Muscle Twitch Lab Report PDF

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Summary

Muscle Twitch Lab Report...

Description

Muscle response summation and twitch amplitude increases with amplified stimulus voltage and frequency

Danielle Temples APPH 3756 - A

April 15, 2019

Abstract Skeletal muscle makes up almost half the body. This muscle is necessary for functions such as shock absorption, maintaining homeostasis, providing energy, and movement. Injuries or illness, such as strokes, can cause motor impairment to these muscles. It can be a difficult process for the effected person to regain the connection between the brain and the damaged muscle. Rehabilitation techniques such as electrical stimulation and stretching can help facilitate this process. The effects of increasing stimulation amplitude, increasing stimulation frequency and changing muscle length of a frog’s Gastrocnemius muscle was studied. The measured results included muscle response amplitude, tetanus probability, and muscle tension. It was hypothesized that as stimulation voltage and frequency increased, muscle twitch amplitude would increase and muscle tetanus would be more likely to occur. It was determined that maximum twitch amplitude was seen at the highest stimulus amplitude tested. Muscle response changed from twitch, to unfused tetanus, to fused tetanus as stimulus frequency increased. In addition, active tension was also discovered to increase until Lo was reached, and then it decreased; passive tension continually increased. These findings support the hypothesis. These conclusions are important for creating, improving, and supporting rehabilitation techniques for damaged muscles. The study supports the significance of electrical stimulation machines for regaining contraction in muscles that experience atrophy. It also stresses the importance of stretching in order to decrease muscle tension (tightness).

Introduction

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Skeletal muscle is imperative for maintaining homeostasis such as shivering when there is a drop in temperature, providing cushion to one’s bones at points of impact, and serving as an energy reserve. About 40% of the human body is made up of skeletal muscle. Skeletal muscle fibers are divided into three classes based on their contractile speed and metabolic outline: fast glycolytic (FG), fast oxidative/glycolytic (FOG), and slow oxidative (SO). Both FG and FOG fibers have a type of myosin with a high ATPase activity which allows them to quickly hydrolyze ATP and thus contract faster. On the other hand, SO fibers have a type of myosin with a low ATPase activity, resulting in a slower contraction speed. Glycolytic fibers obtain most of their ATP from glycolysis, while oxidative fibers derive most of the ATP from oxidative phosphorylation. The amount of force a skeletal muscle fiber can produce is directly related to its cross-sectional area, so the larger the fiber, the more force it can produce. FG fibers typically have the largest diameter so they create the greatest amount of power; however, FG fibers fatigue quickly because glycolysis is not as efficient as oxidative phosphorylation in producing ATP. In contrast, SO fibers have the smallest diameter so they generate the least amount of power, but they are very resilient to fatigue. Because of these differences, long distance runners have a high percentage of SO fibers, sprinters have a high percentage of FG fibers, and aerobic exercise can lead to the conversion of FG fibers to FOG fibers. Skeletal muscle is made up of a mixture of all three of these fibers. A motor neuron in a skeletal muscle, however, only branches to one type of skeletal muscle fiber. Motor neuron activation occurs on a recruitment basis, that is smaller motor units are activated before larger motor units. SO fibers have the lowest threshold so they are recruited first, then FOG fibers, and lastly FG fibers, which have the highest threshold. In addition, motor units with lower thresholds encompass fewer skeletal muscle fibers than motor units with higher thresholds. As the brain

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increases the number of motor units activated, the number of fibers activated in each motor unit also increases. Skeletal muscle contraction begins when an action potential generated by the neuromuscular junction spreads over the sarcolemma. The action potential then travels down the transverse tubules. At the triad, where the transverse tubule is bordered by the terminal cisternae of the sarcoplasmic reticulum (SR), the L-type calcium channel undergoes a conformational change. This L-type calcium channel is connected to the ryanodine receptor, which opens up and allows calcium to flow out of the SR. Calcium then binds to troponin which causes the troponintropomyosin to move, allowing myosin to bind actin. Myosin undergoes the cross bridge cycle and uses the energy from ATP hydrolysis to pull actin to the center of the muscle fiber, causing the muscle to shorten. The Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) uses energy from ATP hydrolysis to pump calcium against the concentration gradient into the SR. Calcium detaches from troponin, the troponin-tropomyosin complex moves back to its original location, and the muscle relaxes. Another factor that plays a role in the contraction force of a skeletal muscle fiber is summation. Summation is the frequency at which a skeletal muscle fiber is stimulated. If a fiber is stimulated by a single action potential, it will briefly generate tension to cause a twitch, then it will relax. If a fiber is stimulated by two action potentials in rapid succession, and the second action potential fires before the fiber is fully relaxed from the first action potential, the second twitch will summate with the first twitch and result in an even larger tension. Lastly, if many muscle fibers are stimulated in rapid succession, the fiber will produce a tetanus which is a smooth increase in tension to a plateau, followed by relaxation.

