|Dr David G. Allen||Professor (in-charge)||University||1989-|
|Lorraine Kerr||Senior Technical Officer (0.5)||NHMRC||1989-|
|Chris Balnave||PhD student||APA||1992-|
|Dr Eva Chin||Postdoctoral Research Fellow||NHMRC||1993-|
|Dr Anna Park||Visiting Scholar||1993-|
Current effective full-time personnel = 4.5
Research in this Laboratory is concerned with the regulation of intracellular ions, particularly calcium, sodium and protons, and with their effects on cell function.
The distribution of ionic and myofibrillar changes in muscles damaged by stretch
Muscles which are stretched during contraction are susceptible to a form of injury which involves reduced force and a delayed onset muscle stiffness and soreness. With Chris Balnave the Laboratory has developed a single fibre model of stretch-induced muscle injury and it was shown that resting intracellular calcium is raised, while the intracellular calcium during tetani is reduced. It was shown that the reduced tetanic calcium is part of the cause of the reduced force in muscles injured by stretch. The distribution of these changes provides information about the potential mechanisms involved - for instance if localized damage to the surface membrane causes the elevated resting calcium, then the distribution of the elevation might also be localized. Therefore, the distribution of both resting and tetanic calcium was measured by digital fluorescence microscopy before and after a series of stretches which caused injury. It was found that both changes were uniformly distributed over the fibre. This result suggested that the injury mechanism is relatively uniformly distributed over the whole muscle fibre. In other approaches to this issue a collaboration with Dave Davey is examining the distribution of myofibrillar damage in the electron microscope. In addition, the distribution of sarcomere irregularities in fixed single fibres is being examined using protein fluorescence in the confocal microscope.
The mechanism of action of caffeine in skeletal muscle fibres
Caffeine is well known to increase tetanic force in skeletal muscle and to slow relaxation. The increased force is attributed to increased calcium release from the sarcoplasmic reticulum, while the mechanism of slowing of relaxation is not well understood. With Håkan Westerblad who was on a short visit from the Karolinska Institute in Sweden, it was shown that caffeine increases intracellular calcium, increases the calcium sensitivity of the contractile proteins and slows the sarcoplasmic reticulum calcium pump. Using the model, developed by the Lab., of intracellular calcium movements and force development, it was shown that these mechanisms acting in concert were adequate to explain the slowing of relaxation which occurred in the presence of caffeine.The cause of reduced calcium release in fatigued skeletal muscle
In previous work by the Lab it was shown that during muscle fatigue the intracellular calcium release declines and that this makes an important contribution to the reduced force which characterizes muscle fatigue. Most of the reduced calcium release recovers quickly but a small component fails to recover and causes a long-lasting form of fatigue, known as low frequency fatigue. With Eva Chin, possible mechanisms for this long-lasting reduction in calcium release have been investigated. The starting hypothesis was that it might be caused by the elevation of intracellular calcium which inevitably accompanies the increased muscle activity which leads to fatigue. Some recent biochemical studies have suggested that a calcium-activated protease might selectively damage the calcium release channel in the sarcoplasmic reticulum when calcium is elevated. To test this hypothesis caffeine was used to increase intracellular calcium to about the same level as occurred in fatigue, leading to low frequency fatigue. The results showed that elevated calcium alone is capable of producing low frequency fatigue; so the hypothesis was supported.The causes of slowing of relaxation in fatigued frog muscle
Repeated tetani in skeletal muscle lead to a reduction in force and a slowing of relaxation. In previous studies in mouse muscle the Lab showed that most of the slowing of relaxation was caused by slowing of crossbridge kinetics. In the present study the causes of slowing of relaxation in fatigued Xenopus single muscle fibres have been reinvestigated. Brief tetani were repeated at short intervals until force had declined to 85% of control and relaxation was substantially slowed. The figure shows intracellular calcium, Ca2+-derived force and force for the 1st (continuous line) and the 25th (intermittent line) tetanus. It is clear that the rate of decline of [Ca2+]i was substantially slowed in the 25th tetanus. Because the relation between calcium and force is conditions. These two relations were then used to convert the control and fatigued calcium signals to Ca2+-derived force. Measurements of time to 50% relaxation suggested that half of the slowing of relaxation was apparent in the Ca2+-derived force and was caused by the reduced rate of decline of intracellular calcium. The other half of the slowing of relaxation was seen only in the force records and represented changes in crossbridge kinetics.
Intracellular [Ca2+]i , Ca2+-derived force and force from Xenopus single muscle fibre. Continuous line shows a control tetanus; dashed line shows the 25th tetanus in a series when the muscle was fatigured.
The role of intracellular sodium and pH in ischaemic muscle damage in the heart
The Lab has shown that intracellular calcium is elevated in ischaemia and is associated with muscle damage when ischaemia is prolonged. With Anna Park, a visiting scholar from Gyeongsang National Univ., Korea, the Lab is currently exploring the mechanism of the rise of calcium. One hypothesis is that acidosis, caused by lactic acid accumulation, triggers sodium entry to the cell in exchange for protons and that subsequently calcium entry occurs in exchange for sodium. This hypothesis has been tested by measuring both intracellular pH and sodium during ischaemia. Sodium did not rise in ischaemia, despite a large acidosis, so it appeared that the sodium-hydrogen exchanger was blocked during ischaemia. Conversely, during reperfusion, sodium showed a transient rise which was inhibited by blockers of the sodium-hydrogen exchanger.
The mechanism of failure of calcium release in fatigue
Earlier work by the Lab showed a correlation between failure of calcium release and the time at which intracellular ATP starts to decline. Using caged ATP, the Lab will explore whether a reduced ATP concentration causes the failure of calcium release by suddenly increasing ATP.Long term fatigue studies
All current work has been concerned with muscle fatigue which is induced by intense muscle activity and takes only 5-10 min to develop. Many forms of human activity lead to slower development of fatigue in which the main metabolic change is gradual consumption of glycogen. A series of experiments are planned to explore the cause of this slowly-developing muscle fatigue.
The Laboratory is located in rooms 348A-H of the Anderson Stuart Building. Prof. Allen's office is 348D. Four independent projects can be accommodated and each is based around a fluorescent microscope and accessory equipment. One microscope is adapted for digital fluorescent imaging. The Lab also has access to the confocal microscope located in Max Bennett's Laboratory. A comprehensive range of stimulating, recording and analysis equipment and software is available.
|NHMRC||The cause of failure of calcium release
in fatigued skeletal muscle
|NHMRC||Intracellular calcium in skeletal
muscle during length changes
|NHF||Role of changes of intracellular ions
during myocardial ischaemia
|NHF||Regulation of intracellular ions
during cardiac ischaemia
Total for 1994: $138,935 + share of confocal equipment grants
Total for 1995: $141,108
|Med 2||Med 3||MedSc 3
|Practical classes (no.)||24||(8)||-||-||-||24|
|Practical discussion sessions||8||-||-||-||8|
Additional time was devoted to lecture preparation, setting and marking of exams and discussions with colleagues on curriculum development.