Proceedings of the Australian Physiological and Pharmacological Society (2004) 34: 1-11 http://www.apps.org.au/Proceedings/34/1-11 ©D.G. Allen 2004 Skeletal muscle function: the role of ionic chang es in fatigue,damage and disease D.G. Allen School of Biomedical Sciences and Institute for Biomedical Research, University of SydneyF13, NSW 2006, Australia Summary Introduction 1. Repeated activity of skeletal muscle causes a Ionic changes are central to the activity of muscle. variety of changes in its properties; muscles become weaker The action potential is caused by rapid movements of Na+ with intense use (fatigue), may feel sore and weak after into the cell and K+ out of the cell. The action potential in repeated contractions involving stretch, and can degenerate the T-tubules triggers rapid release of Ca2+ from the in some disease conditions. This reviewconsiders the role sarcoplasmic reticulum into the myoplasm where it binds to of early ionic changes in the development of each of these troponin initiating cross bridge cycling. An early source of conditions. energy is the anaerobic breakdown of glycogen whose 2. Single fibre preparations of mouse muscle were products are lactate and protons. Thus changes in the used to measure ionic changes following activity-induced intracellular concentrations of Na+,Ca2+ and H+ all occur as changes in function. Single fibres were dissected with part of normal muscle activity.Inthe studies described in intact tendons and stimulated to produce force. Fluorescent this reviewweare concerned with the changes in muscle indicators were micro-injected into the fibres to allow function which accompanyrepeated activity.Weshowthat simultaneous ionic measurements together with mechanical each of the above cations can change during repeated performance. muscle activity and analyse howthis changes contribute to 3. One theory to explain muscle fatigue is that it is muscle function. caused by accumulation of lactic acid producing an Our approach to these issues has been to develop the intracellular acidosis which inhibits the myofibrillar single mammalian muscle fibre preparation first described proteins. In contrast we found that during repeated tetani by Lännergren and Westerblad1.Single fibres are dissected there was little or no pH change but failure of calcium from the flexor brevis muscle of the mouse, clips are release was a major contributor to fatigue. Currently it is attached to the tendons at either end and the muscle fibre proposed that precipitation of calcium and phosphate in the can then be attached to a tension transducer and a motor to sarcoplasmic reticulum contributes to the failure of calcium impose length changes. Electrodes running parallel to the release. fibre allowstimulation. Normally fibres are continuously 4. Muscles can be used to shorten and produce force perfused by a physiological salt solution with pH buffered - or theycan be used to deaccelerate loads (stretched or by HCO3 /CO2.These fibres can be penetrated with eccentric contractions). Aday after intense exercise microelectrodes and microinjected with fluorescent dyes or involving stretched contractions muscles are weak, sore and manyother substances e.g. ions, drugs, peptides, proteins, tender and this damage can takeaweek to recover. Inthis DNAplasmids. At the end of the experiment fibres can be condition sarcomeres are disorganised and there are fixed for light or electron microscopyorsubject to increases in resting intracellular Ca2+ and Na+.Recently we immunofluorescence. The attractions of this approach are demonstrated that the elevation of Na+ occurs through a that anysequence of stimulation (twitches, tetani, repeated stretch-activated channel which can be blocked by either in anypattern) or contraction type (isometric, shortening or + gadolinium or streptomycin. Preventing the rise of [Na ]i lengthening) can be imposed on the fibre and the force and with gadolinium also prevents part of the muscle weakness fluorescence can be monitored from a single cell during after stretched contractions. activity.Inthe experiments described in this reviewwe 5. Duchenne muscular dystrophyisalethal have used fluorescent Ca2+,Na+ or pH indicators to allow degenerative disease of muscles in which the protein continuous measurements of these ions. With use of an dystrophin is absent. Dystrophic muscles are more imaging microscope the distribution of these ions within a susceptible to stretch-induced muscle damage and the single cell can also be determined. stretch-activated channel seems to be one pathway for the increases in intracellular Ca2+ and Na+ which are a feature Muscle fatigue of this disease. We hav e recently shown that blockers of the It is a common experience that the performance of stretch-activated channel can