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Skeletal muscle contraction calls for an increase in the calcium ions in the muscle cell cytoplasm (myoplasm). As long as the calcium concentration stays high, the muscle will contract, and when the concentration decreases, the muscle will begin to relax. When a single action potential fires on a fiber, the myoplasmic calcium concentration increases very briefly, which is why only a twitch is seen. When stimulation frequency increases, however, calcium and force begin to fuse, which leads to an increase in myoplasmic calcium concentration to a plateau and allows time for the contractile proteins to become fully activated and generate maximal force, resulting in a tetanus. Skeletal muscle force also depends on the length of the muscle. The length tension curve is the relationship between the length of the muscle and the amount of tension produced. When the skeletal muscle is resting at a very short length, there is little tension created when the muscle is activated. As the resting muscle is lengthened towards Lo, the optimal muscle length that yields maximum tension, the generated tension increases. Tension upon activation decreases when the muscle is lengthened past its Lo, but the tension generated by the resting muscle (passive tension) increases. A muscle’s total tension is the sum of its active tension and passive tension. Passive tension is caused by stretching structural proteins within the fiber and connective tissue surrounding the fiber. Skeletal muscles appear straited to the presence of sarcomeres which are organized bands of actin and myosin. The length dependence of active tension is function of sarcomere length because it is determined by the amount of overlap between the thin and thick filaments. At long sarcomere lengths (>2.2 μm), there is less overlap, fewer cross bridges can bind to actin, and force decreases. On the other hand, at shorter sarcomere lengths (2 - 2.2 μm), there is optimal

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overlap, more cross bridges can bind to actin, and force increases. At very short sarcomere lengths, however, actin filaments on opposite ends of the sarcomere collide, so force decreases. Motor impairment, as typically seen following a stroke or injury, is unfortunately very common in today’s world. One’s muscles can go into atrophy and forget how to contract. On the other hand, people can also suffer from constant contraction, as seen in muscle spasms. We believe that contraction/stimulation techniques and stretching can help mediate these disabilities. We hypothesize that as stimulation voltage and frequency increases, muscle twitch amplitude will increase as well as muscle tetanus likelihood. These findings can be used to improve rehabilitation techniques, whether it is electrical stimulation or classic physical therapy.

Methods The overall experimental design involved testing the amplitude of muscle twitches on a frog’s Gastrocnemius muscle at different stimulation amplitudes as well as calculating the amount of stimulation frequency it took to produce a fused tetanus. In addition, the active and passive tension in a frog’s Gastrocnemius muscle was also determined as several muscle lengths. As described in the Muscle Twitch Summation and Length Tension Protocol, a frog’s Gastrocnemius muscle was separated and removed from the lower leg and placed in amphibian ringer’s solution. A thread was then attached to the Achilles tendon below the muscle and hung on the hook of the force transducer. Two stimulating electrodes were attached to a femur clamp and positioned so that each one was touching the muscle, but not each other. The “zeroing knob” on the force transducer was adjusted properly to make the baseline equal to zero.

