Lab #9: Muscle Physiology

Transcription

1 Background Overview of Skeletal Muscle Contraction Sarcomere Thick Filaments Skeletal muscle fibers are very large, elongated cells (Fig 9.1). Roughly 80% of the content of each muscle fiber consists of long bundles of protein called myofibrils. The myofibrils, in turn, consist of two types of myofilament (Fig 9.2). One type of myofilament, called the thick filament, is composed of hundreds of molecules of a protein called myosin. The other type of myofilament, the thin filament, contains three different proteins: a structural protein called actin that can form bonds with myosin, a protein called tropomyosin that regulates binding between myosin and actin, and the calciumbinding troponin which regulates the position of tropomyosin. The two myofilaments are arranged in the myofibrils in distinctive repeated structures called sarcomeres. Each sarcomere contains a series of thin filaments at either end that partially overlap with thick filaments found in the center. Muscle contracts through an ATP-driven Myosin Troponin Tropomyosin Actin Thin Filaments Fig 9.2. Arrangement of myofilaments into sacromeres within a myofibril (above) and the structure of thick and thin filaments, illustrating the proteins that make up each. interaction between actin and myosin called crossbridge cycling (Fig 9.3). First, the globular head of a myosin molecule extends laterally and binds with a complementary binding site on an actin molecule to form a bond called a crossbridge. Then, in a process called a power stroke, the globular head bends inward towards the center of the sarcomere, pulling the thin filament with it. The crossbridge then breaks, and the globular head of the myosin unbends, preparing the myosin molecule to repeat the process. As a result of many myosin molecules alternately binding the thin filaments and pulling them inward, the thin filaments are pulled over the thick filaments toward the center of the sarcomere, thus shortening the overall length of Myosin Head Unbends Myosin Binds to Actin, Forming Crossbridge Fig 9.1. A micrograph of segments of skeletal muscle fibers. N = nucleus, CT = connective tissue, M = myofibrils. Note the alternating light and dark banding pattern created by the repeated sarcomeres along the lengths of the myofibrils. Image is from /atlas/ histology/08/ah08202.htm Myosin Releases Actin, Breaks Crossbridge Fig 9.3. An outline of crossbridge cycling Power Stroke Pulls Thin Filament over Thick p.1

2 Without Calcium Crossbridges Cannot Form Fig 9.4. The sliding filament mechanism of muscle contraction. Myofibrils contract by the thick filaments pulling the thin towards the center of the sarcomere, increasing the degree of overlap between the thick and thin filaments. each sarcomere and, in turn, the length of the muscle (Fig. 9.4). Crossbridge cycling, and hence muscle contraction, can only occur under specific conditions. This is because normally troponin positions tropomyosin on top of myosin-binding Myofibrils Motor End Plate Thin filaments Somatic Motor Neuron Thick filaments Transverse Tubule Sarcoplasmic Reticulum Sarcolemma Fig 9.5. Excitation of a skeletal muscle fiber. The sarcoplasmic reticulum has been removed from the left side of the illustration to show the arrangement of the thick (myosin) and thin (actin) filaments in the sarcomeres of the myofibrils. Skeletal muscle excitation typically occurs in the following series of events enumerated in the illustration: 1) The binding of acetylcholine from a somatic motor neuron to chemically gated ion channels on the motor end plate (subsynaptic membrane) triggers an action potential in the sarcolemma. 2) The action potential propagates down the length of the muscle fiber. 3) When the action potential reaches the openings of transverse tubules, the depolarization is conducted down these tubules and into the interior of the cell. 4) The depolarization of the transverse tubules induces the opening of ion channels in the sarcoplasmic reticulum, and is released into the cytosol. With Calcium Crossbridges Form Fig 9.6. triggers skeletal muscle contraction. In the absence of, troponin positions tropomyosin on the thin filament in such a way that it blocks myosin s globular heads (thick filament) from binding with complementary sites on the actin of the thin filament. When is released into the cytosol (yellow circles), it binds to troponin (light blue ovals) inducing a conformation change in this protein. As troponin changes shape, it alters the position of tropomyosin, (purple ribbons) exposing the binding sites on the actin molecules and allowing crossbridges to form. sites on the actin molecule. This blocks crossbridges from forming, and hence no contraction can take place. Only if troponin repositions tropomyosin to expose the myosin binding sites can crossbridge cycling occur. This occurs in response to an action potential being triggered in the skeletal muscle fiber, which leads to a series of events collectively called excitation-contraction coupling. The contraction of a skeletal muscle fiber is triggered by an action potential occurring in the sarcolemma (plasma membrane) of that muscle fiber (Fig 9.5). The action potential propagates down the sarcolemma and is conducted down transverse tubules into the interior of the cell. This, in turn triggers the release of from the sarcoplasmic reticulum (a modified endoplasmic reticulum) into the cytosol. The binds to troponin on the thin filament and causes it to undergo a conformational change (Fig. 9.6). This change in the shape of troponin shifts the position of tropomyosin on the thin filaments, exposing binding sites for myosin on the underlying actin and enabling crossbridge formation (the bonding of myosin on the thick filaments to actin on the thin filaments) to commence. p.2

