Smaller motor units with smaller motor neurons are typically activated first in vivo, then larger motor units (Belanger, 2004), but the results differ in vitro. Usually smaller motor units are associated with slow-twitch muscle fibers, while the larger ones are associated with fast-twitch fibers (Belanger 2004). In this experiment we expect the time between the start of the contraction and maximum tension to increase with increasing stimulus frequency as more small motor units with slower-conducting, smaller axons become activated.
One important part of skeletal muscle function is regulation of skeletal muscle tension through temporal summation (Belanger, 2004). Stimuli that reach the muscle before it has had the chance to relax from a previous stimulus will add upon one another and lead to maintained tension (Belanger, 2004). As new CAPS begin to arrive before the one before it is over, a fusion of signals is shown. This fusion eventually leads to tetanus, where one signal is indistinguishable from any others (Belanger, 2004). We believe that this experiment will show that higher stimulus frequency will produce greater tension, then fusion and, eventually, tetanus.
The length-tension curve shows that the maximum tension is produced in skeletal muscle at intermediate sarcomere lengths because this is where the myosin and actin overlap maximally and is most conducive to cross-bridge cycling (Belanger, 2004, Huxley and Peachey, 1961). Stretching the muscle in this experiment will increase the length of the individual sarcomeres (Belanger 2004). We believe that this experiment will show tension to increase with muscle length up until a point when the muscle fibers are torn, after which it will decrease.
Methods and Materials
The method followed was that outlined in the Fall, 2004 BIOL 4161 Laboratory Manual. The only change was that the section entitled “Regulation of muscle tension” was performed before the section called “Effect of muscle length on twitch strength” to prevent damage to our specimen before we could perform all parts of the experiment. Also, due to a failed first attempt, our group did not perform the second half of the experiment in which students were to test their own hypotheses.
Results
Recording the muscle twitch
Pulse amplitude was varied from -0.01V to -2.0V. The resulting electromyogram (EMG) was recorded (Fig.1), as well as the force amplitude of the muscle contractions that were produced (Fig.2). A chart of these values may be found in the Appendix. Muscle contractions began to occur at a pulse amplitude of -0.16 V. The EMG produced increased from 2.421 mV at a stimulus of -0.16 V to 3.001 mV at -0.32 V. It then decreased to 1.012 mV at -0.64 V before increasing to 2.650 mV at -1.28 V at which point it appeared to plateau. There was no clear pattern in our recordings of the force of contractions produced over increasing stimulus strengths. The maximum force of 10.54 mV was produced at -1.28 V stimulus. The minimum of 1.236 mV was produced at
-0.64 V. The CAP recordings on our computer were not functioning properly, so we have no data on the CAPs produced by increasing stimulus strengths.
The delays between the stimulus and the start of the EMG and the contraction were also recorded, as was the duration of the twitch and the time from start of twitch to maximum tension. A table of these values may also be found in the Appendix. The delay between the stimulus and the start of the EMG was 5 ms in every instance at which a contraction was produced. The delay between the stimulus and the start of the contraction (Fig.3) increased slightly from 6 ms at -0.16V to a maximum of 9 ms at -0.32 V then decreased at higher voltage stimuli until it leveled off at 3 ms at a -1.28 V stimulus. Twitch duration (Fig.5) varied from 71 ms at a stimulus of -1.28 V to 84 ms at -0.16 V, but showed no pattern. The time from the start of the twitch to the maximum tension (Fig.4) began at a maximum of 39 ms at a -0.16 V stimulus then decreased to a minimum of 27 ms at -1.28 V. It then increased again to 33 ms at -2.0 V.
Effect of muscle length on twitch strength
A stimulus intensity of -1.28 V was used for this portion of the experiment. The initial length of the muscle was 4.1 cm. The change in baseline tension and force amplitude were recorded at changes in length from 0 mm to 7 mm. A table of values for this portion of the experiment may be found in the Appendix. The change in baseline tension showed no pattern at changes in length from 0mm to 2 mm, where it went from 0.057g up to 0.923g then back down to 0.090g (Fig.6). From 2mm to 6mm, it increased to a maximum of 2.418g. At a stretch of 7mm it dropped to 0.616g. The contraction force increased with increasing change in muscle length from 4.657 mV at 0mm to a maximum of 53.676 mV at 5 mm (Fig. 7). It then dropped slightly and leveled off at approximately 50 mV.
