Muscle Cell Organization and Contraction: Skeletal Muscle Structure and Function, Lab Reports of Biology

Download Muscle Cell Organization and Contraction: Skeletal Muscle Structure and Function and more Lab Reports Biology in PDF only on Docsity! I. Introduction. Muscle Cell organization.1 About 40% of the total body mass of a human is skeletal muscle. Skeletal muscle is intimately associated with the skeletal system and, combined, they are responsible for supporting and moving the body. Skeletal muscles are composed of numerous multi-nucleated fibers (cells). The fibers lie in parallel and run the length of the muscle (Fig. 1). Single muscle fibers are made up of numerous myofibrils which, when examined under the light microscope with appropriate optics, can be seen to have a banded or "striated" appearance. A B Figure 1. A. Structure of skeletal muscle. B. Schematic of the filaments in the Sarcomeres. The myofibrils are bundles of protein filaments, which are laid down in a series of repeated units called sarcomeres. It is the sarcomeres that give striated muscle its banded appearance. Sarcomeres are bound by Z lines (Fig. 1) and contain thin (Fig. 2A) and thick (Fig. 2B) filaments. The Z-bands function as the “anchor points” to connect sarcomeres together so that as the filaments slide across each other the fiber shortens. The thin filament (Fig. 2A) is composed predominately of actin. Thick filaments (Fig. 2B) are composed predominately of myosin, a large protein with a long rod like “tail” and two “heads” which each contain an actin binding site and ATP hydrolysis site. Muscle Physiology Lab #12 MCB 403 Fall Page 1 of 25 1 See the URL on WWW: http://ortho84-13.ucsd.edu/MusIntro/Over.html A. B. Figure 2. A. Thin filament (Actin). B. Thick Filament (Myosin). Sliding Theory of Muscle Contraction. Muscle contraction is produced by a simultaneous shortening of all sarcomeres within the muscle - i.e. the length of all sarcomeres decreases. The theory that underlies how each sarcomere shortens is called the "SLIDING FILAMENT THEORY" (Fig. 3). Figure 3. Sliding Filament Theory. According to this theory, the shortening of the sarcomeres is produced by an increase in the amount of overlap (produced by the sliding of filaments) between thick and thin filaments in the sarcomere. Thin filaments project from the Z-line and overlap with thick filaments in the center of the sarcomere. During a contraction, the filaments do NOT change length, rather, they slide over one another. The force producing this sliding results from the cyclical interaction of the myosin heads with molecules (Fig. 2B) of the thin filaments. Muscle Physiology Lab #12 MCB 403 Fall Page 2 of 25 Mechanics of muscle contraction5. When the nerve controlling a muscle is stimulated, the resulting action potentials in the muscle fibers set up the sliding interaction between the filaments of the individual myofibrils in the muscle. This sliding generates a force that tends to make the muscle fibers, and therefore the muscle as a whole, shorten. Whether or not the muscle actually shortens, however, depends on the load attached to the muscle. While we might attempt to order the muscles in our arms, to lift an automobile, it is unlikely that the muscles would be able to shorten against such a load. The force developed in an activated muscle is called the muscle tension, and only if the tension is great enough to exceeded the weight of the load will the muscle shorten and lift the load. We can distinguish between two kinds of responses to activation of a muscle. If the muscle tension is less than the load, the contraction is said to be isometric ("same length") because the length of the muscle does not change even though the tension increases. That is, the force exerted on the load by the muscle is not sufficient to move the load, so the muscle cannot shorten. An isometric contraction is diagrammed in Figure 7. In the figure, an isolated muscle is attached to a load it cannot lift. When the muscle is activated, the resulting tension is registered by a strain gauge that measures the minuscule flexing of the rigid strut to which the muscle is attached. A single activation of the muscle triggers a transient increase in tension lasting typically about 0.1 sec. You can easily feel the tension developed in an isometric contraction by placing your palms together with your arms flexed in front of your chest and pushing with both hands, one against the other. Figure 6. Measures of muscle length and muscle tension during Isometric contractions. At the upward arrow, the nerve innervating the muscle is stimulated, causing