3 Stimulus Intensity Muscle Tension Subthreshold Submaximal Supramaximal Threshold Stimulus Muscle Twitch Parameters Maximal Stimulus Fig Whole muscle contraction in response to stimuli of different strengths. A twitch is a muscle contraction that occurs in response to a single, rapid stimulus that evokes a single, isolated action potential in a muscle fiber. Although single, isolated twitches are not in and of themselves very useful for generating controlled, coordinated movements needed for maintaining homeostasis, observations of twitch contractions present invaluable insights into the basic physiology by which muscle fibers generate tension. Because the action potential is an all or none response, the contraction of a muscle fiber in response to a single action potential is likewise an all or none response. Therefore, there is a minimum stimulus strength that must be applied to the muscle fiber in order to reach threshold, evoke the action potential and, in turn, induce the contraction. Once the action potential occurs, though, no further increase in stimulus strength will increase the strength of contraction, as the gates in the sarcoplasmic reticulum are open for a fixed amount of time once opened. Individual muscle fibers respond to isolated stimuli in an all or none fashion. However, a muscle organ, such as the gastrocnemius muscle, is composed of many individual muscle fibers. By varying the number motor units (groups of muscle fibers innervated by a singe somatic motor neuron) contracting at a given time, the amount of tension generated by the whole muscle can vary. In one of the experiments we are performing today, you will note that the strength of the contraction varies with the strength of the stimulus applied (Fig 9.7). This does not violate the all or none principle. Rather, as stimulus strength is being increased, progressively more muscle fibers reach their thresholds and contract. Thus, the change in tension is due to the number of contracting muscle fibers, not a change in how much tension the individual fibers are generating. Note that stimuli below the minimum strength needed to trigger any of the muscle fibers to reach threshold and undergo an action potential (i.e., subthreshold stimuli) will not trigger any contraction in the muscle. Threshold is considered to be the level of stimulation required to trigger the smallest measurable contraction resulting from the excitation and contraction of the first few muscle fibers. If stimulus is increased above threshold into a range of stimulus intensities called submaximal stimuli, contraction strength will increase with stimulus intensity as progressively more and more muscle fibers in the muscle undergo contraction. Finally, when stimulus strength is increased above a certain level (maximal) no further increase in tension occurs, as all muscle fibers in the muscle are contracting. A rather complex series of events occurs within the time course of a single twitch. The action potential is evoked upon application of the stimulus. That action potential, in turn, propagates down the length of the muscle fibers and triggers the excitation-contraction coupling process (release of from the sarcoplasmic reticulum, binding of to troponin, etc.). Once crossbridge cycling ensues, the muscle fibers contract, generating tension. Tension peaks, but then decreases as the activity of pumps in the sarcoplasmic reticulum reuptake from the cytosol, lowering the ability of actin and myosin to form crossbridges, and reducing tension generation as the fibers stretch back to their original length. These three basic stages (excitation-contraction coupling, tension generation, and relaxation) correlate with three different time phases during the twitch (Fig 9.8). During the latent period (the time between the p.3

