Muscle Physiology

Bill Sellers Email: wis@mac.com This lecture can be found at:

http://mac-huwis.lut.ac.uk/~wis/lectures/

In order to understand how skeletal muscles work to produce to movement in the body so called macro-properties it is necessary to understand what happens at a microscopic level. In this lecture I will describe the microscopic anatomy of muscle and start to explain what we think is going on at a cellular level to bring about muscle contraction.

Figure 1 Macro Muscle Structure [McGinnis 1999] The basic structure of muscle is shown in figure 1. The muscle belly is a bundle of bundles: the bigger bundles are called fascicles and each fascicle is a bundle of muscle fibres. The coverings of each bundle also have names with the epimysium covering the muscle itself, the perimysium covering the fascicle and the endomysium covering the fibres. Unlike most tissues in the human body the muscle fibre does not consist of a large number of cells. Instead the cell membranes have fused together to produce one large supercell or syncytium containing multiple nuclei. The remaining outer cell membrane is called the sarcolemma and the contents are called sarcoplasm. Within the fibre individual contractile units are called myofibrils.

Figure 2 Light Microscopy [Alexander 1992] When viewed using light microscopy muscle fibres appear striated as in figure 2. This suggests some underlying structure at the sub-cellular level. The striations can be seen more clearly if viewed under polarised light but to get a really good look you need to use electron microscopy.

Figure 3 EM Medium Power [Keynes & Aidley 2001] In figure 3, a medium power electron micrograph, we can see these details. The different sections are identified with letters. The dark and light bands that make up the striations are know as I (isotropic) and A (anisotropic) bands. Within the A band there is a mid-tone H (heller) zone and dark lines can be seen in the middle on the bands: the Z (Zwischenscheiben) and M (Mittelscheibe) lines. The L zone can sometimes be seen as a slightly lighter area around the M line. This structure repeats along the length of the fibre and the distance between repeats (actually the distance between neighboring Z lines) is called a sarcomere (usually a few m). This arrangement has been interpreted as a set of interdigitating filaments as shown in the diagram below the micrograph.

Figure 4 EM High Power [Keynes & Aidley 2001] When viewed at higher power (figure 4) there is more support for this interpretation. In addition cross-linking can be seen running between the filaments. These are know as crossbridges.

Figure 5 Filament molecules [Keynes & Aidley 2001] Furthermore it is possible to identify the proteins responsible for this structure with actin filaments in the I band (light) and myosin filaments in the A band (dark). These two proteins seems to be primarily responsible for producing contractions. Nebulin and titin seem to be there to help maintain the arrangement.

Figure 6 Structure Overview [McGinnis 1999]

Figure 6 shows an overview of the probably intracellular organisation or a muscle fibre based on our current state of knowledge. What we want to know is how this complex arrangement of molecules actually brings about musclular contraction. The standard theory nowadays is called the sliding filament theory which suggests that the actin and myosin filaments themselves do not change length but slide over each other, increasing the amount of overlap during contraction and reducing the amount of overlap during relaxation.

Figure 7 Tension & Sarcomere spacing [Keynes & Aidley 2001] Figure 7 shows some experimental evidence to support this theory. It shows the maximum force that a muscle can produce (in this case a frog gastrocnemius muscle that has been dissected out and put in a test rig) when it is stretched to different lengths. As the length of the muscle is altered so the sarcomere length changes and this can be measured by observing the distance between striations. As you can see from the graph the tension falls to zero for very short and very long muscle and is highest in the middle.

Figure 8 Myofilament Arrangements [Keynes & Aidley 2001] Figure 8 shows how this sarcomere length translates to degree of overlap of the filaments. At maximum stretch there is no overlap and no tension. At minimum length there is overlap but the myosin filaments are pushed up against the Z lines so the muscle cannot shorten any

further and no external force is generated. At mid-lengths the force generated varies due to the amount of overlap changing and to interference between the actin strands.

Figure 9 Myosin Molecule [Keynes & Aidley 2001] It is a good theory but somehow we need to come up with a molecular method for producing this sliding movement. The force generated seems to be a function of the cross-bridges which seem to be primarily associated with the myosin molecules. Figure 9 shows a more detailed structural analysis of the myosin molecule. It consists of 4 peptide subunits and forms complex chains as indicated.

Figure 10 Actin Molecule [Keynes & Aidley 2001] As we can see from figure 10, actin chains are much simpler. Actin is a globular molecule and forms spiral filaments in association with two other proteins: troponin and tropomyosin. When actin and myosin are mixed they produce a viscose complex called actomysion and this complex acts as an ATPase that is it splits ATP (adenosine triphospate) into ADP (adensosine diphospate) and P (phosphate) with the normal concomitant release of energy. ATP ADP + P is the standard chemical reaction that provides energy for cellular activity so this is a promising finding. This reaction seems to be controlled by calcium ion concentration (with no Ca2+ actomyosin disassociates).

Figure 11 In vitro actin & myosin [Keynes & Aidley 2001] This experiment can be extended by producing a lawn of myosin on a glass plate which can be observed to move actin filaments under the influence of ATP (figure 11).

Figure 12 S1 lever arm [Keynes & Aidley 2001] Detailed analysis of the myosin components has suggested that the S1 part swings when ATP binds and that this movement acts like a ratchet pulling the actin filament along the myosin filament. The calcium dependence of the chemical reaction appears to be controlled by the tropomyosin and troponin proteins since in their absence (if pure solutions of actin and myosin are used) actomysin will split ATP molecules without calcium. Thus the evidence for this mechanism for muscle contraction is very good. However it is still only half the story. We still need to be able to control the muscle contraction.

