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Properties of Skeletal Muscle

Michelle Chen

Jesse Dingle, Jenna Niggli, Sendy Vo, Derek Yip

NPB101L Section 09: Krista Sowell

April 25, 2013

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Introduction

The largest group of tissues in the body is muscle, and it can be found in various organs

throughout the body. Muscle tissue’s main function is to generate force for movement. Skeletal

muscle alone makes up about 40% - 50% of total body weight in men and women (Sherwood,

2010, p. 256). It is composed of groups of muscle fibers bundled together by connective tissue

and attached to bones by tendons. A motor unit is a single motor neuron and all of the muscle

fibers it innervates. One motor neuron is able to innervate multiple muscle fibers, but each

muscle fiber is controlled by only one motor neuron (Sherwood, 2010, p. 258). The number of

muscle fibers supplied by one motor unit and the number of motor units per muscle vary widely

throughout the body, depending on the specific function of the muscle. For weak contractions,

which are responsible for fine, delicate movements such as blinking, only a few motor units are

activated. For stronger contractions, which are responsible for coarse movements such as

kicking, a larger number of motor units are recruited (Sherwood, 2010, p. 270). According to

Henneman’s findings, which came to be referred to as “The Size Principle,” motor unit

recruitment follows an orderly pattern. In response to greater stimulation, motor units are

recruited from the smallest to the largest (Hodson-Tole et. al, p. 58)

The overall series of events that link muscle excitation to muscle contraction is referred

to as excitation-contraction coupling. Muscle excitation occurs when an action potential is

present in the muscle fiber. Muscle contraction occurs when cross-bridge activity causes the thin

filaments to slide closer together to produce a shortening in the sarcomere (Sherwood, 2010, p.

264). The sarcomere is the functional unit of skeletal muscle, which means it is the smallest

component able to carry out the function, in this case contraction (Sherwood, 2010, p. 258).

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In order for contractions to be carried out, there are a number of important compounds

involved. Calcium plays the role of linking excitation and contraction. Skeletal muscles are

stimulated to contract when acetylcholine (Ach) is released at the neuromuscular junction. The

binding of Ach with the motor end plate of a muscle fiber causes the permeability in the muscle

fiber to change, and an action potential is delivered throughout the cell membrane (Sherwood,

2010, p. 264). Two structures located near the surface membrane of the muscle fiber that play an

important role in this excitation-contraction coupling are traverse tubules and the sarcoplasmic

reticulum.

The goals of this experiment are to: see the effects of direct stimulation, analyze the

effects of stimulus intensity and stimulus frequency on contraction, and analyze the effects of

acetylcholine (Ach) receptor blockade on muscle activity.

The post synaptic neuron can expected to be brought to threshold by either spatial or

temporal summation. The effects of spatial summation can be expected when stimulus intensity

is applied to the sciatic nerve. As stimulus intensity increases, so will the tension produced.

When an increase in stimulus frequency is applied, an increase in tension will be expected as

well. In both situations, a gradual increase is expected, and eventually the twitches will fuse and

result in a plateau in the amount of tension generated. Once tubocuare, which is a skeletal muscle

relaxant, is applied, muscle paralysis is anticipated. Direct versus neuronal stimulation will also

be observed and analyzed, and it is hypothesized that stimulation of the nerve will produce

greater tension, even at lower voltages.

Materials and Methods

The subject of this experiment was frogs. Details about the materials and methods used

can be found in the NPB 101L Systemic Physiology Lab Manual under the section labeled

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“Exercise 2” (Bautista et al. 2008 pp 9-17). The gastrocnemius of a frog was first isolated and

then the sciatic nerve using blunt dissecting glass probes according to the procedure outlined in

the manual. The frog was then connected to a force transducer that delivered electrical pulses.

Muscle contraction was first induced through the stimulation of the sciatic nerve. Muscle tension

was measured along with changes in stimulus voltage and frequency. The effect of tubocuare on

muscle activity was also observed. Lastly, muscle contraction was induced through direct

electrical stimulation. Throughout the experiment, the muscle and nerve of the frog was kept

moist using Ringer’s solution, and the rest of the frog was kept moist with water. It is important

to note that the data collected to compile the results presented in Figure 1 as well as the nerve

stimulation recorded in Table 1 came from the Morgan group. Therefore, the experiments of

these sections were conducted on a different subject. The data presented in Figures 2, 3, and the

direct stimulation recorded in Table 1 are still from the subject used by our group (Selye).

Results

In order for the following experiments to be consistent, a baseline tension of about 20 g

was maintained throughout because the amount of stretch on the gastrocnemius changes the

amount of force produced.

