Pathophysiological Effects of ACL (Agkistrodoncontortrix laticinctus) Myotoxin on Mammalian Skeletal

Muscle in vitro

A Senior Paper Presented to

The Department of Biologyof Oral Roberts University

In Partial Fulfillment Of the Requirements for the Degree

Bachelor of Science

by Genard HajdiniDecember 1999

Dr. Steffan Anderson

1

IntroductionACL myotoxin is a protein isolated from the venom of the broad-banded

copperhead, Agkistrodon contortrix laticinctus. It is myotoxic, meaning that it has "direct

and specific action" on skeletal muscle (Fletcher et al. 1997). Snake venom myotoxins

are a large group of toxins that cause several types of necrosis in skeletal muscle cells

(Johnson and Ownby 1994). Snake venom myotoxins structurally belong to a larger

group of proteins, the PLA2 (phospholipase A2) family (Selistre de Arajo et al. 1995), but

ACL myotoxin lacks the properties necessary to be part of that class (Johnson and

Ownby 1993). ACL myotoxin has a pI greater than 9, is a single polypeptide (fig 1),

having 126 amino acid residues (fig 1 & 2), and has a molecular weight of 14.5

kilodaltons (Johnson and Ownby 1993; Li et al. 1993).

ACL myotoxin is a phospholipase A2 like protein. It lacks characteristic PLA2

enzymatic activity, but has similar structure and resultant necrosis after intramuscular

injection. PLA2 myotoxins are split into two different groups, based on the presence or

absence of an aspartic acid residue at position 49 (Fletcher et al. 1997). Position 49 is

highly conserved among the more enzymatically active PLA2 molecules (Selistre de

Araujo et al. 1996). When a lysine (K) is replaced for the aspartic acid (D) at position 49

it results in a large reduction, or elimination of the enzymatic activity of the protein

(Fletcher et al. 1997). ACL myotoxin has substitution of D by K in position 49 (fig 3).

Some other researchers say that this substitution completely stops enzymatic activity, and

that any small amount of observed enzymatic activity can be contributed to

contamination by D49 PLA2's in the same venom (Fletcher et al. 1997). Ca2+ is not

present in the active site of the K49 substituted protein, which is in contrast to the D49

protein. It is also proposed that the K49 substitution hinders Ca2+ from binding to the

active site of the protein. This inability to bind may be because of steric hindrance of

K49, or the electrostatic repulsion of Ca2+, due to the same positive charge of the K.

2

Aspartate, however attracts the Ca2+ because of its negative charge. In addition, it does

not sterically hinder Ca2+, because of its small size, relative to K.

.

Figure 1. Space filling model of ACL myotoxin. (Selistre de Araujo et al. 1996)

3

Figure 2. Single polypeptide structure of ACL myotoxin, showing K49 subsitution of the

D49 residue. (Selistre de Araujo et al. 1996)

This toxin has an interesting way of disabling the muscle into which it is injected.

Myonecrosis, or localized muscle cell tissue death, is evident within five minutes after

the injection of the toxin (Johnson and Ownby 1993). The predominant pattern of

myonecrosis induced by ACL myotoxin is characterized by clumped myofilaments in

damaged muscle cells. However, it is possible to see two other pathological changes,

which are: clear vacuoles, and disorganized myofibrils (mottled appearance) (Johnson

and Ownby 1994) (Fig 3). The main pathological change, which is a pattern of densely

clumped myofibrils in the muscle cells, is the result of hypercontraction of myofilaments

(Johnson and Ownby 1994). The hypercontraction suggest elevated levels of free

intracellular calcium (Harris and MacDonnel 1981). Mechanisms to explain these high

calcium levels are proposed later in this paper. The second pathological change, the

appearance of clear vacuoles is what is visible on the light microscopic level. A closer

look through electron microscopy, reveals that this is characterized by swollen

4

Surce: Fl In: Venom M., Ed., 1997

sarcoplasmic reticulum. The swollen sarcoplasmic reticulum and disorganized myofibrils

are likely due to an influx of water into the cytoplasm which may be due to osmotic

swelling (Johnson and Ownby 1993). The disruption of the sarcolemma may eliminate

the permeability barrier of muscle cells, allowing for an influx of various ions, such as

sodium or calcium (Johnson and Ownby 1993). This influx of ions is what is probably

responsible for the osmotic swelling that causes the swollen sarcoplasmic reticulum.