Exercise 1:

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The aim of this exercise was to determine the relationship between the stimulus strength and the muscle twitch response. As described in the Muscle Twitch Summation and Length Tension Protocol, the pulse amplitude was set to 0.000 V, the number of pulses was set to 1, the pulse width was set to 10ms, and the frequency was set to 0.5 Hz. The muscle twitch response was recorded, and the pulse amplitude was increased by a value of 0.5 until it reached 5.0 V. The Muscle Twitch Amplitude, the difference between the baseline tension of the muscle and the tension at the peak of the twitch, was calculated by placing one cursor at the beginning of the twitch and the second cursor at the peak of the twitch and recording the V2-V1 value for each pulse amplitude. The Contraction Time, the time between the beginning and the peak of the twitch, was measured by keeping the cursors in the same place, but instead recording the T2-T1 value for each pulse amplitude. The ½ Relaxation Time, the time required for twitch tension to fall from the maximal twitch tension to ½ maximal twitch tension, was calculated by placing one cursor at the peak of the twitch and the second cursor at ½ the peak tension on the falling side of the twitch and recording the V2-V1 value for each pulse amplitude. The Latency, the time it takes the muscle to start responding to a stimulus, was calculated by placing one cursor at the beginning of the stimulus pulse and the second cursor at the beginning of the muscle twitch and recording the T2-T1 value for each pulse amplitude. The muscle was then remoistened by the ringer’s solution. These values were used to determine the answer for our hypothesis regarding muscle twitch amplitude.

Exercise 2: The aim of this exercise was to measure the amplitude of contraction produced in a muscle that is stimulated with repeated pulses delivered at progressively higher frequencies. As

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described in the Muscle Twitch Summation and Length Tension Protocol, the pulse amplitude was set to 5.0 V because it caused the maximal muscle response, the number of pulses was set to 15, and the frequency was set to 0.5 Hz. The muscle twitch responses were recorded, and the stimulus frequency was increased to 1, 2, 3, 4, 5, 10, 20, and 30 Hz. The Amplitude of First Muscle Twitch, the difference between the baseline tension of the muscle and the tension at the peak of the first twitch in the series, was measured by placing one cursor at the beginning of the first twitch and the second cursor at the peak of the twitch and recording the V2-V1 value for each stimulus frequency. The Maximum Amplitude in Summation/Tetanus, the difference between the baseline tension of the muscle and the tension at the peak of the tallest twitch in the series, was calculated by placing one cursor at the beginning of the first twitch and the second cursor at the peak of the tallest twitch in the series and recording the V2-V1 value for each stimulus frequency. The Change in Unfused Tension, the difference between the baseline tension of the muscle and the tension at the highest relaxation point between the twitches in the series, was calculated by placing one cursor at the beginning of the first twitch and the second cursor on the highest relaxation point between any pair of twitches in the series and recording the V2-V1 value for each stimulus frequency. The muscle response was then determined to be a twitch, unfused tetanus, or fused tetanus. The muscle was then remoistened by the ringer’s solution. These values were used to determine the answer for our hypothesis regarding muscle tetanus probability.

Exercise 3: The aim of this exercise was to determine the relationship between the muscle length and the amount of tension the muscle produces. As described in the Muscle Twitch Summation and

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Length Tension Protocol, the length of the muscle was shortened until wrinkles appeared. Then, the distance from the base of the ring stand to the bottom of the femur clamp was measured. The stimulus mode was set to pulse, the pulse amplitude was set to 5.0 V because it caused the maximal muscle response, the number of pulses was set to 1, the pulse width was set to 10ms, and the frequency was set to 0.5 Hz. The muscle twitch responses were recorded, and the muscle length was increased by 2-4mm for a total of five trials. The Total Tension (the tension produced at the peak of the twitch), the Passive Tension (the tension generated prior to stimulating the muscle), and the Active Tension (the Total Tension minus the Passive Tension) was calculated for each twitch.

Results Results were collected from Exercise 1, 2, and 3. Overall, as stimulus amplitude increased twitch amplitude increased, as stimulus frequency increased maximum amplitude generally increased, and as length increased total tension increased, to an extent. Data Table 1 shows the amplitude and times of muscle twitches at different stimulus amplitudes. It was seen throughout the experiment that as Stimulus Amplitude increased, the Twitch Amplitude also increased (displayed by Graph 1). The ½ Relaxation Time and Contraction Time both stayed relatively consistent throughout the different stimulus amplitudes. The Latency value decreased as Stimulus Amplitude increased. Data Table 2 shows the strength of muscle contractions during mechanical summation and tetanus. It was generally seen that as Stimulus Frequency increased, both the Amplitude of First Twitch and Maximum Twitch Amplitude increased (displayed by Graph 2). Unfused

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Tension did not appear until the Stimulus Frequency reached a value of 4.0 Hz. The muscle response was a twitch for Stimulus Frequencies 0.5 – 3.0 Hz and an unfused tetanus for Stimulus Frequencies 4.0 – 30.0 Hz. A fused tetanus was actually not presented until the Stimulus Frequency was set to 40.0 Hz. Data Table 3 shows the relationship between muscle length and active, passive, and total tension. It was seen that as the muscle length was stretched past Lo, total tension and passive tension increased while active tension decreased. These trends are displayed in Graph 3.

