Classic Experiment 18.

Looking at Muscle Contraction

T
he contraction and relaxation of striated muscles allow us to perform all of our daily tasks. How does this happen? Scientist have long looked to see how fused muscles cells, called myofibrils, differ from other cells that cannot perform powerful movement. In 1954, Jean Hanson and Hugh Huxley published their microscopy studies on muscle contraction, which demonstrated the mechanism by which it occurs.

Background
The ability of muscles to perform work has long been a fascinating process. Voluntary muscle contraction is performed by striated muscles, which are named for their appearance when viewed under the microscope. By the 1950s, biologists studying myofibrils, the cells that make up muscles, had named many of the structures they had observed under the microscope. One contracting unit, called a sarcomere, is made up of two main regions called the A band, and the I band. The A band contains two darkly colored thick striations and one thin striation. The I band is made up primarily of light-colored striations, which are divided by a darkly colored line known as the Z disk. Although these structures had been characterized, their role in muscle contraction remained unclear. At the same time, biochemists also tried to tackle this problem by looking for proteins that are more abundant in myofibrils than in other non-muscle cells. They found muscles to contain large amounts of the structural proteins actin and myosin in a complex with each other. Actin and myosin form polymers that can shorten when treated with adenosine triphosphate (ATP). With these observations in mind, Hanson and Huxley began their study of cross striations in muscle. In a few short years, they united the biochemical data with the microscopy observations and developed a model for muscle contraction that holds true today.

The Experiment
Hanson and Huxley primarily used phase-contrast microscopy in their studies of striated muscles that they isolated from rabbits. The technique allowed them to obtain clear pictures of the sarcomere, and to take careful measurements of the A and the I bands. By treating the muscles with a variety of chemicals, then studying them under the phase-contrast microscope, they were able to successfully combine biochemistry with microscopy to describe muscle structure as well as the mechanism of contraction. In their first set of studies, Hanson and Huxley employed chemicals that are known to specifically extract either myosin or actin from myofibrils. First, they treated myofibrils with a chemical that specifically removes myosin from muscle. They used phase-contrast microscopy to compare untreated myofibrils to myosin-extracted myofibrils. In the untreated muscle, they observed the previously identified sarcometic structure, including the darkly colored A band. When they looked at the myosin-extracted cells, however, the darkly colored A band was not observed. Next, they extracted actin from the myosin-extracted muscle cells. When they extracted both myosin and actin from the myofibril, they could see no identifiable structure to the cell under phase-contrast microscopy. From these experiments, they concluded that myosin was located primarily in the A band, whereas actin is found throughout the myofibril.

With a better understanding of the biochemical nature of muscle structures, Huxley and Hanson went on to study the mechanism of muscle contraction. They isolated individual myofibrils from muscle tissue and treated them with ATP, causing them to contract at a slow rate. Using this technique, they could take pictures of various stages of muscle contraction by using phase-contrast microscopy. They could also mechanically induce stretching by manipulating the coverslip, which allowed them to also observe the relaxation process. With these techniques in hand, they examined how the structure of the myofibril changes during contraction and stretch. First, Huxley and Hanson treated myofibrils with ATP, then photographed the images they observed under phasecontrast microscopy. These pictures allowed them to measure the lengths of both the A band and the I band at various stages of contraction. When they looked at myofibrils freely contracting, they noticed a consistent shortening of the lightly colored I band, whereas the length of the A band remained constant (see Figure 18.1). Within the A band, they
Z disk Stretched 120% A band Relaxed 100% I bands

observed the formation of an increasingly dense area throughout the contraction. Next, the two scientists examined how the myofibril structure changes during a simulated muscle stretch. They stretched isolated myofibrils mounted on glass slides by manipulating the coverslip. They again photographed phasecontrast microscopy images and measured the lengths of the A and the I bands. During stretch the length of the I band increased, rather than shortened, as it had in contraction. Once again, the length of the A band remained unchanged. The dense zone that formed in the A band during contraction, became less dense during stretch. From their observations, Hanson and Huxley developed a model for muscle contraction and stretch (see Figure 18.1). In their model, the actin filaments in the I band are drawn up into the A during contraction, and thus the I band becomes shorter. This allows for increased interaction between the myosin located in the A band and the actin filaments. As the muscle stretches, the actin filaments withdraw from the A band. From these data, they proposed that muscle contraction is driven by actin moving in and out of a mass of stationary myosin molecules.

S 2.8 A 1.5 I 1.3

Discussion
By combining microscopic observations with known biochemical treatments of muscle fibers, Hanson and Huxley were able to describe the biochemical nature of muscle structures and outline a mechanism for muscle contraction. A large body of research continues to focus on understanding the process of muscle contraction. Scientists now know that muscles contract by ATP hydrolysis driving a conformational change in myosin that allows it to push actin along. Researchers are continuing to uncover the molecular details of this process, while the mechanism contraction proposed by Hanson and Huxley remains in place.

S 2.3 A 1.5 I 0.8 S 2.0 A 1.5 I 0.5 S 1.8 A 1.5 I 0.3

Contracted 90%

Contracted 80%

Contracted 60%

S 1.5

v FIGURE 18.1 Schematic diagram of muscle contraction

and stretch observed by Hanson and Huxley. The lengths of the sarcomere (S), the A band (A), and the I band (I) were measured from 60 percent contraction (bottom) to 120 percent stretch (top). The lengths of the sacromere, the I band, and A band are noted on the left. Notice that from 120 percent stretch to 70 percent contraction the A band does not change in the length, whereas the length of the I band can stretch to 1.3 microns, then contract to 0.3 microns. At 60 percent contraction, the I band disappears, and the A band shortens to the overall length of the sarcomere. [Adapted from J. Hanson and H. E. Huxley, 1955, Symp. Soc. Exp. Biol. Fibrous Proteins and their Biological Significance 9:249.]