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Investigation of Frog Gastrocnemius Muscle Contraction.

Abstract:

Skeletal muscle’s remarkable property of elasticity and contractibility brings forth the ability for us to move around and interact with the world around us. In this experiment we studied the contractile behavior of the frog’s gastrocnemius muscle to be able to extrapolate the studies and gauge an idea of perhaps how the human skeletal muscle’s properties. The first part of the this experiment dealt with measuring the muscle twitch contraction in presence of a single electric stimulus of increasing intensity starting from 0 volts to 5 volts with 0.25 volt increments. With each increment in stimulus intensity, the contractions were more powerful till 2.25 volts—after which any further increase in intensity had no corresponding increase in contraction. The second part of the experiment investigated how the muscle contraction occurs in presence of multiple stimuli close in time to each other with constant 2 volt stimulus. The phenomenon of summation and tetanus were observed at frequencies of 4 and more hertz, with complete tetanus happening at 30Hz.

Introduction:

The skeletal muscle system is an essential and characteristic part of the vertebrates. The key benefit of this system is that it gives ability to move in addition to protection, defence and aggression (to prey, for example). The skeletal muscles are composed of skeletal muscles cells (also known as muscle fibres).The skeletal muscles are voluntarily controlled by nervous system using neuronal innervations of the muscle fibre. Skeletal muscle fibres are long, slender, contains several nuclei near the sarcolemma (the plasma membrane of the muscle cells) (Hoehn & Marieb, 2007). The muscle fibres are in turn made of myofilament structure of actin and myosin, which is the functional unit of muscle fibre known as sarcomere (Hoehn & Marieb, 2007). The actin and myosin filaments slide against each other to form contraction of sarcomeres leading to muscle contraction in presence of an electrical stimulus which causes action potential across the muscle fibre. The action potential triggers the release of Ca2+ ions from sarcoplasmic reticulum (Moyes and Schulte, 2008). The Ca2+ ions bind to troponin which causes change in tropomyosin positioning and exposes the myosin bind sites on actin filament. The binding of myosin on actin along with the usage of ATP molecule causes myosin head to pull on actin to cause power stroke (Moyes and Schulte, 2008). Combination of such power strokes are the functional basis of contraction seen in the whole muscle (Lombardi et al., 1992).

The amount of force generated is dependent on the length of the muscle fibre and the load put on it.  There is an optimal load which stretches the muscle at an optimal length, allowing the muscle to perform the maximum work (Wassenbergh et al.2007). At the optimal length muscle, in sarcomeres, the actin and myosin cross-bridging is such that neither excess overlapping nor too little of it occurs to ensure sufficient contractile space (Moyes and Schulte, 2008). Hence stretched muscle will have better chance of producing higher contractile force than unstreched muscles as stretching may cause fibres to be placed optimally at optimal lengths compared to an unstreched muscle (Dou et al. 2008).

There are two types of muscle contractions: Spatial and Temporal. Spatial summation of the twitch contractions increases the amplitude or strength of contractions by recruiting more fibres (Staud et al., 2007). Higher intensity causes increased amount of Ca2+ to be released and hence increasing number of crossbridges being formed (Kargo and Rome 2008). Temporal summation is achieved by increasing frequency of the stimulation which causes increase in amplitude of contraction and successive decrease in relaxation phase (Staud et al., 2003).

The purpose of this experiment is to examine response of the muscle by changing variables of strength and frequency of stimulus. The latency, contractile, relaxation phases and amplitude of the twitch is the information that may enable us to understand muscle response with reference to variables of stimulus. The muscle will also be tested to see if there is spatial and temporal summation in terms of its contractions.

Methods and Materials:

Physiology Lab 1: Skeletal muscle lab manual was followed to conduct the experimental procedures. There were no deviations or modifications made from the prescribed laboratory procedure as given in the lab manual.