Figure13 Resting and Action Potentials [Keynes & Aidley 2001] Muscle fibres are excitable cells just like nerve cells. This means that at rest the inside of the cell is 100mV negative compared to the outside of the cell as shown in figure 13. When stimulated, which can be chemical, electrical or even physical, there is a brief change in the local electrical potential: the cell membrane becomes depolarised and the inside becomes 20mV positive with respect to the outside. This effect is local to the stimulus but the change in potential stimulates the surrounding membrane so that part of the cell becomes depolarised. However immediately after depolarisation the membrane is briefly no longer sensitive to further stimulus so secondarily stimulated areas cannot stimulate the area that stimulated them. They do stimulate previously unstimulated neighboring areas and this way a pulse of depolarisation propagates throughout the whole muscle fibre.

Figure 14 Extracellular recording [Keynes & Aidley 2001] Figure 14 shows how this wave of depolarisation looks to external electrodes. This shows how the waveform looks rather different. This waveform is from a single muscle fibre and recordings from a whole muscle is a summation of many individual fibre electrical signals and so is a much more complex shape.

Figure 15 Ion concentrations [Keynes & Aidley 2001] The electrical potential is generated by differences in ionic concentrations. These ion concentrations are electrogenic when the membrane is selectively permeable. The resting potential is due to the selective permeability of the membrane to potassium ions and can be calculated using the Nernst equation (at room temperature EK = 25loge[K]o/[K]i mV). The values do not quite add up because the cell membrane is not perfectly selectively permeable but experiments varying the external potassium concentration suggest that the principle is correct.

Figure 16 Ion channels [Keynes & Aidley 2001] The difference in concentration needs to be produced in the first place (and maintained because of leaks) and this is achieved by a sodium/potassium exchange pump (figure 16). This pump pumps out 3 Na+ ions for every 2 K+ ions it pumps in. Thus the pump is itself electrogenic. It is also active, requiring ATP to function. As well as this active pump the membrane has two electrically gated channels that are important for the action potential.

Figure 17 Action potential processes [Keynes & Aidley 2001] Figure 17 illustrates the processes that occur during an action potential. The membrane potential switches rapidly from approximately the Nernst potential for potassium to approximately the Nernst potential for sodium (depolarisation) then back again. Remembering that the potential across a membrane depends on the ions it is selectively permeable to, this is therefore associated with a brief increase in the sodium permeability due to the activation of the voltage dependent sodium channel. This channel activates very quickly but only stays open for a very short period causing the positive voltage pulse. The potassium channel opens and closes rather more slowly and causes a period of hyperpolarisation (i.e. a more negative potential) just after the positive spike. This potassium effect allows the potential to return to normal more quickly but is not strictly necessary. The local circuits show how the action potential in one area causes the depolarisation of neighboring areas.

Figure 18 Calcium effects [Keynes & Aidley 2001] The action potential still needs to cause muscle contraction. Experiments measuring the internal calcium concentration of a muscle fibre using the jelly fish protein aequorin (figure 18) indicate that depolarisation leads to an increase in calcium level and we already know that this triggers the actin and myosin binding. However the calcium ions do not appear to come from outside the cell.

Figure 19 Muscle T system [Wheater et al. 1979] Muscle fibres have a system of transverse tubules (figure 19) invaginations of the surface sarcolemma that run into the fibre. These tubules are closely associated with the sarcoplasmic reticulum (a series of membranous structures visually similar to the endoplasmic reticulum in normal cells). This system would seem ideally suited to transmit the action potential signal.

Figure 20 Ryanodine receptor [Keynes & Aidley 2001] The T tubule membrane is closely associated to the sarcoplasmic reticulum by structures known as feet. These consist of two sets of receptors (DHP and ryanodine) and it is believed that the action potential causes a conformational change to the foot complex which leads to the release of calcium from the sarcoplasmic reticulum cellular compartment into the sarcoplasm where it leads to contraction. This calcium is eventually transferred back into the sarcoplasmic reticulum by the active calcium pump as indicated in figure 20.

Figure 21 Motor end plate [Wheater et al. 1979] The only area still to cover is the coupling of the nerve to the muscle. This occurs at the motor end plate (figure 21). This is effectively a synapse with the neurotransmitter acetylcholine being released by the nerve endings. Note how one nerve contacts several muscle fibres this is a motor unit since these fibres will always contract together.

Figure 22 EM of frog neuromuscular junction [Keynes & Aidley 2001] In figure 22 you can see the vesicles (V) containing acetylecholine ready for release into the synaptic cleft (C). Note the prominent folds (F) in the sarcolemma and the numerous mitochondria (Mi).

Figure 23 End-plate potential [Keynes & Aidley 2001] The acetylecholine activates specific channels on the sarcolemma which are believed to allow both sodium and potassium ions through. This causes a small change in the resting potential

(end-plate potential: see figure 23) which is just sufficient to trigger the voltage gated sodium channels and start the required action potential. In summary: Force is generated by filaments of actin and myosin sliding over each other This is triggered by calcium ions The calcium is released from the sarcoplasmic reticulum by an action potential Action potentials are due to the change in membrane permeability from potassium to sodium and back again This is initially triggered by acetylecholine receptors which change the resting potential by increasing both the potassium and sodium permeability

Bibliography Alexander RMcN. Exploring Biomechanics. 1992 New York: Scientific American Library. Keynes RD, Aidley DJ. Nerve and Muscle. 2001 Cambridge: Cambridge University Press. McGinnis PM. Biomechanics of Sport and Exercise. 1999 Chamapaign, IL: Human Kinetics. Wheater PR, Burkitt HG, Daniels VG. Functional Histology. 1979 Edinburgh: Churchill Livingstone