activation of the muscle fibers. Muscle Physiology Lab #12 MCB 403 Fall Page 5 of 25 5 Most muscle contractions are a combination of isometric and isotonic contractions Figure 7. Measures of muscle length and muscle tension during Isotonic contractions. At the upward arrow, the nerve innervating the muscle is stimulated, causing activation of the muscle fibers. If the tension is great enough to overcome the weight of the load, the contraction is said to be isotonic ("same tension”) because the tension remains constant once it reaches the level necessary to move the load. This situation is diagrammed in Figure 7. The strain gauge again records the increase in tension, as with the isometric contraction. When the tension reaches the level necessary to lift the load, it levels off and the muscle begins to shorten as the load is lifted. During the change in muscle length, the tension remains constant and equal to the weight of the load. This is because it is this weight—hanging from the muscle and support strut—that determines the flexing measured by the strain gauge. Thus, while the muscle is changing length the contraction is isotonic. In an isotonic contraction, the force developed by the sliding filaments in the myofibrils making up the muscle produce work in the form of moving the load through space. One difference between isometric and isotonic contractions can be seen in the different delays between muscle activation and the occurrence of a measurable change in either muscle tension (isometric) or muscle length (isotonic). The tension begins to rise within a few milliseconds, the time required for the effect of the excitation-contraction process to take hold. However, if muscle length is measured instead there is a pronounced delay between activation of the muscle and beginning of shortening. This delay is the time required for the tension to rise to the point where the load is lifted, which will depend on the size of the load. Thus, with light loads the shortening begins quickly, but with heavier loads the onset of shortening is progressively delayed. With sufficiently heavy loads there is no shortening at all and the contraction becomes isometric. In addition, with heavier loads the duration of shortening will be less and the maximum speed of shortening will be slower. In a sense, the measurement of tension during an isometric contraction gives a more direct view of the contractile state of the muscle. Muscle Physiology Lab #12 MCB 403 Fall Page 6 of 25 Control of Muscle Tension Recruitment of Motor Neurons.6 A muscle typically receives inputs from hundreds of motor neurons and neurons innervate several cells each. Thus, tension in the muscle can be increased by increasing the number of these motor neurons that are firing action potentials; the tension produced by activating individual motor units sums to produce the total tension in the muscle. A simplified example is shown in Figure 8. The increase in the number of active motor neurons is called recruitment of motor neurons and is an important physiological means of controlling muscle tension. When motor neurons are recruited into action during naturally occurring motor behavior, such as locomotion or lifting loads, the order of recruitment is determined by the size of the motor unit. As the tension in a muscle is increased, motor units containing a small number of muscle fibers are the first to be recruited; larger motor units are recruited later. Motor Neurons Muscle Fibers Figure 8. A simple muscle consisting of four motor units of different sizes. It shows isometric tension in response to simultaneous action potentials to various combinations of the motor neurons. Thus, when there is little activity in the pool of motor neurons controlling a muscle and the tension in the muscle is low, small motor units are recruited to produce an increase in tension. This insures that the added increments of tension are small and prevents large jerky increases in tension when the tension is small. As tension increases, however, further increases in tension must be larger in order to make a significant difference; thus, larger motor units are added, resulting in larger increments of tension when the background tension is already high. This behavior is referred to as the size principle in motor neuron recruitment. In Figure 8, for example, it would be expected that tension would be increased by adding the smaller motor units (l and 2) first and the largest unit (4) last. Muscle Physiology Lab #12 MCB 403 Fall Page 7 of 25 6 See a description of motor units at http://nmrc.bu.edu/mul/motunit/motunit.htm#MotorUnit1 