18.9: Ca++ Ions Regulate Skeletal Muscle Contraction

Typically, the neurotransmitter acetylcholine released by a motor neuron binds to receptors on muscle cells to initiate contraction. Early experiments had already revealed that \(\rm Ca^{++}\) was required, along with ATP, to get glycerinated skeletal muscle to contract. It was later shown that \(\rm Ca^{++}\) ions were stored in the sarcoplasmic reticulum (the smooth endoplasmic reticulum) of intact muscle cells. As we have seen, an action potential generated in the cell body of a neuron propagates along an axon to the nerve terminal, or synapse.

The action potential at a neuromuscular junction that initiates contraction is summarized in the illustration in Figure 18.24 (below).

Figure 18.24: Innervation leads to \(\rm Ca^{++}\) release from sarcoplasmic reticulum to regulate contraction.

In a similar fashion, an action potential generated at a neuromuscular junction travels along the sarcolemma (the muscle plasma membrane) to points where it is continuous with transverse tubules (T-tubules). The action potential moves along the T-tubules and then along the membranes of the sarcoplasmic reticulum. This propagation of an action potential opens \(\rm Ca^{++}\) channels in the sarcoplasmic reticulum, releasing \(\rm Ca^{++}\) to bathe the myofibril sarcomeres where they bind to one of three troponin molecules; this will allow skeletal muscle contraction (i.e., to allow filaments to slide). The three troponins and a tropomyosin molecule are bound to actin filaments.

Experiments using antitroponin and antitropomyosin antibodies localized the three proteins in electron micrographs at regularly spaced intervals along actin filaments, as modeled in Figure 18.25 below.

Figure 18.25: Thin skeletal muscle filaments consist of actin associated with troponins and tropomyosin, which play a role in regulating actin-myosin interactions during contraction.

In resting muscle, tropomyosin (a fibrous protein) lies regularly spaced along the actin filament, where it covers up the myosin-binding sites of seven G-actin monomer subunits in the microfilament. The negatively stained electron micrograph in Actin Bound to Tropomyosin is consistent with the model illustrated in Figure 18.25. The cross section drawn in Figure 18.26 illustrates how troponin T (tropomyosin-binding troponin) and troponin I (inhibitory troponin) hold the tropomyosin in place, and how the binding of \(\rm Ca^{++}\) ions to troponin C regulates contraction.

Figure 18.26: \(\rm Ca^{++}\) ions bind troponin C to Initiate the uncovering of myosin-binding sites on G-actin

A chain reaction of conformational changes begins when \(\rm Ca^{++}\) ions bind to troponin C. The result is that the three-subunit troponin complexes that are bound to tropomyosin will shift position along the filament to expose the myosin-binding sites on the G-actin subunits. Only after this shift can ATP-bound myosin in turn bind to actin and initiate the microcontraction cycle discussed earlier. The regulation of contraction by \(\rm Ca^{++}\) is animated in the following link.

337 Regulation of Skeletal Muscle Contraction by Calcium

18.9.1 Muscle Contraction Generates Force

Contraction by ATP-powered sliding of thin along thick filaments generates force on the Z-lines. In three dimensions, the Z-lines are actually Z-discs, to which the actin filaments are attached. The protein \(\alpha\)−actinin in the Z-discs anchors the ends of the actin filaments to the discs. That way when the filaments slide, force transduction draws the Z-discs closer, shortening the sarcomeres. Another Z-disc protein, desmin, is an intermediate filament organized around the periphery of the Z-discs. Desmin connects multiple Z-discs in a myofibril. With the Z-discs being kept in register, the muscle cell—and ultimately whole muscle contraction—is coordinated. Finally, actin filaments at the ends of the muscle cell must be connected to the plasma membrane so that force transduction will cause the muscle cell to shorten during myofibril contraction.

Several proteins, including syntrophins and dystrophin (an intermediate filament protein) anchor the free ends of microfilaments coming from Z-discs to the cell membrane. Still other proteins anchor the cell membrane in this region to the ECM (extracellular matrix) tendons, which are in turn attached to bones! The force generated by myosin hydrolysis of ATP and the sliding of filaments in individual sarcomeres is thus transmitted to the ends of muscles to effect movement. If the name dystrophin sounds familiar, it should! The gene and its protein were named for a mutation that causes muscular dystrophy, resulting in a progressive muscle weakening.