I. The Effect of Stimulus Intensity on Muscle Activity: Graded Response

In this first experiment, threshold and maximum voltage were determined and then the

effect of stimulus intensity on muscle activity was observed. Figure 1 demonstrates that as

voltage increases, tension increases as well, producing a graded response. The smallest voltage to

elicit a twitch, 0.1 volts, is referred to as Vthreshold and produced 33.65 g of tension. From this

point onwards, voltage was increased in increments of 0.01 volts until 0.2 volts was reached,

which produced 101.78 g of tension. Twice the amount of the initial voltage produced about

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three times the amount of tension. An increasing trend between voltage and tension continued

until a voltage, known as Vmax, and any subsequent voltages applied after this point, did not

lead to any further increases in tension. Vmax for our data occurred at 0.14 volts, which

produced 97.75 g tension. From this point onwards, tension remained constant at about 99-100 g.

Despite the minor dip that occurs at 0.13 volts, the trend still shows a steady increase and an

eventual plateau.

Figure 1. Voltage versus tension of a frog gastrocnemius, under stimulation of the sciatic nerve.

Frequency was held constant and tension was set at a baseline of 20 g at the start of the

experiment. Voltage was increased by 0.1 volts for every twitch observed.

II. The Effect of Stimulus Frequency on Muscle Activity: Summation

In the next experiment, the effect of stimulus frequency on muscle activity was observed.

Figure 2 provides us with a graphical representation of the correlation between frequency (pps)

and tension (g) – as frequency increases, tension also increases. The data collected from the

BioPack for this portion of the experiment provides a visual representation of summation at 2.0

pps and tetani at 8.0 pps. Frequency was increased in increments, beginning with 0.5 pps for 15

0

20

40

60

80

100

120

0.1 0.12 0.14 0.16 0.18 0.2 0.22

Ten

sio

n (

g)

Voltage (V)

Voltage vs. Tension "Graded Response"

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seconds producing a tension of 212.5 g. From 0.5 pps onwards, each increase in frequency was

run for 30 seconds, and the highest frequency applied was 25 pps, which produced a tension of

515.95 g. Once again, despite a small dip at 2 pps, the trend shows that as frequency increased,

tension also increased, but eventually began to plateau in between 15 and 25 pps, where tension

only increased from 475.15 g to 515.95 g.

Figure 2. Frequency versus tension of a frog gastrocnemius, under stimulation of the sciatic

nerve. Voltage was held constant at Vmax of 2.4 volts. Frequency was increased starting from

0.5 pps for 15 seconds, then 1, 2, 4, 8, 15 and 25 pps, each for 30 seconds. Tension was set at a

baseline of 20 g at the start of the experiment.

III. The Effect of Tubocuare on Muscle Activity: Paralysis

In the third part of our experiment, muscle activity after the injection of 0.25 mL

tubocuare was observed. Figure 3 deals with Tubocuare and how it relates to tension over time.

This figure represents data after the point of injection, which occurred at around 89 seconds and

24.35 g of tension. The stimulator was kept running for 5 minutes (300 seconds), and eventually

the tension dropped down to 10.11 g at 359 seconds. Measurements were recorded every 30

0

100

200

300

400

500

600

0 5 10 15 20 25 30

Ten

sio

n (

g)

Frequency (pps)

Frequency vs. Tension

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seconds after the point of injection, and the biggest drop in tension occurred between 89 and 119

seconds, from 34.95 g to 23.5 g. From that point onwards, the decrease in tension was more of a

gradual decline, about 2 g every 30 seconds.

Figure 3. Muscle activity of a frog gastrocnemius after injection of tubocuare for around 5

minutes (300 seconds). Measurements of tension recorded every 30 seconds after point of

injection. Tension was set at a baseline of 20 g at the start of the experiment.

IV. The Effect of Direct Electrical Stimulation on Muscle Activity

In the last portion of our experiment, we observed the effects of direct stimulation and

compared these findings to stimulation of the nerve. Table 1 compares and contrasts the tensions

produced at Vthreshold and Vmax. The voltage for Vmax of direct stimulation is 10 times

greater than the voltage at Vthreshold. We expected to see that the tension produced from direct

stimulation would be less than stimulation of the sciatic nerve, even at voltages greater than

Vmax of the nerve. Due to the use of two different test subjects, our results did not agree with

our expected findings. The tension produced via direct stimulation at Vmax (165 g) still ended

up being greater than the tension produced at Vmax via stimulation of the nerve (97.65 g). It is

0

5

10

15

20

25

30

35

40

85 135 185 235 285 335 385

Ten

sio

n (

g)

Time (seconds)

Tubocuare

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important to note that Vmax for the direct stimulation (10 volts) is 71 times greater than Vmax

for the nerve stimulation (0.14 volts), a much larger voltage to produce only 67.35 g more

tension. Vthreshold for direct stimulation needed to be increased by 10 times to elicit 164.2 g

more tension, while Vthreshold for nerve stimulation only needed to be increased by 1.4 times to

elicit 91.32 g more tension.