4 Stimulus Muscle Tension Peak Tension ½ Peak Tension Baseline Tension Tension Stimulus Tension Latent Period Contraction Time ½ Relaxation Time Stimulus Total Relaxation Time Fig Time intervals of a twitch contraction. application of the stimulus and the onset of contraction), excitation-contraction coupling takes place. During the contraction time (the time from the onset of contraction to peak tension), crossbridge cycling occurs at a high enough rate that the muscle fibers shorten. During the relaxation time (from peak tension to the point when tension returns to baseline), is being pumped back into the sarcoplasmic reticulum, and the muscle is stretching back to its original length. Since the duration of the total relaxation time is often difficult to calculate (since it is difficult to determine exactly when tension returns to baseline levels), researchers commonly use an alternate measurement, the ½ relaxation time, which is the duration it takes for tension to drop from peak tension to ½ of peak tension. Summation and Tetanus Observations of twitch contractions within single muscle fibers or within whole muscle organs can yield important insights into the basic cellular processes involved in converting an electrical signal into a mechanical response by the muscle fibers. However, with a few exceptions, twitch-types of contractions are not the typical type of contraction that skeletal muscles inside the human body produce. That is Tension Stimulus Fig Contractile response of muscle stimulated at varying frequencies. Note the fusion of contractions and the overall increase in tension generated with high frequency stimulation. because in order to enable coordinated body movement and the maintenance of balance and posture (the primary function of most skeletal muscles), tension must be sustained beyond the fraction of a second generated by a twitch. Therefore, most skeletal muscle contractions in the body are tetanic contractions. The basis of tetany, or tetanus (not to be confused with the disease commonly called lock jaw caused by the bacterium Clostridium tetani) within a skeletal muscle organ can be somewhat confusing. Some authors believe that tetany is due solely to the generation of twitches by different groups of motor units occurring asynchronously, so that as one group of motor unit enters its relaxation phase, another is in its contraction phase, etc., so that the sum of these different units contracting is a smooth, steady level of tension. This, however, presumes that individual muscle fibers (or single motor units, for that matter) are incapable of generating sustained tension, which is incorrect isolated individual muscle fibers are able to generate sustained levels of tension with high frequency stimulation. To understand how, we need to keep in mind that the electrical excitation of the p.4

5 skeletal muscle (i.e., the action potential) and the mechanical response of the muscle (tension generation) do not have the same time courses. It takes ~10 msec for an action potential to be propagate down the length of a skeletal muscle fiber in the frog gastrocnemius, whereas the total time for a twitch contraction of the gastrocnemius may be ~150 msec. Thus, many action potentials can occur in the amount of time needed for a single twitch. If a muscle that is relaxing from a contraction is stimulated before it fully relaxes, the sarcoplasmic reticulum will release more, and the cell will begin to contract again without fully relaxing (See Fig 9.9). In effect, then, the twitches partially fused together. If stimulated at progressively higher frequency, the amount of relaxation that occurs in between each twitch is progressively reduced, until a steady state of tension (tetanus, or tetany) is generated. The tetanic contractions generated in today s experiment are caused by sustained, steady levels of tension generated by individual muscle fibers stimulated electrically at high frequency. In most of the tetanic contractions in the body, however, complete tetanus (contraction without any relaxation) is not common. Most sustained contractions are generated by a combination of twitches and partial-tetanic contractions by different motor units whose motor neurons are stimulating the fibers at different intervals and at different frequencies. Interestingly, the amount of tension generated during a tetanic contraction is often substantially higher than that of a maximal twitch. There are several reasons for this. First, when a muscle begins to contract, some of the tension generated by the muscle is absorbed by stretching elastic elements within the muscle s attachments. This can reduce the total tension generated on the attachments in a twitch contraction whereas tetany, these elastic elements are fully stretched and more tension is exerted directly on the attachments. Secondly, recall that each time the muscle fibers undergo action potentials is released from the sarcoplasmic reticulum. The sarcoplasmic reticulum begins to reabsorb this almost as soon as it is released, but it does take time to fully recover all of the. If the sarcoplasmic reticulum is induced by another action potential to release before it has fully recovered all of the previously released, then there will be overall more in the cytosol during the second contraction, more interaction between actin and myosin, and a stronger resultant contraction. Thus action potentials generated in rapid succession can have a summation effect on the strength of the contraction. Functional Contraction Types The contractions generated by skeletal muscles are used for two basic functions: movement of the body and maintaining position and orientation of the body. Isotonic contractions are those that result in the muscle shortening in length, generating movement of a body part. In order for an isotonic contraction to occur, the muscle must contract with enough force to overcome the load applied to the muscle. Isometric contractions, in contrast, are contractions where the muscle is contracting and generating tension, but the muscle does not shorten in length as the force generated by the muscle is equal to the load place on the muscle. Muscles that allow you maintain posture generate isometric contractions to counteract the force of gravity. Electromyograms The action potentials generated by contracting muscle alter the electrical charge in the surrounding extracellular fluid. These electrical changes are conducted through body fluids, and can be detected from the surface of the skin using electrodes applied to the skin. A variety of instruments can detect the differences in charge between the electrodes, amplify them, and generate recordings of these electrical changes called electromyograms (EMGs). EMGs are used diagnostically to detect damage to muscle or to the neural pathways responsible for triggering muscle contractions. p.5