IIB. PowerLab Setup. You will be stimulating the gastrocnemius muscle. You will be recording the muscle contraction (initially) using a displacement transducer. IIB1. Setup (for Isotonic contractions). Make connections to the PowerLab as illustrated in Figure 14. You will stimulate the muscle with the integrated stimulator, and record the muscle contraction using the displacement transducer using the Scope program. Fill the chamber with Ringers solution taking care not to douse the transducer with fluid. Clamp Pulley Banana leads to electrodes Three- fingered clamp Movable Stage Electrodes for muscle stimulation Muscle Mounting Rod Thread from muscle to transducer Gastrocnemius BNC Lead Displacement Transducer Hook for weights Ring Stand Figure 14. Displacement transducer setup to measure Isotonic contractions. IIB2. Transducer Mounting. 1. Mount the displacement transducer in a clamp mounted on a ring stand. 2. Attach a string to the hook on the transducer (do so by making a loop in the end of a piece of string, not by tying it directly to the hook). 3. Pass this thread over the pulley and attach to the Gastrocnemius. 4. Move the stage to take up tension on the thread. If need be hang a weight on the hook attached to other end of the displacement transducer to keep tension on the thread. Muscle Physiology Lab #12 MCB 403 Fall Page 10 of 25 IIB3. PowerLab Connections. 1. Connection the Stimulating electrodes to the PowerLab (Fig. 14). 2. Connect the displacement transducer to the DIN-8 connector for Channel #1 on the PowerLab. 3. Connect the output of the Stimulator to Channel #3. IIB4. Setup of the Data Acquisition software. 1. Open the Scope file “MuscScopeDisp40308”. 2. Check that the initial Input Amplifier settings for Channel #1 (Displacement) are Differential 3. Check that the initial Range is 200 mV. 4. Check that the initial Input Amplifier settings for Channel #3 (Stimulus) are are Single ended 5. Check that the initial Range is 10 V. IIB4. Calibration of the Displacement Transducer. 1. Make sure all the cables are connected as is shown in Figure 14. 2. Choose the Input Amplifier for Channel #1 3. Using the movable stage to place the displacement transducer to the center of its throw. Remember that the thread must be taunt. Use a weight if needed (Fig. 14). 4. Click on the Units.. button to go to the Units Calibration dialog box and highlight the waveform. 5. Click on the upper arrow (not triangle) and the value of the signal will be transferred to the left hand box. 6. Type 0. into the right hand text box. 7. Click on the OK button to accept this and return to the Input Amplifier dialog box. 8. Move the transducer a known distance. 9. Click on the Units.. button to go to the Units Calibration dialog box and highlight the waveform. 10. Click on the lower arrow (not triangle) and the value of the signal will be transferred to the left hand text box. 11. Type in the distance that you moved the transducer into the right hand text box. 12. Click on the OK button to accept this and return to the Input Amplifier dialog box. 13. Move the transducer a couple of times a known distance each time checking that the digital display matches the distance you are moving the rod. If not re-calibrate until it does. 14. Selected the appropriate units while in the Units Calibration dialog box (such as cm). Muscle Physiology Lab #12 MCB 403 Fall Page 11 of 25 III. Protocols. Isotonic Contractions IIIA. Stimulus-response relationship. Threshold and Maximal Contraction (Single twitch). IIIB. Work done by Muscles. IIIC. Summation and Tetanus. Isometric Contractions IIID. Length Tension relationship. Muscle Physiology Lab #12 MCB 403 Fall Page 12 of 25 Maximal Stimulus. Record the Twitch amplitude, stimulus and input amplifier settings to that Page Comments. and indicate this is the Maximal Twitch. Save the file (but don’t close it) and go to the next page. 6. Reduce the stimulus strength to Twitch Threshold. Moving up in increments of 25% up to 125% of Maximal Stimulus. Record the Twitch amplitude, stimulus and input amplifier settings to that Page Comments. and go to the next page. Activate the Overlay option as you proceed so that you can watch the gradual increase (Fig. 17) of the Displacements with increasing stimulus levels. Record three replicates at each stimulus level. This data will be used to construct a Stimulus/Response Curve (Fig. 18). IIIA6. Calculations. Determine the relationship between strength of stimulus (V) and amplitude of the Twitch (cm). Is it linear? Can it be described in a simple linear equation? Hint: try the line fit functions in Cricket