This kind of swelling leads to the final observed pathological change. The swelling of

the cell may expand the cytoplasm surrounding the myofibrils to the point where

myofibrils are physically forced apart. Such a swelling may also produce shear forces

that result in breakage of some of the myofibrils (Johnson and Ownby 1993). The

forcing apart and breakage of the myofibrils result in the observation of myofibril

disorganization.

Figure 3. Electron microscopic observed changes induced by ACL myotoxin. CM

shows where disorganization of the myofibrils has occurred. V shows where it is

vacuolated, and M shows the myofibril clumping. (Fletcher et al. 1997)

5

The morphology observed is quite similar to the action of other snake venom

myotoxins (Johnson and Ownby 1994). ACL myotoxin's molecular weight, amino acid

composition, lack of phospholipase A activity, and the type of myonecrosis it induces, is

similar to myotoxin II from Bothrops asper venom, and a myotoxin from the venom of B.

nummifer (Johnson and Ownby 1993). This means that they may be chemically similar

and therefore have a similar mechanism of action. Notexin, crotoxin, and myotoxin I

cause the same observed myofibril clumping as ACL myotoxin (Johnson and Ownby

1993). Bothropstoxin, from the venom of Bothrops jararacussu is also similar to ACL

myotoxin in molecular weight, number of amino acid residues, type myonecrosis it

induces, and that it lacks phospholipase A activity (Rodrigues-Simioni et al. 1995).

Myotoxin a, another non-phospholipase A2 toxin, shows similar pathophysiological

effects upon its injection into skeletal muscle (Hong and Chang 1985; Homsi-

Brandeburgo 1988).

A mechanism has been proposed, which explains the pathological action of ACL

myotoxin. Current research suggests that the general mechanism of action is that there is

an unregulated influx of Na+ into the sarcoplasmic reticulum, which causes a continuous

efflux of Ca2+ from the sarcoplasmic reticulum into the cell. Specifically, the unregulated

Na+ influx is proposed to be because the ryanodine receptors and dihydropyridine

receptors are adversely affected by ACL myotoxin. Finally, the high levels of Ca2+

possibly lead to the activation of intracellular proteases or cytoplasmic enzymes.

The general mechanism for the pathological action of ACL myotoxin is that there

is an unregulated influx of Na+, into the SR (sarcoplasmic reticulum), which leads the

continuous release of Ca2+ from the SR into the cell. As stated previously, the

observation of hypercontraction of the myofibrils suggests that Ca2+, normally stores in

large amounts in the SR (Ruegg 1988), is at an elevated level inside the cell. This Ca 2+ is

released into the cell through the terminal cisternae of the SR (Marieb 1998). It is

6

released upon depolarization of the SR, by the arrival of an action potential, from the T-

tubule system of the muscle cell (Marieb 1998). In normal situations the depolarization

of the SR involves a transient increase in its permeability to Na+ (Marieb 1998). The

possible, uncontrolled release of Na+ into the cell, due to the disruption of the SR by ACL

myotoxin, may depolarize the sarcolemma and prevent its later repolarization. This leads

to an unregulated release of Ca2+ from the SR (Johnson and Ownby 1994). The release of

the Ca2+ would be continuous, and thus leading to constant contraction of the myofibrils,

which is the observed myofibril clumping. The influx of Na+, drawing water with it into

the SR, causes the swelling, which is the observed clear vacuoles. As stated earlier the

swelling leads to disorganization of the myofibrils. Ultimately these pathological

changes caused by the proposed mechanism kill the myocyte.

The proposed mechanism for the large influx of Na+ into the cell is that ACL

myotoxin disrupts the ryanodine and dihydropyridine receptors. Fletcher et al. (1997)

suggested that these two membrane-bound ion channels (ryanodine receptors and

dihydropyridine receptors) could be affected by ACL myotoxin through three different

mechanisms. (1) There is a binding site on the channel protein for the toxin or a product

of enzymatic activity (e.g. fatty acids) (2) There is a specific fatty acylation site (e.g. a

cysteine residue) on the ion channel involved in acyltransferase-mediated modulation of

channel function (note that the Na+ channel is an acylated protein (Bizzozero et al. 1994))

(Fletcher et al. 1997). (3) Finally, a possible indirect action of the toxin or lytic products

with the membrane, that is transmitted sterically to the ion channel (Fletcher et al. 1997).