338 Contraction Generates Force Against Z-Discs and Cell Membranes

18.9.2 The Elastic Sarcomere: Do Myosin Rods Just Float in the Sarcomere?

In fact, myosin rods do not “float” in sarcomeres but are instead anchored to proteins in the Z-discs and M-lines. In 1954, R. Natori realized that when contracted muscle relaxes, it lengthens beyond its resting state, then shortens again to its resting length. Natori proposed that this elasticity must be due to a fiber in the sarcomere. Twenty-five years later, the elastic structure was identified as titin, a protein that holds several molecular records! The gene for titin contains the largest number of exons (363) of known proteins. After actin and myosin, titin is also the most abundant protein in muscle cells. At almost \(4 \times 10^6\) Da, the aptly named titin is also the largest known polypeptide, much larger than even myosin! As it extends from the Z-discs to the M-line of sarcomeres, titin coils around thick filaments along the way. The Titin is anchored at Z-discs by \(\alpha\)−actinin and telethonin proteins. At the M-line, titin binds to myosin-binding protein C (MYBPC3) and to calmodulin, among others (myomesin, obscurin, skelamin…). Some if not all of these proteins must participate in keeping the myosin (thick) filaments positioned and in register in the sarcomere. This is like how desmin binds Z-discs to each other to keep sarcomeres in register. The location of titin and several other sarcomere proteins is illustrated in Figure 18.27.

Figure 18.27: Structure of titin and its location in the sarcomere.

Coiled titin molecules (red in the illustration) extend from the Z to M lines. The colorized electron micrograph of one extended titin molecule in the middle of the illustration should convince you of the length (35,213 amino acids!) of this huge polypeptide. Titin’s elastic features are largely in a region labeled P in the electron micrograph—between the Z-discs and the ends of the myosin rods. The many domains of this P region are shown expanded at the bottom of Figure 18.27. With all the binding (and other) functions, you might expect that titin has many domains. It does! They include Ig (immunoglobulin) domains, fibronectin domains (not shown here), and PEVK and N2A domains, some of which help bind titin to \(\alpha\)−actinin in Z-discs. Which ones and how many of the Ig and/or PEVK domains are present in a particular muscle depends on which alternative splicing pathway is used to form a titin mRNA. Over a micron long, titin functions as a molecular spring, as Natori predicted. Its coiled domains compress during contraction, passively storing some of the energy.

When a skeletal muscle relaxes, \(\rm Ca^{++}\) is withdrawn from the sarcomere. ATP can still displace ADP from myosin heads, breaking actin-myosin crossbridges. When actin and myosin heads dissociate in the absence of \(\rm Ca^{++}\), the troponins and tropomyosin reverse their allosteric changes, once more covering myosin-head binding sites on F-actin. The muscle then stretches, typically, under the influence of gravity or an opposing set of muscles. But during contraction, 244 individually folded titin domains had been compressed, so during relaxation, these domains decompress. The stored energy of compression thus also helps to power muscle relaxation. At the same time, titin connections limit the stretch so that a potentially overstretched muscle can “bounce” back to its normal relaxed length. In a particularly elegant experiment, Wolfgang A. Linke (et al.) provided a visual demonstration of myofiber elasticity consistent with the coiled-spring model of titin structure. They made antibodies to peptide domains on either side of the PEVK domain of titin (N2A and I20-I22) and attached them to nanogold particles. The gold particles were electron-dense and would appear as black granules in transmission electron microscopy. In the experiment, individual myofibers were stretched to different lengths, fixed for electron microscopy, and treated with the nanogoldlinked antibodies. The antibodies localize to and define the boundaries of the titin PEVK domains in myofibers, stretched to different lengths (Figure 18.28, showing electron micrographs with simulated localization of nanogold particles, which reflect the actual results).

Figure 18.28: Skeletal-muscle elasticity shown by the location of anti-N2A- and anti-120/122-linked nanogold particles in sarcomeres. Note the increased separation of the PEVK domains targeted by the antibodies as a muscle fiber is increasingly stretched. This titin elasticity facilitates skeletal muscle relaxation.

In the experiment, increased stretch lengthened the I-bands on either side of the Z-lines of sarcomeres (blue bars under each pair of micrographs). Likewise, titin PEVK domains have also lengthened, as seen in the increased distance between the nanogold-linked N2A and the 120/122 antibody localization flanking the PEVK domains. This demonstration of titin (and thus, sarcomere) elasticity is consistent with a storage of some of the free energy of contraction when the titin is compressed, and a passive release of that energy during relaxation. Titin tethers thick filaments to Z-discs and M-lines and thus also limits sarcomere stretch during relaxation. Titin elasticity is animated at Elasticity of Titin Domain.

CHALLENGE

It seems that the many myofiber nuclei don’t behave the same. Myofiber Nuclei Express Different Genes; How can this be so?