taylor (1958): These authors raised the question, which part of the sarcomere is involved in activation of contraction? They mounted a single frog fiber under the microscope, immersed in physiological salt solution. Electrical pulse was applied through a very narrow pipette to the fiber, so that the tip of pipette was placed either to the center of A band or I band. The I band shortened when the pipette was applied to I band, whereas no response was obtained when the pipette was applied to A band (Fig. CA1). It was shown later that in frog muscle the junction of transverse tubules and SR are located in the I band, thus these experiments supported the concept that electrical stimulation of the tubules in muscle releases Ca2+ from SR.


Fig. CA1. Local activation of a twitch fiber from the semitendinosus muscle of Rana temporaria. (From Huxley and Taylor, 1958). In the upper pictures, the pipette was applied to A band, in the lower pictures, the pipette was applied to I band.

Sarcoplasmic Reticulum

The SR is the Ca2+-storage compartment of muscle. It forms a network in the sarcoplasm so that each fiber is surrounded by the reticulum and thereby has easy access to Ca2+. The SR widens at its two ends forming terminal sacs, called cisternae. A functional unit is a triad, consisting of two cisternae belonging to two adjacent SR and of one transverse (T) tubule in between (Fig. SR1). The distance between the terminal cisternae and the T-tubule is 10-15 nm. This gap is spanned by "foot structures". In frog muscle the transverse tubules are located at the level of the Z line, and in mammalian muscles they are located at the edge of A and I bands.

Fig. SR1. Electron micrograph of T-tubule and SR junction. (From Chu et al., 1987). The transverse tubule (TT) is flanked by two terminal cisternae of SR. The arrows point to the foot structures. LT, longitudinal tubule of SR.

Fig. SR2 is a scheme of the SR and T-tubule junction. The T-tubule is shown invaginating from the sarcolemma. The foot structure spans the gap between the terminal cisternae and T-tubule. Within the SR, Ca2+ is bound to calsequestrin, a protein that is anchored to the inner membrane of the terminal cisternae. Upon muscle stimulation, Ca2+ leaves the terminal cisternae and binds to troponin-C in the sarcoplasm to initiate the contraction process (see also Fig, EC2). Until the muscle is stimulated, Ca2+ is continuously released from the terminal cisternae. However, a part of the Ca2+ in the sarcoplasm is recaptured by the longitudinal part of the SR; the Ca2+ pump is responsible for the Ca2+ uptake and ATP supplies the energy. Once Ca2+ is inside the longitudinal tubules, it diffuses back to the terminal cisternae, where it is bound to calsequestrin, at the storage site. A 30-kDa calsequestrin-binding protein (Yamaguchi and Kasai, 1998) regulates the binding of Ca2+ to calsequestrin.

Fig. SR2. Scheme of T-tubule and SR junction (From Paul and Heiny, 1993).


The terminal cisternae contain the Ca2+ release channels of the SR. That channel is a very large protein (MW about 2 million) that spans the entire SR membrane thickness and protrudes up to the T-tubule. The protruding hydrophilic domain of the channel was first visualized in electron micrographs as foot structures. The Ca2+ release channel, also called as the junctional foot protein, was identified as a ryanodine receptor. It contains a central axial channel and four radial conduits. Ca2+ from SR enters the sarcoplasm at the junction via the four conduits. As the Ca2+ channel opens, Ca2+ leaves the SR, calsequestrin releases Ca2+, and more Ca2+ moves out into the sarcoplasm.

Signal transduction between T-tubule and SR-junction: The dihydropyridine (DHP) receptors of the T-tubule and the Ca2+-release channels of the SR terminal cisternae are participating in signal transduction. The DHP receptors act as voltage sensors: as an action potential propagates over the T-tubules (see Fig. EC2) charged regions of the receptor molecule rapidly move and cause a conformational change in the receptor. The DHP receptors and the Ca2+-release channels are in direct apposition at the junction. The conformational change in the DHP receptors may lead to the opening of the Ca2+-release channels. Several hypotheses have been proposed to connect the measured charge movements in the DHP receptors to Ca2+-release from SR.

Excitation - Contraction Coupling

Excitation-contraction coupling (EC) describes the events that lead from electrical stimulation of muscle to the initiation of muscle contraction. The time course of Ca2+ release from the SR during muscle contraction is of great interest. Ashley and Ridgway (1968) were the first to study this relationship. They monitored changes in Ca2+ concentration during muscle contraction by injecting aequorin, a Ca2+-binding bioluminescent protein, into muscle fibers. Upon Ca2+-binding aequorin emits light that can be measured; following the emission of light, aequorin is inactivated and the bound Ca2+ is released.

An aequorin-injected muscle fiber was electrically stimulated and first the action potential was registered. This was followed by light emission reflecting changes in intracellular Ca2+ concentration. When the Ca2+-mediated light output reached its peak, tension developed, by the time maximal tension was produced the Ca2+-mediated light output died away (Fig. EC1).


Fig. EC1. Time relationship of action potential, Ca2+ transient and tension. (From Ashley and Ridgway, 1968).

Currently, fluorescent Ca2+ indicators, such as fura-2 and quin-2, are used to measure changes in intracellular Ca2+ concentrations in the nanomolar to micromolar range. These indicators are excited at slightly longer wavelength when they are free of Ca2+ than when in their Ca2+- bound form. By measuring the ratio of fluorescent intensity at two excitation wavelengths the free Ca2+ concentration can be calculated.

The release of Ca2+ from SR when studied in frog muscle by confocal microscopic imaging of the fluorescent indicator, fluo-3, reveals that the release occurs largely in the form of discrete events, termed Ca2+ sparks. It is not clear whether the sparks are the results of opening of individual channels or of concerted opening of channel clusters (Rios et al., 1999).

Sequence of events: Upon stimulation of the muscle, an action potential propagates over the sarcolemma, travels through the T-tubules and elicits Ca2+ release from the SR into the sarcoplasm. Ca2+ binds to TN and the inhibition of actin-myosin combination that prevails at rest is lifted, and contraction ensues. Ca2+ is the link between excitation and contraction. Fig. EC2 shows that in the resting muscle (A) the membrane is negative on the inside and positive on the outside (left part of the Figure). In the shortened muscle (B), one notes the reversal of polarity upon stimulation and Ca2+ release from the terminal cisternae of SR toward the filaments (right part of the Figure).

 Fig. EC2. Sequence of events during excitation-contraction coupling. (From Keeton, 1972).


Ashley, C.C. and Ridgway, E.B. (1968). Aspects of the relatioship between membrane potential, calcium transient and tension in single barnacle muscle fibers. J. Physiol. 200, 74-76P.

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Yamaguchi, N. and Kasai, M. (1998). Identification of 30 kDa calsequestrin-binding protein, which regulates calcium release from sarcoplasmic reticulum of rabbit skeletal muscle. Biochem. J., 335, 541-547.

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