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In this experiment we used electrical stimulation to study muscle contraction. Intact Gastrocnemius muscle from frog hind leg, innervated by sciatic nerve, was used as a model to investigate skeletal muscle contraction properties namely, muscle twitch, tetanus and fatigue.
Skeletal muscles are responsible for voluntary movements. The structure of muscle cell was deduced almost 300 years ago based on the initial observations of Leeuwonhoek and Croone1. Striated muscle fibers are long and slender. These are rich in mitochondria, glycogen granules and myoglobin to yield energy. The contractile fibers of skeletal muscles are composed of thick myosin and thin actin filaments2. These are arranged as shown in the figure 1.
In 1791, Luigi Galvani observed that frog muscle contracts when an electric charge is applied to it3. This set the stage for the next 300 years of research on electrophysiology. The early 19th century saw an explosion of muscle biochemistry with the isolation and characterization of myosin, actin and the reticular system of muscle which was later identified as the T-tubule1. The detailed structure of a sarcomere was identified by Gustav Retzius4 and the sarcoplasmic reticulum by Veratti5 . Detailed structures were seen in the late 20th century with the advent of the electron microscope. The importance of Ca2+, Na+ and K+ in muscle contraction was shown by Sydney Ringer who observed that isolated hearts of frog and eel showed good spontaneous beats which lasted for a longer time compared to perfusion with only normal saline. This led him to conclude that calcium, sodium and potassium is needed in correct proportions (as present in Ringer's solution used in our experiemnt) for normal muscle activity1. In contrast the skeletal muscle was not was not dependent on the present of extra cellular calcium. Later on it was confirmed that the difference is due to the inability of the heart muscle to retain its calcium store in a Ca2+ poor perfusion medium6. The last 40 years have seen a lot of information about the cell processes involved in muscle contraction. In the 1960s nothing was known of the molecular mechanism of muscle contraction, it was thought that a "switch" allowed the action potential to travel down the tubular invaginations of the sarcolemma to release Ca2+ from the SR7. It was established in the 1970s that these Transverse or T-tubules are flanked on either side by terminal cisternae and are named "Triad Junction"8. The movement of charge was associated with the release of Ca2+ from the SR of fast skeletal muscles and is initiated by the depolarization of the transverse tubules9. At the same time it was discovered that EC-based coupling was sensitive to dihydropyridine-based compounds10;11. A hypothesis was formed stating DHPR as the voltage sensor for EC coupling. In the early 1980s the SR calcium release channel was seen to have affinity for ryanodine, a plant alkaloid12. The DHPR hypothesis was proven with the identification of a naturally occurring DHPR a1S-null dysgenic mouse at birth13. Recently whole-cell studies have been done to locate sites of precise molecular interactions between the DHPR and RyR using novel techniques14. This Ca2+ release in the cytosol initiates the cross bridge cycle resulting in contraction of the skeletal muscle. The Ca2+ binds to troponin15. Troponin moves tropomyosin which in turn allows the formation of cross bridges and results in muscle contraction16. When the muscle relaxes, the Ca2+ ions are pumped back into the sarcoplasmic using ATP energy. Muscles contract on the all-or-none principle but graded muscle contractions can be obtained by varying the electrical stimulus. The response of a muscle to a single action potential is called a muscle twitch. Successive action potentials results in an increase in mechanical response known as wave summation. Under repeated summation, the tension developed in the muscle eventually decrease even under the application of a stimulation. This phenomenon is known as muscle fatigue. Muscle fatigue is a reversible phenomenon and is observed as a progressive decline of muscle performance17.
Frog was sacrificed, presumably painlessly, and legs were removed for the experiment by cutting the femur close to the pelvis. The skin was pulled off using forceps; Gastrocnemius muscle was identified and removed from the tibia. A doubled thread was firmly tied around the Achilles tendon and cut close to the bottom of the foot. The tibia was cut away just below the knee and the lower leg was separated.
The preparation was mounted on the ring stand using the femur clamp and the thread from the Achilles tendon was run through the hole and tied on the end of the blade of the FT-104 force transducer. The femur clamp was adjusted to remove the slack in the thread. The A-BST-100 stimulating electrodes were pushed into to gastrocnemius muscle, almost midway between the knee and the tendon. Ringer solution was used to keep the preparation moist throughout the course of the experiment, provide the ions the skeletal muscle needed and also to adjust the pH.
Single stimulus was given with various voltage levels using the iWorx IWX/214 to determine the threshold voltage. Varying pulses were applied of the threshold voltage to observe summation, tetanus and fatigue.
