MUSCLE CELL FUNCTION LABORATORY

David G. Allen

Research in this laboratory is concerned with the regulation of intracellular calcium and other ions and their effects on cell function.

RESEARCH in 1993

Intracellular calcium in stretched muscles

Eccentric muscle damage is the pain and weakness which develops in muscles which have been forcibly stretched during contraction: for instance, in walking down a mountain. With Chris Balnave the Laboratory has developed a model of this condition in isolated single fibres of mouse muscle. In this technique, the fibre is set up in an apparatus which allows muscle length to be changed in a controlled manner and the muscle fibre is also injected with the fluorescent indicator indo-1, which allows estimates to be made of intracellular calcium. It was found that if the fibre is given a series of 10 tetani, each with a stretch applied in the middle of the tetanus, then the subsequent isometric force production is permanently reduced and, in addition, there is a greater reduction of force at low stimulus frequencies (called low frequency fatigue). Both of these findings are characteristic of the condition in humans. The novel findings were that intracellular calcium during tetani is reduced after the stretch series and that caffeine, which increases intracellular calcium, can overcome this reduction when it is mild, but not when it is severe. This showed, for the first time, that reduced calcium release is the cause of part of eccentric muscle damage. It was also shown that resting intracellular calcium is raised after the stretch series and this may be the cause of the some of the longer term damage which develops in this condition.

Mechanism of the slowing of relaxation after a long tetanus

It has been known for at least 50 years that a very long tetanus relaxes more slowly than a brief tetanus. Relaxation is caused by three processes in series: (i) reduction of intracellular calcium, (ii) dissociation of calcium from its binding protein troponin, and (iii) detachment of crossbridges. It follows that any of these processes might be slowed after a long tetanus and cause the slowing of relaxation. One current hypothesis is that another binding protein, parvalbumin, which is present in high concentration in this (WHICH?) muscle, might contribute to the lowering of calcium after a brief tetanus. After a long tetanus, however, it would become saturated with calcium and no longer contribute to relaxation. Experiments conducted in 1993 reject this hypothesis by showing that the rate of decline of calcium is no greater after a long tetanus than a short one. It follows that one of the latter processes described above, probably the detachment of crossbridges, is slowed after a long tetanus and causes the observed effect.

Effect of intracellular injection of phosphate

The decline of force in fatiguing muscle is, at least in part, caused by the accumulation, inside the fibre, of phosphate, which comes from the breakdown of phosphocreatine and, to a lesser extent, ATP. In skinned fibres phosphate is known to inhibit force production and reduce calcium sensitivity. The Laboratory has also proposed that phosphate might inhibit the sarcoplasmic reticulum calcium pump and slow crossbridge detachment rates. Experiments undertaken with Dr Hakan Westerblad, who was visiting from Sweden, were to inject sodium phosphate into the fibre and see whether the four effects described above could be observed. The results were that none of the above were observed. Instead, a moderate reduction in intracellular calcium and force, which was not caused by damage associated with the injection, were seen. Thus either the Laboratory's hypotheses are incorrect or injected phosphate is rapidly ejected from the fibre or sequestered in some intracellular site. The latter is thought to be more likely and the Laboratory is considering two possibilities: (i) phosphate combines with free creatine to form phosphocreatine, (ii) phosphate enters the sarcoplasmic reticulum where it precipitates with calcium (since in this site the solubility product of calcium phosphate is probably exceeded).

Intracellular sodium during ischaemia

Two overseas visitors, Alan Groves and Anna Park, have further developed the use of the fluorescent indicator, SBFI, which measures intracellular sodium. Methods for loading SBFI into the perfused heart have been improved and the Laboratory now has sufficient signal-to-noise ratio to measure the intracellular sodium during ischaemia. The figure shows that during a 10 min period of ischaemia intracellular sodium rose by only 1-2 mM, whereas during reperfusion there was a much larger rise, amounting to 6-8 mM. The Laboratory are currently investigating the mechanism of this rise by using inhibitors of the Na/H exchanger and by preventing acidosis during ischaemia by prior depletion of glycogen.