There are two proposed mechanisms for how continuous Ca2+ efflux contributes

to myonecrosis. Note that the efflux of Ca2+ into the cell is from the large influx of Na+

into the SR. This elevated level of Ca2+ results in the first mechanism proposed, which is

that it causes excessive excitation-contraction coupling of the myofibrils. This may be

observed microscopically as the clumped myofibrils. The other suggested result of

7

excessive intracellular Ca2+ is that it activates intracellular proteases or cytoplasmic

enzymes that contribute to the destruction of the muscle cell (Fletcher et al. 1997).

Studying the mechanism of action of ACL myotoxin is useful for a number of

reasons. If the mechanism of action is conclusively discovered, it may lead to improved

treatment of snakebite. ACL myotoxin's high degree of homology to other myotoxins is

important because it means that it may be possible to develop an antivenom capable of

neutralizing the tissue damage produced by a large number a snake species. In addition,

it may be possible to antagonize the myonecrosis produced by this toxin using a non-

antibody treatment, if its mechanism of action is determined. Another reason to study the

mechanism of action of ACL myotoxin is that it parallels the effects of muscular

dystrophy. Muscular dystrophy is a genetically acquired disease that is characterized by

the slow death of the muscle tissue. Although a much greater amount of time is needed

for it to occur, in muscular dystrophy the muscle cell goes through changes similar to that

produced by ACL myotoxin. ACL myotoxin's mechanism of action could be used a

model for the mechanism of action of muscular dystrophy. If the two mechanisms really

are similar, it means that finding a way to neutralize one, possibly gives the way to stop

the other. Research on the mechanism of action of ACL myotoxin and other myotoxins

is conducted with much greater ease than that of muscular dystrophy. This makes them a

valuable research tool for revealing the cure to this disease.

Our physiological research on muscle that has been injected with ACL myotoxin

was done because it had not been previously experimentally determined that it results in a

physiological change of that muscle. It is our hypothesis that ACL myotoxin alters the

physiological characteristics of skeletal muscle. This hypothesis was selected because

current research indicates histological changes in the muscle tissue. The histological

changes to the muscle cell are clear vacuoles, disorganized myofibrils, and clumping of

the myofibrils. The clumping of the myofibrils is the most important change, however

the clear vacuoles indicate a change that is nearly as important. The relative merit of

8

these changes arise from the way they support the proposed mechanism of action.

Structure and function are directly related, which means that if the structure of the muscle

cell is altered, there must be an alteration of function. Our experimental results further

supported the hypothesis. Muscle treated with ACL myotoxin showed a lower threshold

stimulus, a shorter latent period, and a smaller maximal stimulus. All three of these

characteristics imply a change in the resting membrane potential of the cell. A change in

the resting membrane potential, means there will be physiological change.

To study the pathophysiological effects of ACL myotoxin, and to hypothesize on

the mechanism of toxin, an optimal muscle group must be selected. The muscle that was

used for this study was the soleus/gastrocnemius. This muscle group was selected

because it is sensitive to the toxin, relatively easy to dissect, and fairly large in

comparison to other muscles in the frog. Since the soleus/gastrocnemius is large relative

other frog skeletal muscles, weighing 3.5g or more, it gives a more visible response to

electrical stimuli. Small responses are difficult to work with, so a large response allows

greater ease of observation. The disadvantage of using the soleus/gastrocnemius was that

it makes it necessary to use an excess of toxin. In addition to the soleus/gastrocnemius,

the EDL (extensor digitorum longus), or rectus femoris could have been used. Some

researchers have used the EDL (extensor digitorum longus) because of its great

sensitivity to the toxin, however the EDL was not chosen because it is difficult to isolate.

The rectus femoris has venom sensitivity comparable to the soleus/gastrocnemius, and is

similar in size, however it is not as easily isolated.

This study involved testing the physiological effects of the toxin on the muscle.

The specific physiological change that we were looking for was an alteration of the

resting membrane potential. To observe this, four different tests were run on the muscle.

They were the determination of threshold voltage, maximal stimulus, latent period, and

tetanic frequency. These tests are designed to test the hypothesis by revealing whether or

not the membrane potential is different.

9

A test of the threshold stimulus indirectly reveals the resting membrane potential

of the cell. The threshold voltage is the minimum voltage that must be applied to the

muscle, to evoke a minimal twitch tension from the muscle (West 1990). If the proposed

mechanism for the action of ACL myotoxin is correct, the Na+ channels in the membrane

will be opened and the resting membrane potential will be closer to the threshold voltage.

