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Analysis of the Glycerinated Muscle Fiber

The goal for this lab was to determine the conditions that demonstrate what chemicals in muscle fibers are necessary for contraction and which ones prevent muscle contraction from occurring in a simplified system in order to determine the minimum requirement for contraction.

Methods

In order to prepare muscle fibers, a single thread was obtained from a mass of glycerinated muscle fibers which was about 0.5 mm in diameter. The muscle was a rabbit psoas muscle in 50% glycerol (stored at -10 degrees C) obtained from Carolina Biological. In order to have an observation of what was taking place with the muscle, Nikon E400 was used. The fiber was placed on a slide in the presence of 0.05 M KCl and 0.005 M K phosphate buffer (pH 7). The first step was to detect the minimum requirement solution for muscle contraction to take place. This was done by using the following solutions: 0.001 M MgCl2, 0.001 M CaCl2, and 0.1 M ATP. The requirement was determined after observing the changes taking place when each of the solutions were added to the muscle separately, in combinations of two, and all three solutions together.

After the minimum requirement was determined, chelators were used to see whether they inhibited contraction in the presence of the solution that caused contraction. The chelators were 0.002 M EDTA and 0.002M EGTA. The EDTA binds Ca ++ and Mg ++. EGTA binds only Ca++. The chelating agents enhance the solubility of magnesium and calcium and allow them to spread out of the muscle fibers. This causes the removal of the ions from the actin and myosin environment. It was important that the chelators were added before the contracting agents when inhibition was being tested. Otherwise, it would be impossible to detect any changes once the contraction has taken place; contraction is not reversible once it occurs in a simplified system.

Then Solution A and Solution B were used to examine the localization of myosin and actin in the myofibrils. Solution A’s function was to solubilize and remove myosin in the form of monomers from its loci which is in the myofibril. Solution B had the same behavior as the actin. The Solutions were placed on the myofibril to see changes that took place and observations were made.

Table 1: Solutions used to determine the minimum requirement for muscle contraction and the observations made

Solutions

Contraction Occurring

One dimensional

Mg2Cl2

No

- the fiber stayed the same in size, color, and shape

No change

CaCl2

No

No change

ATP

Yes

The fiber shortened in length and became darker yellow- brownish color

Not one dimensional

MgCl2 + ATP

Yes

Fiber shortened and color changed to very dark brown

Very fast

Faster than ATP

Seemed to be faster than Ca2Cl2+ATP

Not one dimensional

Ca2Cl2 + ATP

Yes

Fiber shortened and color changed to dark brown

Slower than MgCl2 + ATP; but still fast

Faster than ATP

Not one dimensional

ATP+ Mg2Cl2 + CaCl2

Yes

Fiber shortened in length and color became darker

Fast contraction

Not one dimensional

Table 2: Using the minimum requirement, ATP, and the chelators, EDTA and EGTA, determine whether inhibition would be caused in presence of contraction initiating solution (ATP)

Chelators

Inhibition

One Dimension

EDTA + ATP

No

Contraction still took place

Not one dimensional

EGTA + ATP

No

Contraction occurred

Not one dimensional

EDTA + EGTA

No

Contraction occurred but was slower than when ATP was present

Not one dimensional

Table 3: Solution A and Solution B effects on actin and myosin

Solutions

Observations

Solution A

- 0.6 M KCl

- 0.1 M K phosphate buffer, pH 6.5

- 0.001 M Na pyrosphosphate

- 0.001 M MaCl2

No contraction

The color became lighter yellow

Solution B

- 0.6 M KI in 0.1 M Phosphate Buffer pH 6.5

No contraction

The color became lighter

Discussion and Conclusion

During this lab, using the microscope we examined the changes that took place when certain solution were introduced to the rabbit psoas muscle fibers. The solutions caused contraction, inhibited contraction to occur, or had no effect on the sarcomeres at all. We used glycerinated myofibrils from rabbit psoas muscle which is a type of striated muscle. Rabbit psoas muscle was a good model to use for this lab since the fibers are long and straight. Also one other advantage was that there were not a lot of connective tissues connecting the muscle fibers together. This was an in vitro model meaning that experiment was completed outside the living organism.