Graph and be able to explain how well the line fits the data. IIIA7. Data Presentation. Present the data in format similar to that in Figure 18, supporting what is shown in each. IIIA8. Topics that should be addressed in the report: Does the muscle react in a similar manner as you observed in the NeuroMuscular lab? How does your data compare to other groups’ data? 1.31.21.11.00.90.80.70.60.50.40.30.20.10.0 -1 1 3 5 7 Stimulus Threshold and Maximal Levels. Stimulus (mV) D is pl ac em en t (c m ) Twitch at Threshold Stimulus Maximal Contraction Supra-Maximal Contractions Threshold Stimulus Figure 18. Plot of Stimulus-Response (displacement) Curve. Muscle Physiology Lab #12 MCB 403 Fall Page 15 of 25 IIIB. Work done by Muscles. IIIB1. Introduction. The purpose of this section is to determine the amount of Work done under various loads. Although energy is used in both isometric And isotonic muscle contractions only in isotonic contractions can any work be done. Work is defined as weight (load) times the distance it is moved (Remember that weight is equal to mass * gravity, and that we are using units for mass i.e.. grams). We will examine the interrelationship of work versus load and the concomitant idea of power. ∆D Muscle Contraction with a 10 gram weight attached. ∆ D = 0.31 mm, thus Work = 0.31 mm * 10 gram Work = 3.1 gram mm Figure 19. Trace of stimulus and muscle contraction indicating the parameters to be measured in order to calculate work. Muscle Physiology Lab #12 MCB 403 Fall Page 16 of 25 IIIB2. Set up. 1. Using the Isotonic setup shown in Figure 14, record muscle displacement on Ch #1 and Stimulus on Ch #3. Set the stimulus to elicit a 80% Maximal twitch. 2. 1.00ms 0.210V5.00ms Figure 20. Example of stimulus parameters for Work determination. IIIB3. Example of Data. ∆D ∆T Velocity (mm/sec) = ∆D/∆T Figure 21. Trace of muscle contraction and stimulus. Indicated on the contraction trace the area used to determine muscle contraction velocity. IIIB4. Variables. Load Work Velocity Muscle Physiology Lab #12 MCB 403 Fall Page 17 of 25 IIIC3. Example of Data. Figure 25. Example of fused tetanus. IIIC4. Variables. Summation Inter-Stimulus Interval Incomplete Tetanus Inter-Stimulus Interval Complete Tetanus Inter-Stimulus Interval Muscle Physiology Lab #12 MCB 403 Fall Page 20 of 25 IIIC5. Procedures. 1. After checking to see that the set up is correct, record the multiple stimuli and muscle twitch by pressing the Start button. 2. If the trace does not show any sign of summation decrease the inter-stimulus interval until you get summation (un-fused tetanus). 3. Upon getting summation (Fig. 23) record the stimulus settings to the Page Comment Notes. and indicate this is Incomplete Tetanus. Go to the next page. 4. Decrease the inter-stimulus interval. At a certain interval the amount of contraction will become greater than that seen at lower intervals - this is Incomplete Tetanus. 5. Upon getting un-fused tetanus record the stimulus settings to that Page Comment Notes and indicate that this is Incomplete Tetanus. Go to the next page. 6. Decrease the inter-stimulus interval further. Finally, you will not see single twitches, rather a single prolonged contraction - this is fused tetanus. 7. Upon getting fused tetanus record the stimulus settings to that Page Comment Notes. and indicate this is Fused Tetanus. Go to the next page. 8. Reduce the stimulus amplitude to zero (0) and observe the slow relaxation of the muscle. 9. Save the file (but don’t close it) and go to the next page. 10. Repeat steps 1-9, three times. 11. Save the file and close it. IIIC6. Calculations. Determine the inter-stimulus interval needed to produce Incomplete Tetanus. Measure the Peak contraction to Peak contraction time interval for Incomplete Tetanus. Determine the inter-stimulus interval needed to produce Fused Tetanus. Measure the Peak contraction to Peak contraction time interval for Incomplete Tetanus. Determine the inter-stimulus interval needed to produce Fused Tetanus. IIIC7. Data Presentation. Present a table of your data and that of the section as a whole. How does your data compare to the sections as a whole. IIIC8. Topics that should be addressed in the report: Does the inter peak time interval equal that of the inter-stimulus interval? If not Why? How does your data compare to other groups’ data? Muscle Physiology Lab #12 MCB 403 Fall Page 21 of 25 IIC. Setup (for Isometric contractions). Make connections to the PowerLab as illustrated in Figure 26. You will stimulate the nerve with the integrated stimulator and record the force of muscle contraction simultaneously using the Scope program. Fill the chamber with Ringers solution taking care not to douse the transducer with fluid. String from muscle to transducer Electrodes for muscle stimulation Muscle Mounting Rod Clamp Clamp Movable Stage Force Transducer Ring Stand Figure 26. Recording/Stimulating arrangement using a PowerLab and a force transducer. This will allow for measurements of Isometric contractions. IIC2. Transducer Mounting. 