A skeletal muscle is a collection of muscle cells, or muscle fibers. Each muscle fiber is composed of a cell membrane, cytosol, mitochondria, and a whole bunch of myofibrils (bundles of contractile and elastic proteins that carry out the work of contraction). Each muscle cell also contains an extensive sarcoplasmic reticulum for calcium storage, and release.
A myofibril is comprised of several types of proteins: two contractile proteins, myosin and actin, two regulatory proteins, troponin, and tropomyosin, and a few accessory proteins to boot.
The contraction of a muscle is a complex process, involving many ions, proteins, and certain regulatory mechanisms. Myosin is motor protein that converts chemical bond energy from ATP into mechanical energy of motion. Your textbook describes the molecular basis for contraction, the power stroke, a six-step process very well (Please read it). Actin is associated with the regulatory proteins that prevent the myosin heads from completing the power stroke.
Tropomyosin is a long polymer that wraps around the actin filament, directly blocking the myosin head binding site, and inhibiting actin and myosin sliding. Ca++ inhibits troponin so filament sliding can occur. Ca++ plus troponin, cause a tug on tropomyosin, pulling it deeper into the groove between actin strands. This uncovers active sites, which react with cross bridges on myosin strands causing the sliding process (power stroke). ATP breaks these reaction bonds and
forms new ones (power stroke), and is required for relaxation. You should be able to explain how the following components work together to produce a controlled skeletal muscle contraction: Actin, tropomyosin, troponin, myosin, myosin head (crossbridge), Ca++(salts) and ATP. The control of contraction is summarized in Chapter 12 of your text for a full discussion.
Relaxation of the muscle occurs when the sarcoplasmic reticulum pumps Ca++ back into its lumen using a Ca++ -ATPase. As the free cytosolic calcium decreases, Ca++ releases from troponin, tropomyosin slides back to block the myosin binding site and the fiber relaxes.
The basic unit of contraction in an intact skeletal muscle is the motor unit. One motor unit is comprised of one neuron and the muscle fibers it innervates. If the action potentials of a motor unit continue to stimulate the muscle fiber repeatedly at short intervals (high frequency), relaxation between contractions diminishes until a muscle fiber reaches a maximal level of contraction called tetanus. Look at the figures describing summation of contractions leading to maximum tension, and eventually fatigue.
The term fatigue describes a condition in which a muscle is no longer able to generate or sustain the expected power output. Fatigue can be caused by a variety of factors, such as depletion of neurotransmitter, ATP, oxygen, varying ion concentrations, and a build up of lactic acid to name a few.
1.) You will be instructed on how to use the Biopak-computer set up to test your muscles for strength and fatigue.
2.) Observe the glycerinated Psoas muscle for contraction as described below:
Note: All glassware and dissecting tools should be cleaned thoroughly and well rinsed in distilled water before use. Do not contaminate the glycerinated muscle with outside ions such as metal, Ca++ or salts of any kind. Care must be taken to avoid any possibility of cross contamination between the ATP and salt solution vials. Any contamination will lead to ambiguous experimental results. Wash your hands before starting.
a.) Place one drop of glycerol on a microscope slide.
b.) Remove from the test tube one segment 1 cm. long of skeletal muscle and place it into the glycerol on a microscope slide.
c.) Using the modified glass pipettes and a dissecting microscope, gently tease the segment of muscle into very thin groups of myofibrils; single fibers, if obtained,
will demonstrate the greatest contraction. Strands of muscle exceeding 0.2 mm in cross-sectional diameter (about the size of a hair) must not be used.
d.) Mount one of these three strands on separate microscope slide with a cover slip. Using a compound microscope examine one of the strands under low and high magnification. Note the striations in the fibers and the smooth walls.
e.) Place each of the three slides under the dissecting microscope and measure the length of each specimen with a millimeter scale held beneath the slide. Record
the lengths of each.
f.) Using the dissecting microscope, flood the first specimen with several drops of the solution containing KCL and MgCl2. KCl and MgCl2 attach to the troponin
protein which results in an unblocking interaction between actin and the myosin heads (cross-bridges). After 30 seconds or more, observe the reaction of the
fiber, remeasure using the dissection microscope and record.
g.) Using the dissecting microscope flood the second specimen with several drops of the solution containing ATP alone. ATP acts as an energy source causing the
power stroke. After 30 seconds or more, observe the reaction of the fibers, remeasure using a dissecting scope and record.
h.) Again using the dissecting microscope flood the third specimen with several drops of the solution containing ATP plus KCl and MgCl2. After 30 seconds or
more, observe the reaction of the fibers, remeasure and record.
i.) When your group has finished, clean and dry all glassware and return them from whence they came.