Results:

Part 1: Stimulus Response

Table 1: Muscle response in presence of stimulus in terms of its twitch amplitude, contraction, relaxation and latency periods

Stimulus

Amplitude

(V)

Muscle Twitch

Amplitude

(mV)

Contraction time

(ms)

Relaxation time

(ms)

Latency time

(ms)

0

0

0

0

0

0.25

0

0

0

0

0.5

40

55

65

20

0.75

45

50

75

20

1

49

50

90

20

1.25

55

55

100

20

1.5

63

50

115

20

1.75

66

60

125

20

2

73

55

130

20

2.25

73

55

135

20

2.5

75

60

135

15

2.75

76

55

140

20

3

75

55

145

20

3.25

70

60

135

15

3.5

76

60

125

15

3.75

75

60

145

15

4

76

65

135

15

4.25

72

60

125

15

4.5

75

60

140

15

4.75

76

60

160

15

5

78

65

175

10

In this table, muscle twitch amplitude, contraction time, relaxation time and latency time is measured for each respective stimulus voltage increment of 0.25 volt starting from 0 volt to 5 volt. After each stimulus, there was a lag or wait period of about 20-30 seconds before another stimulus is delivered to the muscle to allow the muscle to regain its contractile strength. Figure 1 is representative of muscle twitch in presence of a single stimulus of 2V. The latency period is the period from the initiation of the electric stimulus till the muscle contraction initiation. The contraction period is from the initiation of muscle contraction to the peak of muscle contraction followed by the relaxation phase till the original muscle posture is achieved.

Figure 1: Muscle Twitch & its relevant phases (periods)

Figure 2: Muscle twitch with respect to increasing stimuli with 0.25 V increments from 0 to 5 volts.

Figure 3: Contraction time Vs Stimuli

Figure 2 and 3 represents that with increase in stimulus, there is an increase in twitch amplitude and contraction time.

Part 2: Summation and Tetanus

Table 2: Stimulus with varying frequency of application but constant volt and its effect on amplitude and passive tension

Stimulus

Frequency

(Hz)

Amplitude

1st twitch

(mV)

Maximum

Amplitude

(V)

Change in

Passive tension (V)

Summation/

Tetanus

0.5

68

0.071

0.001

No

1

70

0.072

0.001

No

2

74

0.074

0.005

No

3

68

0.072

0.006

No

4

73

0.073

0.010

Summation

5

74

0.078

0.022

Summation

10

75

0.081

0.069

Incomplete tetanus

20

90

0.098

0.081

Incomplete tetanus

30

97

0.108

0.090

Complete tetanus

Figure 4: Mechanical Summation

Figure 5:Incomplete Tetanus at 2V-20HzFigure 6: Complete tetanus at 2V-30Hz

When more than one stimulus was delivered at a pace of 5 Hz as in Figure 2, the muscle summation was observed relaxation phase was much shorter. With increasing pace of administration of the stimulus, the relaxation phase further decreased to give incomplete and complete tetanus as seen in figure 3 and 4 respectively. This data are represented in Table 2 where increasing stimulus frequency till 3 Hz had no summation or tetanus; 4 and 5 Hz showed summation evident from its change in passive tension and at higher frequencies the tetanus was noticed (incomplete at 10 and 20 Hz; complete at 30 Hz).

Discussion:

The muscle twitch is the basic unit of contraction (Moyes and Schulte, 2008). The twitch observed in our experiment occurs via electrical stimulus administered using an electrode. This stimulus produces the same action potential in the muscle tissue as neuronal excitation would via motor end plate (Hoehn and Marieb, 2007). Just as a neuron requires a certain threshold for an action potential to be generated, muscle, too, needs a certain degree of depolarization before it can fire an action potential across the whole tissue. This also has its biological benefit as energetically it may be expensive for muscles cannot afford to contract at every single stimulus. As observed in figure 1, a single contraction-relaxation twitch has three phases: Latency, Contraction and Relaxation. Latency period is the phase between the initiation of the electric stimulus and the initiation of the muscle contraction. The presence of this lag or delay in muscle contraction can be explained by the time needed for the excitation-contraction (EC) coupling to take place (Moyes and Schulte, 2008). EC coupling is a process involving the entry of Ca2+ into the muscle fibres following a stimulus, and activation of actin-myosin cross-bridges (Moyes and Schulte, 2008). The latency period of the muscle with single stimulus delivery is between 15msec to 20 msec (refer Table 1).