The test of threshold stimulus will reveal this by the way a smaller stimulus is necessary

in order invoke a twitch in the toxin treated muscle, than in the control muscle. If the

threshold stimulus for the treated muscle were greater than the control, this would

indicate a change in the membrane potential away from threshold. The second test to

reveal physiological change in the muscle is the determination of maximal stimulus. In

every muscle there is a maximal stimulus that can be applied, producing an increase in

contractile force (Marieb 1998). The maximal stimulus is the point at which a stronger

stimulus will not cause a stronger contraction. A deviation from the normal value for this

test would reveal that a physiological change has occurred. If the resting membrane

potential has been changed toward threshold, there will be a smaller maximal stimulus.

A lower maximal stimulus also indicates a smaller number of possible motor units. A

larger value for the maximal stimulus is indicative of a change in membrane potential

away from threshold.

The third test of test of the hypothesis is latent period determination. The latent

period is the first few milliseconds following the stimulus when excitation-contraction

coupling is occurring, and no contraction is observed (Marieb 1998). In this test a

maximal stimulus is given to the muscle, and the time period from the administration of

the stimulus to the response, is measured. A shorter latent period is indicative of a

change in the membrane potential toward threshold. The opposite is true when the latent

period is longer. The longer the amount of time it takes for the membrane to

depolarization to reach threshold, the longer the amount of time it will be before

excitation contraction coupling, and shortening of the sarcomere occurs. It will take

10

longer to reach threshold if the resting membrane potential is further away from

threshold, relative to another cell.

The final test of the hypothesis was the determination of tetanic frequency.

Tetanus is a smooth, sustained, contraction, because of rapidly repeating stimuli. This

test is important because its value should not be affected by the resting membrane

potential. The value of the tetanic frequency will change if the ability of the SR to uptake

Ca2+ is altered An increased ability of the SR to uptake Ca2+ from the cell, will result in a

higher tetanic frequency. A decrease in Ca2+ uptake ability, will lead to the opposite

affect. If, upon injection of ACL myotoxin into the muscle, the tetanic frequency

changes, it will indicate a physiological change which is not in agreement with the

proposed mechanism.

ACL myotoxin comes from the venom of Agkistrodon contortrix laticinctus. It is

a PLA2 like protein, has a high degree of homology to other snake venoms, and causes

myonecrosis of the muscle cells it is injected. There is a proposed mechanism for the

action of ACL myotoxin. The mechanism that is proposed is that there is disruption of

the SR, leading to high levels of Ca2+ in the cell. This influx of Na+ is the result of ACL

myotoxins affect on the ryanodine and dihydropyridine receptors. Finally, the high levels

of Ca2+ in the cell leads to the activation of intracellular proteases or cytoplasmic

enzymes. The purpose of this study was to aid in the revelation of a treatment for snake

bites, and to help find the mechanism of action of muscular dystrophy. It is our

hypothesis that there will be physiological changes in the muscle after injection of the

toxin. This hypothesis was tested using four different experiments. They were the

determination of threshold stimulus, maximal stimulus, latent period, and tetanic

frequency.

11

Materials and Methods

Chemicals and Reagents

Dr. Ownby (Oklahoma State University, USA) provided the purified ACL

myotoxin. The ACL myotoxin was diluted in frog ringers solution from the stock to

1g/l. Just prior to the experiment, this solution was further diluted in ringers solution to

the injection concentration of 1g/µl. The injection volume of 100µl was injected into

the soleus/gastrocnemius. The control muscle preparation received the ringers solution,

while the treatment preparation received the toxin dilution. The muscle preparations

were also bathed in ringers solution to prevent them from drying out. The composition of

the frog ringers solution (in mM) was: NaCl 111, KCl 1.9, CaCl2 1.1, NaHCO3 2.4, and

1000ml of DH2O.

Muscle Preparation

The experiment was done on frogs, Rana pipiens, purchased from Carolina

Biological supply company. The frogs were killed using the pithing technique. This

technique was employed because the heart is still beating, providing a normal blood

supply to the muscle for an extended time period. The dissection, isolation, and

mounting of the soleus/gastrocnemius muscle preparations were done following the

technique outlined by the Pflanzer manual (1989).

After the dissection and isolation of the soleus/gastrocnemius muscles from both

legs they were mounted side by side, on two different femur clamps. One was used as a

control and the other for toxin treatment. In addition they are connected to two different

12

leaf-spring force transducers. This allows for the simultaneous conductance of the

experimental tests.