Regulation of muscle tension (Effects of increasing the frequency of stimulation)
A stimulus of -1.28 V was found to produce the maximum muscle contraction and was used for this part of the experiment. The muscle was stimulated at increasing frequencies from 5 Hz to 35 Hz. Baseline tension was recorded and tetanus and fusion were observed. A table of these observations may be found in the Appendix. Fusion began to occur at 12 Hz and tetanus at 32 Hz. The baseline tension increased with stimulus frequency (Fig.8). The increase was only slight at frequencies of 5, 10, and 12 Hz, going from 0g to 0.452g. From 15 to 35 Hz the increase was more dramatic, going from 1.246g
to 7.958g.
Discussion
In our experiment, EMG of a frog skeletal muscle increased slightly, decreased, then increased and leveled off over the course of increasing stimulus strength. This indicates that the increasing stimulus strength first produced stronger CAPs, then weaker ones, then stronger ones again until it reached a maximum CAP strength. We would expect EMG to simply increase with increasing stimulus strength, since the electrical response produced by a muscle is a function of the CAP that produced it. Had we been able to record CAP, we would have most likely have found it to increase with increasing stimulus strength, explaining the EMG trend.
Our recordings of contraction force over increasing stimulus strength were erratic and showed no pattern. The expected response would be for contraction force to increase with increasing stimulus strength because a stronger stimulus is able to activate more motor neurons, therefore causing more muscle fibers to contract (Belanger, 2004, Kuffler and Williams, 1953). The more muscle fibers contracting, the stronger the overall muscle contraction.
The delay between the stimulus pulse and the start of the muscle contraction increased, then decreased and leveled off over increasing stimulus strength. This would indicate that at first, smaller axons are being activated, which conduct the signal to the muscle more slowly (Kuffler and Williams, 1953). At the longest delay, the signal is being carried mostly along small axons; as the delay grows shorter with stronger stimuli, more large axons are being incorporated, leading to a faster muscle response time (Kuffler and Williams, 1953).
The time from the start of the contraction until maximum tension was reached decreased over increasing stimulus strength until it increased some at the end. This goes against our hypothesis that the time from contraction to maximum tension would only increase over increasing stimulus strength as progressively smaller motor units became involved in the response. However, contraction time relies only a little on the axon conductance time and is mostly a function of the type of muscle fiber involved. These data indicate that between stimuli of -0.16 V and -1.28 V, when the time of delay is decreasing, the action of the muscle is shifting from being caused mostly by slow-twitch muscle fibers in the beginning to mostly fast-twitch fibers when the delay is the shortest (Belanger, 2004). The responsibility for the contraction seems to shift back toward slow-twitch muscle fibers at the end when the delay increases.
The duration of the muscle twitch showed no pattern with increasing stimulus strength and is not particularly relevant to our study.
At a stimulus of -1.28 V, the baseline tension of the skeletal muscle increased with increasing stimulus pulse frequency, in agreement with our hypothesis. This is caused by a temporal summation of the pulses. Subsequent pulses arrive at the muscle tissue before it has a chance to relax from previous one. With the muscle “slack” in connective tissues having been taken up by the first stimulus pulse, the subsequent ones are able to build upon one another and produce greater tension, fusion, then tetanus (Belanger, 2004, Randall et al., 2002).
As we had expected, the change in baseline tension of the muscle for the most part increased with increasing change in the length of the muscle before plummeting at the end. Stretching the muscle at first decreases the amount of interference caused by the ends of the sarcomeres and allows the actin and myosin fibers to better interact with one another through cross-bridge cycling (Huxley and Peachey, 1961, Randall et al., 2002). However, the change in baseline tension drops to nearly zero one the muscle fiber is stretch past 6 mm because that is the point at which the muscle tears and the fibers can no longer cross-bridge cycle (Randall et al., 2002).
The contraction force of the muscle also increased with increasing change in muscle length, before dropping slightly then leveling off. This can be accounted for by the same reasons mentioned above: stretching the muscle allows better cross-bridge cycling up until the point when the fibers are too far apart to allow it.
References
Belanger J (2004) Vertebrate Physiology Laboratory Manual 4161, 2004. pp.32-33.
Huxley AF and Peachey LD (1961) The maximum length for contraction in vertebrate
striated muscle. J. Physiol. 156: 150-165.
Kuffler SW and Williams EM (1953) Small-nerve junctional potentials. The distribution
of small motor nerves to frog skeletal muscle, and the membrane characteristics of the
fibres they innervate. J. Physiol. 121: 289-317.
Randall D, Burggren W and French K (2002) Animal Physiology. W.H. Freeman and
Company: New York.