The Phase Contrast with magnification of 10X/ 40X was used during this lab to examine the slide because the cells are transparent and Phase Contrast is the best option to use in order to have a good resolution. Under a microscope the myofibers were striated and they had a repeating pattern of bands and lines. The pattern was caused by parallel organization of protein filaments within the myofibrils. In the myofibril, there are two types of filament- the thick filaments which consist of the protein myosin and thin filaments composed of the protein actin.

As demonstrated on Table 1, Mg2Cl2, Ca2Cl2, and ATP were the solution used to determine the changes taking place with the muscle. All of the three solutions were placed on the slide which consisted of a thread of muscle fiber and contraction of the muscle was observed. We could tell when contraction was taking place because the fiber was short in length and it was easy to see the changes such as the shortening in length and the color change. When Mg2Cl2 and Ca2Cl2 were added individually to the fiber, nothing happened. However, as soon as the ATP was placed on changes were easily observed. ATP caused muscle contraction by itself. The sarcomeres in the muscle fiber shortened in length and the color changed from light yellow to darker yellow.

However, in order to make sure this was the minimum requirement for muscle contraction, we added Mg2Cl2 with ATP and Ca2Cl2 with ATP.

With the MgCl2 and ATP, the contraction occurred immediately as the solutions were added. The contraction was even faster than the ATP alone. Then ATP and Ca2Cl2 solutions were introduced and this also caused contraction. Even though the combination of the two solutions caused contraction to occur faster than ATP by itself, it was slower than the solutions of ATP and Mg2Cl2. As a result, we concluded that ATP was the minimum requirement needed for the cells to contract. All the solutions that caused contraction were not in one dimension because every component of a sarcomere was facing changes except the A band which stayed the same. The I bands, the M line, the Z lines, and the actin and mysosin- they were all decreasing in length in order to cause contraction. At the end, it was determined that ATP was the requirement for glycerinated muscle contraction. When there is no ATP present, the myosin heads in the muscle will not be activated and it would not bind to the actin. In glycerinated tissues, the combination of KCl and MgCl2 with ATP increased the strength of muscle contraction. This was mainly due to myosin’s high affinity for these ions.

Table 2 shows whether contraction was inhibited in the presence of chelating agents, EDTA and EGTA, when it was used with the main solution that caused contraction, ATP. EDTA and EGTA did not inhibit contraction from taking place, but the contraction was slower than when ATP was present. EDTA is a chelating agent that binds Ca++ and Mg++ and EGTA is a chelating agent that binds Ca++. The chelating agents increase the solubility of Mg 2+ and Ca2+ so that they can leave the muscle fibers. With ATP and the chelating agents, contraction occurred and there was no inhibition taking place.

Table 3 shows two different solutions, Solution A and solution B, and their effect on actin and myosin. As shown in the table, solution A had KCl, phosphate buffer, Na pyrophosphate, and MgCl2 while solution B had phosphate buffer. Both of these solutions did not cause any contraction in the muscle based on our observations. However, changes were observable because in both cases the fibers changed color; they became lighter yellow. This meant that the muscles were not contracting. Solution A made the mysosin more soluble and solution B acted in the same manner as the actin.

When comparing the muscle in living tissue, the glycerinated muscle system is different. The glycerination technique eliminates ions and ATP from the tissue and disrupts the troponin/tropomyosin complex. When the complex is interrupted, the available binding sites on the actin fibers are no longer blocked ( Cell and Molecular Biology). As a result, Ca2+ is not one of the requirements to cause contraction. On the other hand, since there is no ATP is in the glycerinated tissue, the myosin heads cannot be activated to cause contraction. After the muscle contracted it did not relax since there were no opposing muscles to pull it. Also, muscle fibers do not contract when there are no stimulations or nerve signals and this was one of the differences with glycerinated muscle and the living cell muscle.

Errors could have occurred in this lab if one used very thick muscle strands. Having thinner strands were better to have good results. Also, it is possible that much of the calcium was still in the sarcoplasmic reticulum of the glycerinated muscle, which would have lead into incorrect results. All in all however, the lab was successful and we have obtained what we were looking for- what solutions cause contraction, which ones inhibit, and what is the minimum requirement for muscle contraction.

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