The muscle physiology of skeletal muscle was observed by using electrical, physical, and neural stimulations of an isolated gastrocnemius muscle from Rana pipiens. The gastrocnemius receives signals from the action potentials of the sciatic nerve. The muscle contraction is caused by the binding of a neurotransmitter once the action potential reaches the neuromuscular junction. Stimulation of the muscle and the sciatic nerve allows for recording and measuring of these properties.
We observed twitch recruitment of the nerve and muscle length-tension relationship of the skeletal muscle, summation of skeletal muscle contractions, tetanus, skeletal muscle fatigue, and the effects of temperature on skeletal muscle contractions. Since motor neutrons can innervate multiple muscle fibers, reaching maximum twitch recruitment needed a lower stimulus voltage and produced a greater contraction amplitude when stimulating the nerve compared to stimulating the muscle directly.
After increasing the gastrocnemius in 1mm increments, maximal muscle contraction was not observed due to the muscle resting length prior to stretching. We found that two stimuli fired in quick succession lead to summation of the muscle contraction. Tetanus was observed after an increase in frequency. Tetanus could not be held forever, so the muscle fatigued at a rate of 3. 57% per second. By increasing the temperature of the ringer’s solution, the contraction amplitude threshold decreased for the gastrocnemius muscle. We found that it was more difficult to produce contractions at cooler temperatures.
By increasing the temperature of the ringer’s solution, the contraction amplitude threshold increased from cold to room temperature and decreased from room temperature to warm temperature. These experiments were performed in order to gain a better understanding of muscle physiology with the skeletal muscle ad neuromuscular functions of the Rana pipiens. Introduction Skeletal muscle is a voluntary striated muscle composed of fascicles which are a bundle of muscle fibers held together by a type of connective tissue, the perimysium.
Muscle fibers are composed of a bundle of myofibrils held together by a type of connective tissue, the endomysium. The myofibrils are composed of sarcomeres that repeat longitudinally and act as a basic functional unit in skeletal muscle (Boswell 2017). Each sarcomere is composed of an A band, an H zone, an I band, a Z disc. The A band’s length remains constant during contractions because its length is determined by the thick filament (myosin filament) which does not change size during contractions. The H zone shrinks during contractions because it is near the middle of the sarcomere where no overlap of actin and myosin occurs.
The I band can be measured by the distance between myosin filaments, and it gets smaller during a contraction. The length of the sarcomere can be measured by the distance between each Z disc. The sarcomere shortens during contractions because the distance between the Z discs is reduced. The amount of motor units stimulated can affect the amplitude of maximal tension that a muscle is able to create. A single motor neuron along with all the muscle fibers it innervates compose the motor unit. With more muscle fibers innervated, there will be a stronger contractile force.
By stimulating a nerve that innervates multiple muscle fibers, a larger contractile force will occur. The gastrocnemius muscle has fewer innervation sites with a larger number of muscle fibers which is expected since no fine-motor skill need for the use of a leg. The tension in a skeletal muscle can be increased by increasing the frequency at which an action potential is fired. Twitch summation and tetanus occur because a second stimulus occurs before the first contraction has time to relax. Tetanus occurs because the muscle fiber stimulated has not relaxed at all.
The action potential of the second twitch will have to occur rapidly enough for a summation of contractions. Tension is at its highest when the muscle is undergoing tetanus. The muscle cannot sustain a tetanic state for an extended period of time without undergoing fatigue and a decrease in tension over time. Understanding the chemicals that affect muscle contractions is vital to understanding skeletal muscle activity. When acetylcholine binds to the nicotinic receptors, excitation contraction coupling begins, and an end plate potential can be generated.
If the end plate potential can reach threshold by depolarizing itself, an action potential can fire. The firing of an action potential causes the release of Ca2+ into the cytoplasm. More calcium is released from the sarcoplasmic reticulum due to the presence of calcium in the cytoplasm. Troponin C gets bound by calcium which causes an allosteric change in the troponin. This allows the tropomyosin to be able to move off of the myosin binding sites on actin. Muscle contractions can be ended by pumps that sequester Ca2+, the presence of ATP, or the presence of acetylcholine.
The body of the Rana pipiens has a temperature at 37°C. We hypothesized that increase temperature would cause an increase in contraction threshold. Methods Experiments 1-5 were performed as outlined in the BIOL 4161 Student Lab Manual (Boswell 2017). In order to test our hypothesis that an increase in temperature would increase the contraction threshold amplitude for the sciatic nerve and gastrocnemius muscle of the gastrocnemius of Rana pipiens, three different temperatures (cold, room temp. , warm) were tested. We followed the protocol for Experiment 1a and 1b while increasing stimulus voltage.