Table 1. Direct stimulation versus sciatic nerve stimulation of a frog gastrocnemius.

Nerve stimulation data transferred over from part 1 of experiment. Direct stimulation collected

by connecting electrodes to muscle directly and finding Vthreshold, then increasing voltage to

10X maximal voltage from part 1.

Direct Nerve

Volts Tension Volts Tension

Vthreshold 1 0.8 0.1 6.33

Vmax 10 165 0.14 97.65

Discussion

Before discussing the results collected from the experiment, further details about how

electrical activity in the nervous system leads to muscle contraction need to be given.

Excitation-contraction coupling begins at the neuromuscular junction, which is the area

where the motor neuron synapses with the muscle fiber (Sherwood, 2010, p. 247). A

depolarization of the neuron occurs and transmembrane molecules fuse, releasing acetylcholine

(Ach) into the synaptic cleft. Ach then binds to nicotinic Ach Receptors (nAchR) on the muscle

fiber, causing these gated cation channels to open (Sherwood, 2010, p. 243). The transverse

tubules bring the action potential to the sarcoplasmic reticulum, and in the process stimulate the

release of calcium ions into the cytoplasm. Specifically, depolarization opens the

dihydropyridine (DHP) receptors on the T-tubule, causing the ryanodine receptors (RyR) on the

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sarcoplasmic reticulum to open as well. This opening allows an efflux of calcium to travel from

the sarcoplasmic reticulum into the cytoplasm, increasing the overall intracellular calcium

concentration, which is responsible for contractions in skeletal muscle (Sherwood, 2010, p. 264).

When skeletal muscle is relaxed, tropomyosin blocks the cross-bridge binding site on actin.

However, now that calcium has been released, it goes to bind with troponin, causing a

conformational change, and tropomyosin is “pulled” off the myosin binding site of actin, thus

allowing binding of the cross bridge (Sherwood, 2010, p. 265). Troponin and tropomyosin serve

as regulatory proteins, and myosin and actin cannot bind if they are in place. The source of

energy for muscle contraction comes from ATP, and repeated cross-bridge cycles produce power

strokes that pull the thin filament inward without shortening the actual length of it (Sherwood,

2010, p. 263). This process continues until calcium has been removed from the cytoplasm,

resulting in the muscle returning to relaxation (Sherwood, 2010, p. 268).

As seen in the first part of the experiment, where stimulus intensity was increased, there

is a positive correlation between voltage and tension, until the point of Vmax is reached. Vmax is

the lowest voltage to cause maximum tension and all motor units to become innervated. From

this information, Vthreshold can also be determined, which is the lowest voltage needed to

depolarize the largest motor unit in the sciatic nerve. The positive correlation between voltage

and tension is due to a phenomenon known as spatial summation, where more and more motor

units are recruited as voltage is increased. Motor unit recruitment is a graded activation of

muscle contraction and involves increasing the number of motor units that participate in muscle

contraction (Hodson-Tole et. al, p. 58). Contrary to Henneman’s Size Principal, which was stated

in the introduction, the results proved the reverse to be true. Since the sciatic nerve was directly

stimulated, the motor neurons were recruited from largest to smallest due to their larger diameter

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and therefore a lower threshold (Hodson-Tole et. al, p. 59). No responses were recorded between

0 to 0.09 volts because these voltages were not high enough to innervate motor units and

occurred before Vthreshold. Once Vmax was attained, all motor units and muscle fibers have

been recruited, which is why any subsequent voltages did not lead to anymore increase in

tension, but instead resulted in a plateau.

Part two of the experiment, where stimulus frequency was increased, demonstrates the

relationship between frequency and tension. Rapid, repetitive excitation from a single persistent

stimulus led to temporal summation and eventually tetanus. Tetanus occurred because the muscle

fiber was stimulated so rapidly that it did not have an opportunity to relax at all in between

stimuli, and a maximal sustained contraction was achieved (Sherwood, 2010, p. 271). Even

though the voltage was already set at Vmax, tension still increased with increasing frequencies

because of two factors. First, as frequency increases, there is less time for calcium to be

reabsorbed into the sarcoplasmic reticulum, leading to an overall increase in cytoplasmic calcium

when the next action potential is carried out. This excess of calcium in the cytoplasm continues

to build up and elicits an increase in cross-bridge formation, which is responsible for producing

more tension (Barclay et al., 2007, p. 3127). Another factor responsible for the positive

correlation between frequency and tension is the series elastic component. At first, not all the

tension is transferred to the bone since some is lost during the stretching of the fibers. However,

as frequency increases, the fibers remain partially stretched since they have not had enough time

to completely recoil all the way, and more force (tension) is transferred to the bone. Less force is

wasted and more can be used in the contraction, which accounts for the increase in tension. The

series elastic component has been found to amplify the amount of work outputted from relaxing

muscle (Barclay et al., 2007, p. 3128).