*** Figure here***

***Legend needed to figure

RESEARCH PLANNED for 1994

The mechanism of failure of calcium release in fatigue

A new postdoctoral researcher, Eva Chin, is currently learning to dissect single mouse muscle fibres. Experiments are planned in which caged ATP will be injected into the fibre. ATP can be suddenly released by an intense flash of UV light and this experiment will test whether the fall of calcium release is a consequence of reduced ATP as has been previously suggested by the Laboratory.

Intracellular sodium during cardiac ischaemia

It is planned to first determine the mechanism of rise of intracellular sodium, since it is thought by the Laboratory that this is the cause of the rise in intracellular calcium which is known to occur in ischaemia and is the cause of arrhythmias and cell damage. Once the mechanism of these changes is known it will be possible to design strategies to minimize the damaging effects of ischaemia on a rational basis.

Intracellular calcium in skeletal muscle following stretches

The next phase of this work will be to determine the site of the elevated resting calcium which has been discovered by the Laboratory. The hypothesis is that there are focal sites of damage, possibly caused by sheared off T- tubules, when the weakest sarcomeres undergo uncontrolled stretch.

PERSONNEL in 1993 and 1994

Dr David G. Allen	Professor (in-charge)		University	1989-

Lorraine Kerr Senior Technical Officer NHMRC 1989-

Chris Balnave PhD student APRA 1992-

Dr Eva Chin Postdoctoral Research Fellow NHMRC 1993-

Dr Anna Park Visiting Scholar 1993-

COLLABORATIONS

Muscle fatigue: Dr Hawkin Westerblad, Karolinska Institute, Sweden (1989-present).

Role of surface membrane pumps: Prof. Basil Roufogalis, Dept of Pharmacy (1992-present).

FACILITIES

The Laboratory is located in rooms 348A-H of the Anderson Stuart Building. Prof. Allen's office is room 348D. Equipment includes four fluorescent microscopes and accessory equipment. These are adapted for intracellular ionic measurements and digital image analysis. A comprehensive range of stimulating and recording equipment is also available.

FUNDING in 1993 and 1994

NHMRC		The cause of failure of calcium 	Allen DG	1993	$74,340

release in fatigued skeletal muscle 1994 $67,300

1995

NHMRC Intracellular calcium in skeletal Allen DG 1994 $36,635

muscle during length changes 1995

1996

NHF Role of changes in intracellular Allen DG 1993 $20,000

ions during myocardial ischaemia 1994 $35,000

Ramaciotti Role of surface membrane calcium Allen DG 1993 $10,000

pump in cardiac muscle

Total for 1993: $104,340

Total for 1994: $138,935

TEACHING in 1993

Medicine 2

Lectures: 18, on the cardiovascular system, including 1 Question session, and 2 Distinction lectures.

Practical classes: 1, of 3 h, repeated to 6 groups, on the cardiovascular system, involving demonstration of blood pressure measurement, ECG and heart sounds; students then design their own experiment to investigate some aspect of cardiovascular function. Each was also given a follow-up session of 2 h, which included student presentations of results, tutorial and multiple-choice questionnaire.

Examination: Involved unseen-data question and set of true/false questions.

Medicine 3 (Clinical Physiology)

Lectures: 4, on clinically-applied cardiovascular physiology.

Examination: One short-answer question and multiple choice questions.

BMedSc: Human Life Science 3

Lectures: 3, on intracellular ionic regulation.

Small group tutorials: 4, discussing ionic regulation in the heart during ischaemia.

Large group tutorials: 2, supervising student presentations on ionic regulation

Examination: long essay question

BMedSc 3/Science 3 (Cardiovascular)

Lectures: 5 on electrophysiology of the heart and the limits to human exercise performance

Tutorials: 5 in which students discuss scientific papers

Examination: long essay questions.