Contraction phase begins at the end of latency period when the action potential passes across the muscle fibre and through the T-tubules releasing the calcium stores from the sarcoplasmic reticulum into the sarcomere (Moyes and Schulte, 2008). The release of calcium causes troponin to change the conformation of tropomyosin and unbinds the actin head for myosin to attach (Moyes and Schulte, 2008). Simultaneously, the myosin or the thick filament uses ATP to get into high energy conformation from low energy conformation. The binding of myosin to actin head and use of ATP ensures that power stroke takes place—moving the actin and myosin across each other and shortening the sarcomere. The combined effect of such shortening of sarcomeres in the muscle fibre causes collective shortening of the muscle. As long as there is availability of calcium in the sarcomere, the muscle will remain contracted due to actin myosin binding.

Relaxation phase begins at the end of contraction phase when the calcium sequestered in the sarcomere is re-taken into the sarcoplasmic reticulum via active transport by Ca2+-Na+ antiporter (Kargo and Rome 2008). The decreased level of calcium in sarcomere causes tropomyosin to resume it’s state of covering the actin binding site and the sarcomere lengthens because of unbinding of myosin and actin filaments. The time needed for most of the calcium re-uptake is reflective of the lengthening of the sarcomere to its normal length—this time needed reflects as relaxation period of the muscle fibre (Kargo and Rome 2008).

Increasing the stimulus increases muscle contraction till 2.5 V. Raising stimulus above 2.5 V had no additive effect on the muscle contraction. This can be explained at the sacromere level and at the muscle level. At low levels of stimulus, e.g. 0-1V, the depolarization is short lived. However, when there are high levels of stimulus, ie above 2.25-5 V, the depolarization is long lived and calcium release is substantial, which is why rate of relaxation for the single high voltage stimulus is increasing. Moreover, with high voltage, the number of muscle fibres employed for the contraction increases. Hence, using high stimulus single administration brings forth stronger, profound contractions. However, muscles has a certain degree of contractility due to the physical barrier of actin-myosin crossbridging (Hoehn and Marieb, 2008). Hence, muscle cannot indefinitely contract at higher stimulus.

Summation of the contraction occurs when there are more than one stimulus administered so close in time that the preceeding relaxation is overlapped with the stimulus to contract again. Such stimuli makes the relaxation phase of the muscle twich shorter and the contractions more powerful (Staud et al., 2007). This was seen when 2 volt stimulus were administered at 4-20Hz where contractions superimposed over each other causing summation and unfused tetanus as seen in figure 4 and 5. At 30 Hz, the contractions are at maximum potential and relaxation is almost zero. This is the condition of fused tetanus as seen in figure 6.

The phenomenon of summation and tetanus can be explained by the contraction initiated by the action potential before the relaxation began. That is, before the calcium re-uptake, a novel stimulus arrived that depolarized reticulum further to release calcium and cease the uptake (Kargo and Rome 2008). Persistent presence of calcium in the fibres executed the contraction to the fullest and resulted in decreased relaxation period. In the summation and additive effect whereby the second round of contraction builds on the first round of contraction or the partial relaxation; thereby increasing the strength of contractions. However, as explained earlier, there is a limit to the maximum capacity of the muscle to contract because of the physical limitation of the actin-myosin crossbridging—after maximal crossbridging, further stimulation of the muscle will have no increase in contractile strength.

The results obtained via this experiment provided a practical insight into how the muscle twitch contraction takes place. It provided practical and quantifiable data to understand about latency, contraction and relaxation period, summation and tetanus of the skeletal muscle fibre. The contraction strength increased as the stimulus strength increased. Mechanical summation eventually fused to form the unfused and fused tetanus and formed a single stronger contraction for prolonged period of time.

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