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During the experimental procedure of observing the gastrocnemius muscle after injection

of tubocuare, a decrease in muscle tension was observed. Tubocuare, which is classified as a

competitive inhibitor, induced paralysis by interfering with the normal signaling system between

nerve and muscle (Nguyen-Huu et. al, 2009, p.1011). Muscle tension decreased after the

injection of tubocuare because tubocuare functions as a competitive inhibitor for the nicotinic

acetylcholine receptor (nAchR). Due to its higher affinity for the nAchR, tubocuare outcompeted

acetylcholine and displaced it (Sherwood, 2010, p. 243). As a result of this competitive

inhibition, depolarization was not able to occur. However, tubocuare is concentration dependent,

so as time progresses, tension should once again start to increase as the injection begins to wear

off. Increasing the concentration of the agonist, in this case acetylcholine, can result in

overcoming this competitive inhibition and preventing muscle paralysis. Such findings were

further explored in a study published in the 2009 issue of Anesthesiology. D-tubocuarine

competes with Ach for occupation of the nAchR. A higher release of Ach at the neuromuscular

junction of the muscle being examined diminishes the effect of tubocuare. A proposed

explanation is that a muscle with higher nAchR density will decrease the neuromuscular

blocking effect of competitive agents (Nguyen-Huu et. al, 2009, p.1014).

Direct stimulation on muscle activity is still able to produce a contraction, as evidenced

by the final segment of the experiment. Neuronal input is not absolutely needed in order to elicit

muscle activity; however, direct stimulation of the muscle will produce contractions at a much

lower force (Rodriguez-Falces et al., 2013, p. 18). Here, the action potential is sent directly to the

muscle fiber and bypasses the neuromuscular junction. Less muscle tension was produced by

direct stimulation of the gastrocnemius since only muscle fibers close to the electrode underwent

contraction. The voltage needed to travel through the muscle fibers instead of the motor unit.

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Skeletal muscle lack gap junctions, so one muscle fiber is not able to signal to its surrounding

muscle fibers. With stimulation of the sciatic nerve, even at small voltages, all motor units can be

stimulated to contract. Even increasing the voltage of direct stimulation above Vmax of the nerve

will produce less tension. The amount of maximal twitch force produced is greater for nerve

stimulation as compared to direct muscle stimulation due to more muscle fibers being activated

in unison (Rodriguez-Falces et al., 2013, p. 16).

Conclusion

Stimulation of the frog gastrocnemius both at varying voltages and frequency affects the

amount of tension produced. Increase in voltage demonstrated the effects of motor unit

recruitment and the achievement of spatial summation within the muscle. Intensification in

frequency demonstrated the effects of temporal summation caused by the increase in cytoplasmic

calcium concentration and the series elastic component. Tubocuare functions as a competitive

inhibitor that binds to the nicotinic acetylcholine receptor, not allowing a depolarization to occur.

Although stimulation of the nerve will produce greater tension, contractions can still be attained

by direct stimulation.

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Works Cited

Barclay CJ and Lichtwark GA. The mechanics of mouse skeletal muscle when shortening during

relaxation. Journal of Biomechanics. 2007;3121-3129.

Bautista, Erwin, and Korber, Julia. NPB101L: Physiology Lab Manual. Second Edition. Ohio:

Cengage Learning, 2009.

Hodson-Tole EF and Wakeling JM. Motor unit recruitment for dynamic tasks: current

understandings and future directions. J Comp Physiol B. 2009;179:57-66.

Nguyen-Huu T, Molgo J, Servent D, and Duvaldestin P. Resistance to D-Tubocuarine of the rat

diaphragm as compared to a limb muscle. Anesthesiology. 2009;110:1011-1015.

Rodriguez-Falces J, Maffiuletti NA, and Place N. Twitch and M-wave potentiation induced by

intermittent maximal voluntary quadriceps contractions: differences between direct

quadriceps and femoral nerve stimulation. “Accepted Article,” doi: 10.1002/mus.23856.

Sherwood, Lauralee. Human Physiology: From Cells to Systems. 7th

ed. Belmont, CA:

Brooks/Cole, 2010.

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Stimulus Response (Group: Morgan)

Frequency Response – summation at 2.0 pps

Frequency Response – tetani at 8.0 pps

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Tubocuare – control and after injection

Direct Stimulation