6 Experiment I. Measurements from Bullfrog Gastrocnemius Muscle Contractions. In this experiment we will take direct measurements from a recording of actual frog muscle contractions using the LabScribe software used to record from the iworx physiography system. A. Effects of Stimulus Strength on Tension Generation in Whole Muscle 1. Go to the computer screen, and be sure the software (LabScribe) is running. The top tracing will display the voltage for the electric stimulus applied to the muscle (Fig 9.10). The lower tracing displays the tension generated by the muscle (here expressed as voltage, but normally this would be converted into some measurement of force). The display time should be set to 20 sec, meaning that the 20 seconds of recording are displayed on the screen at any given time. If it is not, change it to 20 sec by selecting the EDIT menu from the top, then PREFERENCES, then enter the desired display time in the middle box of the top line. 2. Scroll the recording to the right using the scroll bar at the bottom of the screen. Eventually you will see a series of recordings where the stimuli and associated contractions from the muscle become progressively stronger. These are recordings of twitches obtained by shocking the muscle with different voltages. Each stimulus is preceded by a marker bar that crosses both recordings. At the bottom of the screen are labels for each marker that provide the voltage of the electrical stimulus applied to the muscle in each case (See Fig 9.10). Stimulus voltages used to trigger the contractions Fig A series of recordings of twitch contractions recorded with the LabScribe software. The upper tracing records the stimuli applied to the muscle (also marked at the bottom of the screen). The lower tracing records the twitch contractions. Markers between recordings appear as brown vertical lines demarcated by the respective stimulus intensities at the bottom of the screen. Measurement markers appear as blue vertical lines. Note that there is and x on the measurement marker where it intersects the tracing for each recording. The difference in contraction tension (here recorded in volts, V2-V1) is located just above the lower tracing to the right. The difference in time between markers (here recorded in hours:minutes:seconds, T2-T1) is located at the top left corner of the screen just below the menus. p.6

7 3. At the top of the screen is a row of clickable buttons. Click on the second to last button in that row (the one that looks like this: ). You will now have two vertical blue bars crossing the screen. Note that at the points where these blue lines cross the tracings for both the stimulus and muscle tension they form a icon. Also notice that as you change the position of the blue lines numerical values in a couple of sets of readouts on your screen change as well: T2-T1, located at the top screen, provides the time difference between the two points in your recording where you have positioned the blue lines; V2-V1, located at the upper right corner of EACH of the two tracings, gives the difference in voltage (amplitude) between the points demarcated with the blue lines. 4. Using the V2-V1 readout for the lower (Muscle) trace, we will measure the strength of muscle contraction in response to different stimulus intensities. All measurements for this exercise will be taken from the lower tracing. Using the pointer on the screen, left click and hold on one of the two blue lines and drag it until it falls on the baseline area to the right of the recording somewhere between the end of the twitch contraction and the marker for the next recording (See the example in Fig. 9.10). Then left click on the other blue line and drag it until it falls at the peak of the twitch. Record the strength of the contraction (voltage) from the V2-V1 readout for each of the twitches (NOTE: record 0V if there is no visible twitch recorded). 5. Consult your data sheet for additional questions. B. Twitch Time Parameter Measurements 1. Reset the display time to 1 second. Under the EDIT menu at the top, select PREFERENCES, then enter the display time (1 sec) in the middle box of the top line. Notice that this stretches out your tracings so that the spike-like twitches seen earlier now appear as more curved waves (Fig 9.11). Scroll through the tracing until you find a nice, robust twitch recording (e.g., at 1.5 or 2.0 V). Click on the button at the top to bring up the two blue marker lines. 2. Measure the latent period of the twitch by placing one marker at the point on the top (stimulus) recording right at the beginning of the square waveform and the other marker at the beginning of the twitch on the lower tracing. Record the difference in time (T2-T1, top left corner). NOTE: The time difference is given in hours:minutes:seconds. Record your measurements in milliseconds! WE WILL DOCK POINTS FOR NOT EXPRESSING VALUES IN MILLISECONDS!!! Fig Position of measurement markers (in blue) for measurement of the latent period (left) and the contraction time (right) p.7