minimize some of the short- muscle gradually declines when muscles are used term damage in muscles from the mdx mouse, which also repeatedly at near their maximum force. This decline of lacks dystrophin. Currently we are testing whether blockers performance, or muscle fatigue, is reflected in reduced force of the stretch-activated channels givensystemically to mdx production, reduced shortening velocity and a slower time mouse can protect against some features of this disease. course of contraction and relaxation. Of course muscles Proceedings of the Australian Physiological and Pharmacological Society (2004) 34 1 Ionic changes during muscle function Figure 1. pH has minimal role in muscle fatigue. Panel A shows measurement of myoplasmic pH throughout a period of fatigue caused by repeated brief tetani. Data from single mouse muscle fibreat22°C. Note that myoplasmic pH changed little despite the development of fatigue.Data from Westerblad & Allen6.Panel B shows tetanic force from mouse single fibres at three different temperatures (12°C, 22°C and 32°C). At eachtemperaturethree tetani areshown with intracellular pH modified by changes in external [CO2]. From above down, the intracellular pHs arerespectively 0.5, 0 and -0.5 greater than the resting level. Note that the effect of a 1 pH unit changeinintracellular pH is muchgreater at 12°C than at 32°C. Tension or force measurements arenormalized as the force per cross-sectional area of the fibreand arequoted in units of kPawhereaPascal is one Newton metre-2.Data from Westerblad et al.8 (reproduced with the permission of the copyright holder). can be used near their maximal capacity in manydifferent the commonest presenting symptoms in medical activities e.g. maximal continuous isometric contractions consultations2.Ofparticular importance is the fact that all such as lifting a piano, repeated contractions such as elderly humans suffer a gradual loss of muscle mass and the running 100 m or a marathon, repeated stretched consequent weakness and rapid fatigue during every day contractions such as walking down a mountain and it would activities contribute to the loss of mobility and be expected that these different activities would affect independence. Single fibres can be useful in the muscle function in different ways. Equally important many investigation of manyofthese situations by appropriate different diseases cause skeletal muscle weakness e.g. choice of conditions. muscular dystrophies, cardiac failure, renal failure, The present reviewwill consider the muscle fatigue starvation, chronic infections etc. and surveysshowthat caused by repeated short isometric tetani e.g. Figs 1 & 2. complaints about muscle weakness and fatigue are among These figures showthat when short (0.3 s) maximal tetani 2Proceedings of the Australian Physiological and Pharmacological Society (2004) 34 D.G. Allen are repeated every fewseconds, the force produced declines reduces force by about 11%. to 50% within a fewminutes. The time scale of this To sum up, acidosis has little direct effect on the experiment is similar that involved when running 1-2 km or force production in mammalian muscles studied at swimming 200-500 m and it seems reasonable to suppose physiological temperatures (for reviewsee 9). Howeverit that the intracellular mechanisms within the muscles are remains true that production of lactic acid is of great similar. importance in exercise physiology and the training of athletes. When glycogen is consumed anaerobically to Lactic acid as the cause of skeletal muscle fatigue produce lactic acid, the ATP production is 3 ATP per glycosyl unit whereas aerobic metabolism within the Since the pioneering research of A.V.Hill, the mitochondria supplies 39 ATP per glycosyl unit. Thus, the accumulation of intracellular lactic acid has been a glycogen store is more rapidly depleted when large amounts dominant theory of muscle fatigue3.Lactic acid of lactic acid are produced anaerobically and muscle accumulates in manyintense fatiguing regimes and can lead performance is severely depressed at lowglycogen levels. to an intracellular acidosis of about 0.5 pH units. There are Also high levels of lactic acid in the blood contribute to the twomajor lines of evidence that have been used to link this discomfort and breathlessness when performing at close to decline of intracellular pH to the contractile dysfunction in maximum levels of oxygen consumption10. fatigue. First, studies on human muscle fatigue of rapid onset have often shown a good temporal correlation Role of intracellular calcium in skeletal muscle fatigue between the decline of intracellular muscle pH and the reduction of force or power production. Second, studies on Giventhat changes in intracellular pH are not the skinned skeletal muscle fibres