A study of stimulus strength and response, muscle length and tension generated, and contraction strength in gastrocnemius muscle of frogs.
Introduction:
The amount of tension in muscles changes based on how many muscle fibers are contracting. When stimulus strength is increased beyond maximal level, tension does not continue to increase because all of the muscle fibers in the muscle are contracting (Hill, Wyse, & Anderson, 2018). The resting length of skeletal muscles maximizes the muscle’s contraction when stimulated (Hill at al., 2018).
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The objective of stimulus-response exercise was to determine the relationship between the strength of the stimulus and the response of the muscle. The length-tension relationship exercise was performed to determine the relationship between muscle length and tension generated in the muscle. The objective of the summation and tetanus was to measure the amplitude of contraction produced in a muscle that is stimulated with repeated pulses delivered at progressively higher frequencies. The objective of the pre- and post-loaded weight and contractile strength exercises were to measure the strength of contraction while the muscle was lifting pre-loaded and post-loaded weights.
Methods and Materials:
Figure 1: Set-up for weight and work exercises acquired from Animal Muscle -Skeletal Muscle-Weight and Work-SetupIXTA page AM-1-4 used to produce and record contractions from the frog gastrocnemius muscle.
Figure 2: The equipment setup used to induce and record contractions from the frog gastrocnemius muscle related to length and tension of muscle using IXTA data acquisition unit and power supply.
The frogs were placed in an ice bath for ~15 minutes to anesthetize them. The frogs were then pinned down in a dissection tray and their skin was removed from the legs using scissors, forceps, and a scalpel. Steps for frog gastrocnemius were followed from a video titled Frog Dissection on YouTube. Ringer solution was used to moisten the muscle. The femur bone was clamped to the ring stand holder and a fishing hook was used to attach the Achilles tendon to transducer. The stimulating electrodes were positioned against the muscle about partway between the knee and the tendon as illustrated in Figure 2. The FT-302 Force Transducer was calibrated with no weight and 5 grams.
Results:
Table 1: Amplitude of muscle twitches generated by stimulus pulses of different amplitudes.
Stimulus Amplitude (V)
Twitch Amplitude (g)
0.000
0.000
0.250
0.000
0.500
2.769
0.750
10.288
1.000
12.046
1.250
20.750
1.500
28.257
1.750
49.130
2.000
60.014
2.250
63.854
2.500
63.944
2.750
68.599
3.000
70.347
3.250
73.564
3.500
79.990
3.750
81.064
4.000
80.645
4.250
83.224
4.500
85.355
Figure 3: Amplitude of muscle twitches (g) generated by stimulus pulses of different amplitudes (V).
Table 2: Muscle length and muscle twitch amplitude for the frog gastrocnemius muscle.
Muscle Length (mm)
Twitch Amplitude (g)
34
2.267
36
36.86
38
64.749
39
87.307
40
59.11
41
54.914
42
7.202
43
5.494
Figure 4: Muscle length (mm) and muscle twitch amplitude (g) for the frog gastrocnemius muscle. The active tension segment and passive tension segment are labeled, respectively.
Table 3: Amplitude and times of muscle twitches generated by stimulus pulses of different amplitudes.
Muscle Twitch
Stimulus Amplitude (V)
Amplitude (g)
Contract Time (msec)
Relax Time (msec)
Latency (msec)
0.000
0
0
0
0
1.000
99.955
95
160
20
2.000
112.588
100
175
25
3.000
121.634
105
195
25
4.000
124.295
105
180
25
5.000
128.733
105
195
25
6.000
128.766
105
215
25
7.000
128.515
105
210
25
Table 4: Strength of muscle contractions during mechanical summation and tetanus.
Stimulus Frequency (Hz)
Amplitude 1st Twitch (V)
Maximum Amplitude (V)
Change in Passive Tension (V)
Summation/Tetanus
0.5
124.612
126.05
0
No
1
120.376
122.636
0
No
2
116.017
116.812
0
No
3
111.796
118.150
81.975
Summation
4
108.676
120.282
113.929
Incomplete tetanus
5
108.604
122.611
121.033
Incomplete tetanus
10
139.881
143.591
143.098
Incomplete tetanus
20
145.456
145.456
145.456
Complete tetanus
30
146.079
146.079
146.079
Complete tetanus
Table 5: Amplitudes and times of contractions from a post-loaded muscle.
Rod displacement: 19 mm; trace deflection: 1,859 mV; conversion factor: 0.01
Muscle Twitch
Post-load Weight (g)
Amplitude (mV)
Distance (mm)
Contraction Time (msec)
Relaxion Time (msec)
Latency (msec)
Work (Weight x Distance)
Rate of Contraction (Amplitude /Contraction Time)
10
164
1.64
95
65
35
16.4
1.73
20
154
1.54
80
65
35
30.8
1.93
30
138
1.38
90
65
50
41.4
1.53
40
128
1.28
80
60
50
51.2
1.60
50
114
1.14
65
50
35
57.0
1.75
60
106
1.06
75
45
35
63.6
1.41
70
97
0.97
65
45
45
67.9
1.49
Table 6: Amplitudes and times of contractions from a pre-loaded muscle.
Rod displacement: 19 mm; trace deflection: 1,859 mV; conversion factor: 0.01
Muscle Twitch
Pre-load Weight (g)
Amplitude (mV)
Distance (mm)
Contraction Time (msec)
Relaxion Time (msec)
Latency (msec)
Work (Weight x Distance)
Rate of Contraction (Amplitude /Contraction Time)
10
215
2.15
90
65
40
21.5
2.39
20
199
1.99
80
60
35
39.8
2.49
30
195
1.95
95
65
30
58.5
2.05
40
192
1.92
80
65
40
76.8
2.40
50
187
1.87
100
55
45
93.5
1.87
60
177
1.77
100
50
35
106.2
1.77
70
164
1.64
90
60
45
114.8
1.82
Figure 5: Work plotted against load weight, both pre- and post- load.
Discussion and Conclusions:
For the stimulus response exercise, the twitch amplitude increased from 0.500 V stimulus amplitude up to of 3.750 V, and decreased at 4.000 V. This data agrees with the expected results because a small amplitude was produced when a weak stimulus was applied due to the contraction of only some fibers. Additionally, when a strong stimulus was applied, all of the muscle fibers contracted, which caused a large amplitude. At 0.250 V, the twitch amplitude response was 0.000 mV because few channels were open, which was not enough to produce an action potential that reached threshold. The length-tension exercise results were supported by literature. As the muscle length increased to normal resting length for a frog, the overlap of the thick and thin filaments increased optimal cross-bridge binding with actin. Twitch amplitude decreased as length increased past moderate muscle lengths because sarcomeres had less overlap of thick and thin filaments (Hill at al., 2018).
In the mechanical summation and tetanus exercise, once mechanical summation was present, the change in passive tension increased as stimulus frequency increased until the maximum amplitude and change in passive tension were equal. Tetanus was observed at high stimulus frequencies of 20 and 30 Hz because the reuptake of calcium is very fast. An increase in cytosolic calcium levels activates muscle contraction (Kuo & Ehrlich, 2015). Muscle relaxation is slower after tetanus compared to after a single muscle twitch because there is a larger amount of calcium that needs to go back to the sarcoplasmic reticulum.
Literature Cited:
Hill, R. W., Wyse, G. A., Anderson, M. (2018). Animal physiology. Oxford University Press, NY.
Kuo, I. Y., & Ehrlich, B. E. (2015). Signaling in muscle contraction. Cold Spring Harbor perspectives in biology, 7(2), a006023. doi:10.1101/cshperspect.a006023
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