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Juliana Morales April 14th, 2011 GLYCERINATED MUSCLE FIBER CONTRACTION IS DEPENDENT ON ATP CONCENTRATION Comment [D1]: Note the active title INTRODUCTION Over the course of evolution, many forms of locomotion have evolved. The majority of these forms of locomotion use muscles to pull against a skeleton and produce movement (Raven et al., 2008). The mechanism behind muscles contracting to move the skeleton and produce movements is complex and requires a great deal of energy. Muscle contraction is driven by ATP and occurs when actin and myosin filaments slide past eachother (Rayment et al., 1993). The brain coordinates all sensory and motor activities. When the brain sends an impulse to a muscle to contract the impulse is received at the neuromuscular junction. The impulse causes acetylcholine to be released, which binds to Na+ ion channels and opens them. The opening of the Na+ ion channels causes an influx of sodium into the muscle cell. The influx of Na+ ions into the cell causes the sarcolemma (the membrane of the muscle cell) to depolarize. If the depolarization reaches a threshold, then an action potential is produced. The action potential is then carried to the sarcoplasmic reticulum by the T-tubules. Once the action potential reaches the sarcoplasmic reticulum, Ca2+ ion channels are opened. Ca2+ then binds to troponin and causes a conformational change, which causes tropomyosin to move and expose the myosin binding sites on the actin filaments. Before the myosin fiber can bind to the actin filament at the binding site it must become activated. To become activated, and ATP binds to the myosin head and ATPase hydrolyzes the ATP molecule into ADP and an inorganic phosphate (Pi) which Comment [D2]: The citation structure is correct, but I don’t want you to use Raven (the textbook). I want you to find primary articles. both remain attached to the myosin head. The activated myosin head may then bind to the myosin-binding site on the actin filament. When it binds to the actin the cross-bridge is formed, the Pi is released and the power stroke begins. During the power stroke, the myosin head rotates and pulls the actin filament closer to the center of the sarcomere causing it to shorten and the muscle contracts. After the power stroke the ADP is released, but the myosin remains attached to the actin filament until it hydrolyzes another ATP. This cycles continues until the impulse stops and the Ca2+ ion channels close, causing the myosin binding sites to be blocked by tropomyosin again. (Raven et al., 2008) According to Barclay (2003), a muscle shortens approximately 0.2 nm per ATP Comment [D3]: This paragraph is a cogent explanation of the subject matter without going overboard. Comment [D4]: Note how she is referring to the paper but still CITING it properly. used. Considering the fact that to produce movement a muscle must contract significantly more that 0.2 nm and ATP is also used to relax the muscle, a significant amount of ATP is used during contraction. The principle method of ATP production by the majority of muscle fibers is oxidative phosphorylation (Korzeniewski, 1998). When a motor impulse is received at the neuromuscular junction, it not only indirectly triggers the myosin ATPase to hydrolyze ATP it also triggers the production of ATP in order to support the high-energy demands of the contracting muscle (Korzeniewski, 1998). As stated by Cooke and Bialek (1979), varying concentrations of ATP affect the force generating cycles needed for muscle contraction (cross-bridge formation and the power stroke). In order to test how the force generating cycles needed for muscle contractions are affected by ATP concentrations, we exposed glycerinated muscle fibers from pigs to 5 different solutions of varying ATP concentrations. Glycerinated muscle fibers were used so that neural stimulation and Ca2+ were not needed (Cooke and Bialek, 1979). We Comment [D5]: Notice how this part of the introduction begins to get more specific to the reasons for the scientific question. hypothesized that the highest ATP concentration in a salt solution would cause the most muscle contraction because it would provide the most amount of energy. Comment [D6]: Note the clear hypothesis statement that she was able to incorporate with the flow of the paragraph. METHODS Fifteen microscope slides were obtained and labeled according to what solution Comment [D7]: If the first word of your sentence is a number, spell the number out. would be placed on them (3 slides per each solution). The solutions were prepared by mixing the supplied solutions (ATP in salt solution and ATP in distilled water) with appropriate amounts of distilled water in order to make the following solutions: 0.25% ATP and distilled water, 0.25% ATP and salt solution, 0.10% ATP and salt solution, 0.05% ATP and salt solution. A solution with 0.00% ATP in distilled water was also used. A piece of skeletal muscle from a pig was obtained and separated into 15 very thin (approximately 0.2 mm in diameter) strands using a needle. Forceps were used to transfer the muscle strands to the appropriate slides. The length of the muscle strands was measured in centimeters with a ruler and recorded in a table. While the muscle fibers were observed under a microscope, 5 drops of the appropriate solution were added to the muscle fiber and the contraction of the muscle was observed. After 40 seconds the length of the muscle strand was measured again and recorded. This was repeated for all of the 15 muscle strands. Comment [D8]: Note the clarity and explicit descriptions of her methods. RESULTS Muscle Strand 1 Muscle strand 2 Muscle strand 3 0.25% ATP and Distilled water 0.25% ATP and salt solution 0.10% ATP and salt solution 0.05% ATP and Salt solution 0% ATP and salt solution Length Before Solution 5.1 4.4 3.3 3.8 4.7 Length after solution 2.3 4.3 3.1 3.6 4.7 % contraction 54.9% 2.2% 6.0% 5.3% 0.0% Length Before Solution 3.9 4.8 3.5 4.8 5.0 Length after solution 3.2 3.6 3.4 4.6 5.0 % contraction 17.9% 25.0% 2.9% 4.2% 0.0% Length Before Solution 2.6 3.2 5.0 3.5 2.4 Length after solution 2.8 2.6 4.6 3.5 2.4 % contraction -7.3% 18.8% 8.0% 0.0% 0.0% average % contraction Standard deviation 21.8% 0.312859926 15.3% 0.117886951 5.6% 0.025696952 3.2% 0.027970222 0.0% 0 Table 1. Skeletal muscle fibers from pigs were used. Measurements are given in centimeters and were taken 40 seconds after the muscle fibers were exposed to the various solutions. Prior