Physiological Recording Systems

The two physiographs used for recording the response of the muscles were the CB

Sciences PowerLab 200 (ADInstruments, Castle Hill, Australia), and the Gilson Model

56H Polygraph. The software application program used by PowerLab was Chart, which

is designed by (Adams et al. 1998). The polygraph was set up according to the

instructions provided by Ph.D. Richard G. Pflanzer in his manual for both animal and

human physiology (1989). The instruments used to transform the mechanical energy of

muscle contraction to an electrical stimulus, were semi-isotonic leaf spring force

transducers. The model used was model 76613 Force Transducer. Although the muscle

contractions were isotonic, it is helpful to be able to adjust the initial tension using a

Model 76613-T Tension Adjuster (Pflanzer 1989). The stimulator used in this

experiment was a Grass SD4 stimulator. The electrical impulse was given to the muscle

through needle electrodes placed into the muscle, close to the knee.

Test parameters and procedures

In order to conduct the experimental tests, certain parameters had to be set. The

test for threshold stimulus determination was done using the PowerLab. The stimulus

had a duration of 20ms, and paper speed of Chart was 10 mm/s. Single stimuli impulses

were given with an voltage increasing from 0.1- 0.7V, by increments of 0.02V, and from

0.7- 1.0V by increments of 0.5V; until the muscle responded by contracture. The test for

13

maximal stimulus was also done using the PowerLab system. Stimuli and paper speed

conditions were the same as the threshold stimulus test. This test had continuous increase

of the voltage in two, five, or ten volt increments until maximal stimulation was

observed. The latent period test was conducted using the polygraph. This test had its

conditions of voltage set at 10V, paper speed at 100mm/s, and stimulus duration of 20ms.

The single stimulus was given every four seconds. The final test performed was the

determination of tetanic frequency. This test was accomplished using the polygraph, by

setting the stimulus duration at 20ms, and the voltage at 10V. The frequency was

gradually increased starting from 1.0Hz, until tetanic frequency was observed.

14

Results

There were four physiological tests done on the frog skeletal muscle. They were

a determination of threshold stimulus, maximal stimulus, latent period, and tetanic

frequency. The first test, threshold stimulus, is shown in figure 4. This figure shows how

the control shows a greater threshold stimulus than the ACL myotoxin treated muscle.

The statistical results of this test are summarized in table no 1. The control muscle

normally showed a threshold stimulus of 0.631 ± 0.274V, however the toxin treated

muscle showed a lesser value of 0.377 ± 0.104V (n = 7). The students t-test run on the

data showed that these results are significant (P< 0.05). The first t-test is based on the

comparison of the control mean and the experimental mean. The second t-test was

arrived at a little differently. The value for the mean of the % control is based on its

percent of the mean control. However, the value for the % experimental mean is based

on the comparison of the individual experimental results to the mean control. The mean

values for the % control and the % experimental were then compared, which is how the

value for the second t-test was determined.

Table 1. The statistical results of the test for determination of threshold stimulus.(n = 7, P< 0.05)

Threshold stimulus Me

an

S

D

Va

r

T-

test

Control 0.631 0.274 0.075 0.026

Exp. 0.377 0.104 0.011

% Control 99.9999 43.347 1878.95 0.0259

% Exp. 59.73 16.40 268.97

15

control

toxin

Figure 4. An example of threshold stimulus determination. The stimulus is clearly earlier in the toxin treated muscle.

The second test, which was the determination of maximal stimulus did not turn

out. The experimental design for this experiment was good, but the results we obtained

indicated that a mistake had been made in laboratory technique or experimental

procedure. The extreme wide variation of the results, led to the decision that this

particular experiment failed.

Determination of the latent period was the third experiment. It was observed that

is a significant decrease in latent period for the experimental muscle. Figure5 illustrates

how the treated muscle shows the characteristic, shorter latent period. The statistical

results of this test are shown in table no. 2. The mean time for the latent period in the

control muscle is 28.32± 6.78ms, and is 22.64± 9.04ms in the experimental muscle (n =

30). The students t-test run of this data showed that the results are significant (P< 0.05).

16

These statistical tests were obtained by comparison of the means of the control and

experimental values.

Table 2. The statistical results of the determination of latent period. (n = 30, P<

0.05)

Latent

Period

Me

an

S

D

Va

r

T

-test

Control 28.32 6.78 45.977 0.0078

Experimental 22.64 9.036 81.657

17

Control

ACL-treated

Figure 5. Physiograph recording of the latent period of contraction. The bottom straight

line signifies the event marker, and the curved lines are the shortening of the muscle cell.

The final test that was performed was the test to determine the tetanic frequency.