Total distribution (hours of formal teaching)

				Med2	Med3	MedSc3(CV)	HLS3	Total
Lectures			18	4	5		3	30

Prac classes (no.) 24(8) - - - 24

Prac discussion sessions 8 - - - 8

Tutorials 8 - 5 2 15

Total contact teaching time = 77 h.

Additional time was devoted to lecture preparation, setting and marking of exams and discussions with colleagues on curriculum development.

OTHER ACTIVITIES in 1993

Head of Department (1991-present).

University committees

Member, Academic Board.

Chair, University of Sydney Cancer Research Fund.

Faculty of Medicine committees

Member, Board of Directors.

Member, Committee of Management.

Member, Research Committee.

Member, Anderson Stuart Building Refurbishment Committee.

Refereeing

Manuscripts: for Journal of Physiology (3), Circulation Research (6), Pflügers Archiv. European Journal of Physiology (3), Journal of Molecular Cell Cardiology (2), Cardiovascular Research (4), American Journal of Physiology (3), Cardioscience (1), Drugs and Aging (1), Journal of General Physiology (1), Proceedings of the Royal Society, Series B (2).

Grant applications: for NHMRC (4), ARC (2), National Heart Foundation (4).

PhD thesis examined: for Univ. of XXX? (1).

Service to grant giving body

NHMRC Regional Grant Interviewing Committee

International Conferences

International Society for Heart Research, Oita, Japan (invited lecturer) (May)

Seminars

Overseas:

Dept of Physiology, Jikei Medical School, Tokyo (May)

Local:

Dept of Cardiology, Royal North Shore Hospital (Month?)

Dept of Anatomy, Univ. of N.S.W. (Month?)

Department of Pathology, Univ. of Sydney (Month?)

N.S.W. Cardiovascular Club, Sydney (Month?)

5-YEAR RESEARCH PUBLICATIONS

(Appointed in 1989; earlier publications are from research when at University College, London)

JOURNAL ARTICLES

1989

Elliott AC, Smith GL, Allen DG (1989) Simultaneous measurements of action potential duration and intracellular ATP in isolated ferret hearts exposed to cyanide. Circulation Research, 64, 583-591

Lee JA, Ruegg JC, Allen DG (1989) The effects of pimobendan, a novel inotropic agent, on intracellular calcium and tension in isolated ferret papillary muscle. Clinical Science, 76, 609-618

Allen DG, Lee JA, Smith GL (1989) The consequences of simulated ischaemia on intracellular Ca2+ and tension in isolated ferret ventricular muscle. Journal of Physiology, 410, 297-323

Lee JA, Allen, DG (1989) A comparison of the effects of inotropic interventions on isometric tension and shortening in isolated ferret ventricular muscle. Cardiovascular Research, 23, 748-755

Allen DG, Lee JA, Westerblad H (1989) Intracellular calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis. Journal of Physiology, 415, 433-458

1990

Westerblad H, Lee JA, Allen DB (1990) Spatial gradients of intracellular calcium in skeletal muscle during fatigue. Pflügers Archiv European Journal of Physiology, 415, 734-740

Lee JA, Allen DG (1990) Calcium sensitisers: a new approach to increasing the strength of the heart. British Medical Journal, 300, 551-552

Blanchard EM, Smith GL, Allen DG, Alpert NR (1990) The efects of butanedione monoxime on initial heat, tension, and aequorin light output of ferret papillary muscles. Pflügers Archiv European Journal of Physiology, 416, 219-221

1991

Lee JA, Westerblad H, Allen DG (1991) Changes in tetanic and resting [Ca2+]i during fatigue and recovery of single muscle fibres from Xenopus laevis. Journal of Physiology, 433, 307-326

Lee JA, Takai A, Allen DG (1991) Okadaic acid, a protein phosphatase inhibitor, increases the calcium transients in isolated mammalian ventricular muscle. Experimental Physiology, 76, 281-284

Lee JA, Allen DG (1991) EMD 53998 sensitises the contractile proteins to calcium in intact ferret ventricular muscle. Circulation Research, 69, 927-936