to being exposed to the solutions the muscle fibers were glycerinated. Comment [D9]: The student has a clearly labeled table with a succinct caption. The two lines under the table is the caption. Average Percent Contraction Average Percent Contraction In Various Solutions 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% -10.0% -20.0% 0.25% ATP 0.25% ATP 0.10% ATP 0.05% ATP and and salt and salt and Salt Distilled solution solution solution water 0% ATP and salt solution Figure 1. Skeletal muscle fibers from pigs were used. Measurements were taken 40 seconds after the muscle fibers were exposed to the various solutions. Prior to being exposed to the solutions the muscle fibers were glycerinated. Comment [D10]: This is what your graphs should look like aesthetically. Clean, white background, well labeled without unnecessary legends… and labeled FIGURE 1,2 etc… On average, the solution that caused the most percent muscle contraction in 40 seconds was the 0.25% ATP solution in distilled water, but the standard deviation was very high (Figure 1). The 0.25% ATP in salt solution produced the second highest percent of muscle contraction on average, followed by the 0.10% ATP in salt solution, Comment [D11]: In the Results paragraph the student basically does fact stating but refers to the tables and figures when doing so. the 0.05% ATP in salt solution, and 0% ATP in salt solution (Table 1). The 0% ATP in salt solution did not cause the muscle to contract at all (Table 1). DISCUSSION Our results both contradicted and supported our hypothesis that the highest ATP concentration in a salt solution would cause the most muscle contraction because it would provide the most amount of energy. When muscles contract, the force generating cycles require a large amount of energy in the form of ATP (Cooke and Bialek, 1979). In order Comment [D12]: In the case of ambiguous results the student addresses the hypothesis accordingly. to accommodate for the significant increase in ATP usage between a resting muscle and a contracting muscle when a motor impulse is received at a neuromuscular junction the production of ATP by oxidative phosphorylation is triggered (Korzeniewski, 1998). Thus, the amount of force generating cycles and the amount of contraction is limited by the amount of ATP present in a muscle cell when an action potential is generated. Research of this kind is important because it could give us a better understanding of how muscle uses ATP and it could possible give insight to muscle fatigue and diseases affecting the contraction of muscle. Comment [D13]: Good re-explanation of the theory Our results support the majority of the research previously conducted on this topic. In a study done by Cooke and Bialek in (1979) it was shown that the velocity of glycerinated muscle contraction increased with ATP concentration. A study conducted Comment [D14]: Here, the student incorporates her work into other research by He et al. (2000) on human muscle fibers also supports our results. They found that during the shortening phase of muscle contraction the rate of ATP consumption increased proportionally to the shortening velocity. These findings are consistent with our results because when ATP concentration was increased, there was also an increase in the average percent of muscle contraction. This is comprehensible because the only factor limiting muscle contraction in both our system and the system used in Cooke and Bialek’s experiment was ATP due to the muscle being glycerinated and not needing an electrical impulse or Ca+ ions to contract. Therefore, the more ATP present, the more the muscle was able to contract. As previously stated, our results both contradicted and supported our hypothesis. We predicted that the highest concentration of ATP would cause the muscle to contract the most. This part of our hypothesis was consistent with out results because the solution that caused the highest average percent of contraction was the 0.25% ATP in distilled water. We also predicted that the solution that caused the muscle to contract the most on average would be a salt solution. This prediction was not supported by our results. However, the solution that we predicted to cause the most muscle contraction, 0.25% ATP in salt solution, caused the second highest average percent of contraction and had a significantly smaller standard deviation than the 0.25% ATP in distilled water solution. The percent muscle contraction for each trial in the 0.25% ATP in salt solution and 0.25% ATP in distilled water were not precise and they were erratic, while the percent muscle contraction for the 0.10% ATP in salt solution, the 0.05% ATP in salt solution and the 0.0% ATP in distilled water solutions were relatively precise and consistent. The third trial of 0.25% ATP in distilled water actually got longer by 7.3%. The impreciseness of the results of the 0.25% ATP solutions could be attributed to sources of error. One possible source of error is that students prepared the ATP solutions and there could have been contamination or miscalculations while they were preparing the solutions and while the solutions were being applied to the muscle fibers. Our results do allow us to conclude that muscle contraction increases with increasing ATP concentrations, but due to the impreciseness, great variability between trials and relatively small difference between the average percent contraction of both 0.25% solutions, we cannot conclude that the salt solution vs. the distilled water made a significant difference in the results. Comment [D15]: Mentioning of error is important REFERENCES Barclay, C. 2003. Models in which many cross-bridges attach simultaneously can explain the filament movement per ATP split during muscle contraction. International Journal of Biological Macromolecules. 32:139–147. Cooke, R, and Bialek, W. 1979. Contraction of Glycerinated Muscle Fibers as a Function of the ATP Concentration. Biophys. J. 28:241-258. Korzeniewski, B. 1998. Regulation of ATP supply during muscle contraction: theoretical studies. Biochem. J. 330:1189–1195. Rayment, I, Holden, H, Whittaker, M, Yohn, C, Lorenz, M, Holmes, K, Milligan, R. 1993. Structure of the Actin-Myosin Complex and Its Implications for Muscle Contraction. Science. 261:58-65. Raven, P., H., Johnson, G., B., Losos, J., B., Mason, K., A., Singer, S., R. 2008. Biology., 9th Edition. Pearson Education, Inc., CA. Comment [D16]: You can use Microsoft’s reference style, but ensure that you are consistent.