In this experiment, we used electrical stimulation to mimic the neural stimulation in muscle tissue. Skeletal Muscle tissue in vivo is stimulated by motor nerves using Acetylcholine as a neurotransmitter. Acetylcholine binds to the ligand gated Na+ channels to produce an influx of Na+ ions and thus produces an electrical stimulation. Once a threshold electrical stimulation is reached and transmitted down the t-tubules, it results in change in confirmation of the DHPR proteins. These DHPR proteins are closely associated with the ryanodine receptors and the electrical stimulus results in release of Ca2+ and the subsequent muscle contraction. In our experiment we used electrical stimulation to recruit motor neurons. This works in a manner similar to voluntary contraction except that the stimulation comes from the electric battery instead of the central nervous system.
The muscle twitch can be roughly divided into 3 periods (figure 2). The period between the application of the stimulus and the start of the muscle contraction is known as the latent period. During this time Excitation-Contraction coupling takes place and calcium is released into the sarcoplasm. Once the calcium binds to troponin and makes the binding sites on the actin available the contraction phase starts. Upon reaching maximum contraction the fast Na+ channels close while the slow K+ channels open and is observed as the start of the relaxation phase.
In our experiment, the first muscle twitch was observed with a stimulus of 0.75v and below this value no response was obtained from the gastrocnemius muscle tissue. What this meant was that the stimulus of 0.25v and 0.50v was not sufficient to start inducing the release of Ca2+ in the muscle cell and thus unable to recruit motor neurons.
Once we used a stimulus voltage of 0.75v the smallest motor units with the very excitable motor neurons were recruited. With the increase in stimulus voltage, larger motor units with motor neurons having a higher threshold are recruited resulting in greater amplitude of the muscle response. The observation of maximum contraction meant that the motor neurons with the least excitable neurons have been recruited. At this stage any increase in stimulus voltage fails to show an increase in amplitude of muscle response as there are no more motor units left which could be recruited to obtain a greater contraction. This phenomenon is utilized by the CNS to spread the work load across the pool of motor units. This reduces the requirement on a given motor unit18. This property is also used to produce graded motor response. The least fatigable motor units are utilized for lower motor load while the most fatigable and more powerful muscles are used in more demanding tasks19.
The contraction amplitude of single twitches is the same as the calcium channels open and release the calcium which binds to troponin and starts the binding of myosin to actin. The calcium starts getting sequestered back in the sarcoplasmic reticulum as soon as it is released. As the contraction amplitude is dependent upon the increases in concentration and persistence of intracellular calcium, single twitches result in the same Ca2+ ion concentration in the sarcoplasm and therefore show same contraction amplitudes.
Tetanus is seen at high stimulus frequencies as the stimulus builds on the calcium ions released by the previous stimulus resulting in a greater concentration of calcium in the sarcoplasm. This is only observed if the stimuli are close to each other. If the muscle gets enough time to relax between the consecutive stimuli then all the calcium that was released is brought back into the sarcoplasmic reticulum. Based on data obtained in our experiment in table 2, we see that the first wave summation was seen at 3 Hz, this means that at frequency less than that, the muscle has enough time to bring back all the calcium ions into the sarcoplasmic reticulum.
Slow muscle relaxation was also observed after tetanus as compared to a single twitch. This would be due to a higher calcium ion concentration in the sarcoplasm after tetanus as compared to a single twitch.
Based on the data from Table 2, we also observed that the amplitude of the first twitch increased steadily with each stimulation of the gastrocnemius muscle. This increased contraction in response to multiple stimuli of the same strength is known as Treppe or the staircase effect. What this means is that due to previous contractions there is an increased availability of Ca2+ in the sarcoplasm. This combined with the increased efficacy of muscle enzyme systems due to the heat resulting from muscle contractions explains the increased amplitude. As a secondary observation, the first twitch amplitude steadily increased till 3 Hz frequency. This again could be explained with the increased availability of Ca2+ present from previous contractions. In our experiment there was a sharp decline in the first amplitude of the 10 Hz pulse. The most probable explanation for this would be the long wait time we had between the two specific pulses.
The slope of the fused tetanus observed was not as smooth as seen in most journals and text books. There was a lot of baseline noise which could account for the small variations of force seen throughout the curve.
Muscle fatigue was not observed in our experiment as a pulse of 15 was not able to induce muscle fatigue.
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Once this chemical signal is received by the muscle fiber, ligand gated Na+ channel open which results in a change in the membrane potential due to influx of Na+ ions. When the threshold potential, Vm, is reached the cell gets depolarized and the electrochemical current is spread through the Transverse tubules.
Different neurons, in a single nerve, have different thresholds. When we increase the electrical stimulus more neurons are able to recruit more muscle fibers and hence increase the strength of the contraction.