Discussion The data supports the hypothesis because the largest twitch amplitude is recorded at the highest stimulus voltage, and the muscle response increases towards fused tetanus as stimulus frequency amplifies. Data Table 1 and Graph 1 both reinforce that twitch amplitude increases as stimulus pulse amplitude to the muscle increases. This is due to recruitment because as stimulus amplitude increases, the number of motor units activated increases, resulting in an increased number of muscle fibers activated. Contraction Time and ½ Relaxation Time does not have significant changes in response to an increase in Stimulus Amplitude. Although the amplitude of the twitch increases with a larger stimulus, the twitch does not occur any quicker. The latency slightly decreases as stimulus amplitude increases, but overall the change is not significant. Data Table 2 and Graph 2 both back the hypothesis showing that muscle response increases to the point of tetanus as stimulus frequency increases. The particular frog’s Gastrocnemius muscle is shown to have an unfused tetanus threshold at Stimulus Frequency 4.0 Hz. Although it is not documented in Data Table 2, the muscle does not display a fused tetanus until a Stimulus Frequency of 40 Hz was applied. There is a general trend that Amplitude of First Twitch and

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Maximum Twitch Amplitude increases with increased Stimulus Frequency. This is due to the summation of twitches which make the contraction even greater. A Stimulus Frequency of 0.5Hz is shown to have a rather large Amplitude of First Twitch and Maximum Twitch Amplitude compared to the other values, but this error could be due to a technical difficulty such as the stimulating electrodes being too close to each other. A Change in Unfused Tension does not appear until a Stimulus Frequency of 4.0 Hz because that was the first value to have an unfused tetanus with a relaxation point between twitches. Data Table 3 and Graph 3 both show an interesting finding related to muscle tension. The optimal length of the muscle, Lo, is determined to be at 8.9mm because that is the value with the largest Active Tension. As the muscle stretches past its Lo, the Active Tension decreases because there is less overlap in the sarcomeres. However, as the muscle stretches past its Lo, Passive Tension continues to increase because it is based off of the structural proteins inside the muscle fiber and the connective tissue surrounding the muscle fiber. The Total Tension is seen to consistently increase; however, the trend should be an initial increase, then decrease, then increase. This source of uncertainty could be due to technical issues or our measuring being slightly inaccurate. The lowest Stimulus Amplitude that activates a twitch response that we calculated was 0.5 V. We speculate that a Stimulus Amplitude below this value would not generate a muscle response because the voltage would be too far below the threshold to initiate an action potential. The muscle requires a high stimulation frequency to produce a fused tetanus because, to stay contracted, calcium must be constantly flowing in the myoplasm, so action potentials need to keep firing. During tetanus, the muscle is continually contracting because calcium never has the chance to return back to the sarcoplasmic reticulum, so the calcium pump SERCA does not function.

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These findings are significant for discovering ways to better rehabilitate for motor impairment, especially following stroke or injury. After trauma, it is typical for muscles to experience shock and atrophy. It can be a difficult and frustrating process for the effected person to relearn that connection between the brain and the muscle. However, electrically stimulating the damaged muscle can help regain those lost contractions. As electrical stimulation increases, the contraction amplitude also increases, allowing muscle memory to form again. Electrical stimulation machines, such as TENS and NMES, are established techniques that restore motor function through the use of electrical pulses. Electrodes can be placed on nerves that directly target the specific muscles and cause contractions to reappear. Electrical stimulation techniques, such as TENS and NMES can help facilitate this problem by restoring motor function through the use of electrical pulses. Electrodes can be placed on nerves that directly target the specific muscles...