The results indicated that there was no significant change in this value observed in the

18

treated muscle. The control muscle had a mean frequency of 4.9± 0.8 pps (n = 7). The

experimental had a mean of 4.75± 0.689 pps (n = 7), which is very similar to the control

muscle. Table 3 shows the statistical results of this test. The first t-test performed is

Table 3. The statistical results of the determination of tetanic Hz. (n = 7, P< 0.05)

Tetanic Hz Me

an

S

D

Va

r

T

-test

Control 4.9

17

0.

801

0.6

42

0.

354

Experimental 4.7

5

0.

689

0.4

75

% Control 100 1

6.291

26

5.4

0.

357

%

Experimental

97.

096

9.

099

82.

797

based on the comparison of the mean values of the control and experimental muscles.

The second t-test is based on the comparison of the mean values for % control and %

experimental. The comparison of the individual controls to the mean of the controls, is

expressed as the % control mean. However, the comparison of each experimental value

to its control, is expressed as % experimental. The individual % experimentals are

combined to give the % experimental mean.

19

Discussion

The results of the tests conducted conclusively lead to the acceptance of the

hypothesis. The hypothesis was that there would be physiological changes in the muscle

after the injection of ACL myotoxin. The tests to determine threshold stimulus, maximal

stimulus, latent period, and tetanic frequency were conducted in order to test the

hypothesis. The test for the threshold voltage of the experimental muscle supported the

hypothesis. The muscle that was treated with the toxin had a lower threshold stimulus.

This indicates that the membrane was depolarized. As other literature states, this is due

to the way Na+ channels are effected. The proposed mechanism of action of the ACL

myotoxin that the disruption of the SR allows a large influx of Na+, would cause the type

of depolarization that was observed.

The tests we did for the maximal stimulus where a failure. They were designed to

show that the treatment muscle might have a lower resting membrane than the control

muscles. This theory would have been supported by the observation of a lower maximal

stimulus. The results that were obtained did not have any consistency, ranging from

values less than five to more than sixty volts. The final ⅓ of the tests we ran on the

muscle were consistent with each other, and showed that the maximal stimulus is very

likely, less in the treatment muscle. The consistency, compounded with the acquired skill

of running these tests, leads us to believe that the final values obtained were close to the

correct maximal stimuli. They also indicate that the prior tests run failed as a result of

poor laboratory technique or improper experimental procedure. Time prevented the

continued study of maximal stimulus, so this test should be of interest for further

electrophysiological studies of ACL myotoxin on skeletal muscle.

20

The results of the test to determine the latent period also supported our

hypothesis. The shorter latent period is a good indication of a physiological change. It

also indicates that the membrane potential has been lowered toward threshold. This

supports the proposed mechanism of action of ACL myotoxin in that the lowered resting

membrane potential shows that there must be an influx of Na+ into the SR or some other

conducting membrane of the myocyte.

The last test was the determination of tetanic frequency for the control and

treatment muscles. The results we obtained clearly indicate that there is no alteration of

the tetanic frequency of the treatment muscle. This was our hypothesis, because a change

in tetanic frequency would have indicated that the inhibition of Ca2+ uptake by the SR

would be a possible reason for the physiological changes observed. Therefore, the lack

of the change in this value supports the proposed mechanism of action of ACL myotoxin.

Future studies contributing to the uncovering of the mechanism of action of ACL

myotoxin could be the doing more tests similar to the ones we did, such as force of

contracture, maximal stimulus, or fatigue. These tests could reveal more about the

physiological changes of the muscle. Electrophysiological studies to determine the exact

role of ions in the changes in membrane potential related to exposure to this toxin are

another possibility. Finally there needs to be correlation of physiological changes with

the observed histological changes.

There are several main conclusions that can be drawn from this study. They are

that ACL myotoxin induces significant decrease in threshold stimulus of contraction.

Also that ACL myotoxin induces a significant decrease in latent period. Finally, that it

does not cause a change in the tetanic frequency. These all lead to the belief that the

resting membrane potential is changed toward threshold. Our hypothesis that

physiological changes will be induced in skeletal muscle injected with ACL myotoxin

was accepted.

21

Acknowledgements

Thank you Terry Colberg for isolating ACL myotoxin, and Charlotte Ownby for

kindly providing the purified ACL myotoxin and providing technical advice. We would

like to thank our research advisor, Dr. Steffan Anderson. We want to thank Dr. Wendy

Perryman for scientific counsel and for graciously providing the rats for our research. A

thank you goes out the whole biology department for any extra effort they have put into

helping us make this project possible. Most of all, we would like to thank our Savior for

the opportunity and the ability to learn this material.

22

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