1. Mount the force transducer in a clamp mounted on a ring stand. 2. Attach a string to the hook on the transducer (do so by making a loop in the end of a piece of string, not by tying it directly to the hook). 3. Move the stage to take the play out of the string. IIC3. PowerLab Connections. 1. Connection the Stimulating electrodes to the PowerLab (Fig. 26). 2. Connect the force transducer to the DIN-8 connector for Channel #2 on the PowerLab. 3. Connect the output of the Stimulator to Channel #3. Muscle Physiology Lab #12 MCB 403 Fall Page 22 of 25 IIID6. Calculations. Determine the tension produced at each length of the muscle. IIID7. Data Presentation. Plot Tension vs. Muscle length (Fig. 29). T en si on Length In-Vitro In-Vivo Figure 29. Length-Tension curve. Note that the more traditional curve applies to a single fiber (In-Vitro), while what we should be seeing in lab using a whole muscle is shown in the In-Vivo trace. IV. Questions. 1. Why did direct electrical stimulation produce contractions of the muscle? 2. Low-intensity electrical shocks produced no muscle contractions. Why? 3. Increasing the intensity of single shocks applied to the muscle produced contractions that increased in magnitude (within a certain range). Explain this observation. 4. High-intensity electrical shocks produced a "maximum" contraction - i.e. further increases in shock intensity did not produce an increase in contraction magnitude. Explain this observation. 5. Why did the amount of work done initially increase with increased weight? 6. Why did the amount of work done decrease when heavier weights were used? 7. If the amount of contraction is dependent upon the amount and the time course of elevated intracellular calcium concentration, why do single twitches produce the same amount of contraction? 8. Incomplete tetanus is indicated by an increase in the amount of contraction. According to the above question (#7) this may be explained by an elevated intracellular [Ca++]. What causes this and how is stimulus frequency related to this phenomenon? 9. Why is the rate of muscle relaxation much slower after tetanus that after a single twitch? 10. Why does tension depend upon the length of the muscle? 11. What would you predict is the resting length of your frog Gastrocnemius muscle? V. References. Carafoli, E. and J.T. Penniston 1985. The Calcium Signal. Scientific American. November. pp. 50. Cohen, C. 1975. The Protein Switch of Muscle Contraction. Scientific American. November. pp. 36. Murray, J.M. and A. Weber 1974. The Cooperative Action of Muscle Proteins Scientific American. February. pp. 58. Muscle Physiology Lab #12 MCB 403 Fall Page 25 of 25 I. COMPONENTS OF MUSCLE CELL • SARCOMERE - FUNDAMENTAL UNIT OF MUSCLE CELLS - BOUND BY Z LINE/BAND: ANCHOR • THICK FILAMENTS (MYOSIN) - ONE TAIL - TWO HEADS (ACTIN BINDING SITE & ATP HYDROLYSIS SITE) • THIN FILAMENTS (ACTIN) - TROPONIN (KEY) - TROPOMYOSIN (DOOR TO MYOSIN HEAD BINDING SITE) II. SLIDING THEORY OF MUSCLE CONTRACTION "MUSCLE CONTRACTION IS PRODUCED BY SIMULTANEOUS SHORTENING OF ALL SARCOMERS WITHIN THE MUSCLE" • SHORTENING: PRODUCED BY INCREASED AMOUNT OF OVERLAP BETWEEN THICK AND THIN FILAMENTS: SLIDING • FILAMENTS DO NOT CHANGE LENGTH -- ONLY SLIDE • FORCE DUE TO CROSS BRIDGE CYCLING - CROSSBRIDGE = MYOSIN HEAD ACTIN FILAMENT INTERACTION Muscle Physiology Appendix Lab #12 A - 1 of 3 III. MECHANICS OF MUSCLE CONTRACTION • SLIDING FORCE TENDS TO MAKE MUSCLE SHORTEN • MUSCLE TENSION: FORCE DEVELOPED IN ACTIVATED MUSCLE • LOAD: DETERMINES IF MUSCLE SHORTENS AS A WHOLE -TENSION > LOAD => SHORTENING -TENSION < LOAD => NO SHORTENING • TWO ACTIVATED MUSCLE RESPONSES -ISOMETRIC: LENGTH DOES NOT CHANGE, T < L -ISOTONIC: LENGTH SHORTENS, T > L IV. LENGTH - TENSION RELATIONSHIP (1) (2) (3) optimal length -- maximal forcetoo short -- no force too long -- no force crossbridges can form but no room for filaments to move filaments are too exended for crfossbriges to form crossbridges can form, filaments can slide past each other Length Te ns io n • ACTIN FILAMENT OVERLAP - DECREASED TENSION DUE TO NO ROOM TO SLIDE • NORMAL LENGTH - OPTIMAL OVERLAP - MAXIMUM SLIDING POTENTIAL Muscle Physiology Appendix Lab #12 A - 2 of 3