We placed the sciatic nerve and gastrocnemius muscle within the muscle holder and attached the force transducer. The metal clips were attached to the electrodes that were used to stimulate the sciatic nerve. We calibrated the force transducer to show Newtons for the contraction amplitude instead of mV. We set the pulse 1 width of 150µs, a pulse 2 width of 150µs, a pulse 1 height of -80mV (maximal stimulus), a pulse 2 height of 0mV, and a frequency of 1Hz. First, we added cold ringer’s solution (4°C) to the sciatic nerve for two minutes which was then stimulated, and contraction amplitude was taken.
We repeated this step for the gastrocnemius muscle. Next, we added room temperature ringer’s solution (22°C) to the sciatic nerve for two minutes which was then stimulated, and contraction amplitude was taken. We repeated this step for step for the gastrocnemius muscle. Next, we added warm temperature ringer’s solution (22°C) to the sciatic nerve for two minutes which was then stimulated, and contraction amplitude was taken. We repeated this step for step for the gastrocnemius muscle. We did not perform replicates for this experiment.
Results Twitch recruitment after nerve stimulation: After increasing the stimulus in intervals of 10mV starting at 0mV, we observed that stimulus voltages under 60mV did not elicit a response. The threshold stimulus voltage was observed at 70mV with an amplitude of contraction at 0. 4506 N (Fig. 1A). The stimulus voltage continued to increase until a maximal stimulus voltage was observed at 80mV with a contraction amplitude of 0. 667 N (Fig. 1A). Twitch recruitment after muscle stimulation: After increasing the stimulus in intervals of 50mV starting a 50mV, we observed that stimulus voltages under 650mV did not elicit a response.
A threshold stimulus voltage was observed at 700mV with a contraction amplitude of 0. 0202 N (Fig. 1B). The stimulus voltage continued to increase until maximum stimulus voltage was observed at 900mV with a contraction amplitude of 0. 3051 N (Fig. 1B). Length-tension relationship of skeletal muscle: The smallest contraction amplitude of the gastrocnemius muscle was observed prior to stretching with and amplitude of 1. 4304N (Fig. 2). While steadily increasing the stretch by 1mm until a 10mm stretch was reached, a maximum muscle contraction was reached at a 10 mm stretch with an amplitude of 2. 722N (Fig 2).
Summation of skeletal muscle contractions: Using a maximal stimulus voltage of 80mV, we decreased the interstimulus interval (ISI) in increments of 50µs starting at 500 µs. Both contractions started with different amplitudes at 500µs. Contraction 1 (1. 1134N) and Contraction 2 (1. 2124N) (Fig. 3). After reaching 100 µs, we started to decrease the interstimulus interval in increments of 20 µs. Summation started when ISI decreased to 100 µs. Contraction 1 had a contraction amplitude of 0. 12711N and contraction 2 had a contraction amplitude of 1. 1541N (Fig. 3).
Complete summation was observed at an ISI of 60µs when contraction 1 was 1. 4746N and Contraction 2 amplitude was 1. 4746N (Fig. 3). Tetanus of skeletal muscle: Using a maximal stimulus voltage of 80mV, we tested the effects of different frequencies (Hz): 1,2. 5,5,10,20,30,40, and 50. At a frequency of 1 Hz, the peaks started out with an average amplitude of 1. 166133N, and the troughs started out with an average amplitude of 1. 68933N (Fig. 4). As the frequency increased, so did the peak and trough size until tetanus was reached at 20Hz: peak amplitude 1. 611933 + 0. 015366N and trough amplitude 1. 11933 + 0. 015366N (Fig. 4). There was significant difference observed between the trough and peaks contraction amplitudes before tetanus.
There was no significant difference observed between the trough and peaks contraction amplitudes during tetanus. Fatigue of skeletal muscle: The skeletal muscle began in a state a tetanus, and it fatigued at a rate of 3. 57% per second. Varying Temperature: In the nerve for cold temperate the maximum the contraction 0. 7607 N, and the contraction threshold was observed at 130 mV (Fig. 5A). In the room temperature, the maximum contraction amplitude was 0. 065 N, and the contraction threshold was observed at 150 mV (Fig. 5A) The warm temperature maximum contraction amplitude was 0. 6180 N, and the contraction threshold was observed at 100mV (Fig. 5A).
In the muscle, for cold temperature Ringer’s solution, the maximum contraction was 0. 4121N and, the contraction threshold was observed at 550 mV (Fig. 5B) The room temperature the maximum contraction amplitude was 0. 3106N, and the contraction threshold was observed at 350 mV (Fig. 5B). The warm temperature maximum contraction amplitude was 0. 2867 N, and the contraction threshold was observed at 350mV (Fig. 5B)