8 Fig Position of measurement markers (in blue) for measurement of the ½ relaxation time. First, position one marker at the beginning of the contraction and the other at the peak of the contraction (left). Record the tension of the contraction (V2- V1) calculate ½ of the contraction tension by dividing the peak tension by 2. Then, move the left hand marker to the right of the peak until the tension reasing (V2-V1) is as close to the calculated value for ½ contraction tension as possible (right). Then record the resultant time difference (T2-T1), which will be the ½ relaxation time. 3. Measure the contraction time by placing one marker at the beginning of the twitch and the other at the highest point in the twitch. Record the difference in time (T2-T1, top left corner). 4. To measure the ½ relaxation time (Fig 9.12), record the tension (V2-V1) of the contraction from the onset of the contraction to the peak. Divide this number by 2 to calculate ½ peak tension. Then, move the marker from the onset of the contraction and move it to the right of the peak until the V2- V1 reading for the lower trace is as close to ½ peak tension as possible. Record the difference in time (T2-T1, top left corner). 5. Consult your data sheet for additional questions. C. Summation and Tetanus 1. Reset the display time to 10 seconds. Under the EDIT menu at the top, select PREFERENCES, then enter the display time (10 sec) in the middle box of the top line. Scroll the recording to the right using the scroll bar at the bottom of the screen until you reach the 1 Hz marker. The markers from here onwards indicate the frequency (Hz = #events/sec) that a 2V stimulus is being applied to the muscle (Fig 9.13). 2. Place one of your two blue measurement markers in the baseline area to the right of the contraction series between the contraction recording and the brown reference marker for the next recording (see Fig 9.13 and note the position of the measurement markers with the recording for 10 Hz). Move the other measurement marker across the peaks in the contraction and note the change in tension (V2-V1) that occurs. Try to locate the highest point of tension in the recording (usually near the end of the contraction) and record this value. 3. Consult your data sheet for additional questions. p.8

9 Peak tension marker Baseline marker Fig Recordings of muscle contractions evoked by series of stimuli at different frequencies. Experiment II: Electromyogram 1. Place three adhesive disk electrodes in a row down the center of the inside of your lower arm. 2. Attach the EMG electrode cables to the electrode disks in the following order (the disks snap on to the cables): 1. Red (+) most proximal (closest to your elbow) 2. White (-) middle 3. Black (gnd) most distal (closest to your wrist) 3. Hold the dynamometer in the hand of the arm to which you have attached the electrodes. 4. Click START in the upper right corner of the screen. Fig EMG tracing illustrating motor unit recruitment. Note that increasing the number of active motor units (indicated in the EMG recording at the top) leads to an increase in tension generation (from the dynamometer recording below). 5. Squeeze lightly on the dynamometer, then release. Note that a light distortion occurs in the upper tracing (see Fig 9.14). p.9

10 This is the electromyogram a recording of the electrical activity of the muscle. Also note the upward deflection of the lower tracing, which is a recording of the pressure being applied to the dynamometer. 6. Squeeze the dynamometer again, slightly harder than before. Notice that the increased strength of contraction is accompanied by an increase in the amount of distortion in the EMG tracing. This is because of motor unit recruitment you are activating more motor units to increase the overall strength of contraction in the muscle organs, thus creating a larger electrical change in the body fluids. Repeat this procedure several times, increasing how strongly you squeeze each time (Fig 9.14). 7. Put down the dynamometer. Extend the fingers into a relaxed position. Flex each finger individually for one second (generating an isotonic contraction) then extend the finger back to its original position. Does the flexion of some fingers produce an EMG signal whereas flexion of others does not? 8. Produce an isometric contraction by placing your extended fingers against the underside of the countertop. Flex each finger against the countertop. Note that when the muscles contract an EMG signal is produced, even if the muscle itself does not shorten. p.10