Westerblad H, Allen DG (1991) Changes in myoplasmic calcium concentration during fatigue in single mouse muscle fibres. Journal of General Physiology, 98, 615-635

Lee JA, Allen DG (1991) Mechanisms of acute ischemic contractile failure of the heart: role of intracellular calcium. Journal of Clinical Investigation, 88, 361-367

Poronnik P, Cook DI, Allen DG, Young JA (1991) Diphenyl-2-carboxylate (DPC) reduces calcium influx in a mouse mandibular cell line (ST885). Cell Calcium, 12, 441-447 *

Westerblad H, Lee JA, Lännergren J, Allen DG (1991) Cellular mechanisms of fatigue in skeletal muscle. American Journal of Physiology, 261, C195-C209

1992

Cook DI, Wegman EA, Ishikawa T, Poronnik P, Allen DG, Young JA (1992) TEA blocks muscarinically evoked secretion in the sheep parotid by a mechanism additional to its blockade of BK channels. Pflügers Archiv European Journal of Physiology, 420, 167-171 *

Lee JA, Allen DG (1992) Changes in intracellular free calcium concentration during long exposures to simulated ischemia in isolated mammalian ventricular muscle. Circulation Research, 71, 58-69

Westerblad H, Allen DG (1992) Changes of intracellular pH during repeated tetani in single mouse skeletal muscle fibres. Journal of Physiology, 449, 49-71

Allen DG, Westerblad H, Lee JA, Lännergren J (1992) Role of excitation-contraction coupling in muscle fatigue. Sports Medicine, 13, 116-126

Westerblad H, Allen DG (1992) Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle. Journal of Physiology, 453, 413-434

Elliott A, Smith GL, Eisner DA, Allen DG (1992) Metabolic changes during ischaemia and their role in contractile failure in isolated ferret hearts. Journal of Physiology,454, 467-490

Westerblad H, Allen DG (1992) Myoplasmic [Mg2+]i concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade. Experimental Physiology, 77, 733-740

Elliott AC, Cairns SP, Allen DG (1992) Subcellular gradients of intracellular free calcium concentration in isolated lacrimal acinar cells. Pflügers Archiv European Journal of Physiology, 422, 245-252

1993

Cairns SP, Westerblad H, Allen DG (1993) Changes of myoplasmic pH and calcium concentration during exposure to lactate in isolated rat ventricular myocytes. Journal of Physiology, 464, 561- 574

Westerblad H, Allen DG (1993) The influence of pH on contraction, relaxation and [Ca2+]i in intact single fibres from mouse muscle. Journal of Physiology, 466, 611-628

Westerblad H, Duty S, Allen DG (1993) Intracellular calcium concentration during low frequency fatigue in isolated single fibres of mouse skeletal muscle. Journal of Applied Physiology, 75, 382-388

Westerblad H, Allen DG (1993) The role of [Ca2+]i in the slowing of relaxation in fatigued single fibres from mouse skeletal muscle. Journal of Physiology, 468, 729-740

Dinudom A, Poronnik P, Allen DG, Young JA, Cook DI (1993) Control of intracellular Ca2+ by adrenergic and muscarinic agonists in mouse mandibular ducts and endpieces. Cell Calcium, 14, 631-638

Cairns SP, Westerblad H, Allen, DG (1993). Changes in [Ca2+]i and tension during b-adrenoreceptor activation of single, intact fibres from mouse skeletal muscle. Pflügers Archiv European Journal of Physiology, 425, 150-155

Allen DG, Duty S, Westerblad H (1993) Uniformity of activation in muscle during fatigue. (Letter) Journal of Muscle Research and Cell Motility, 14, 543-544

Early 1994

Elliott AC, Smith GL, Allen DG (1994) The metabolic consequences of an increase in the frequency of stimulation in isolated ferret hearts. Journal of Physiology, 474, 147-159

Westerblad H, Allen DG (1994) The role of sarcoplasmic reticulum in relaxation of mouse skeletal muscle; the effects of 2.5-di(tert-butyl)1,4-hydroquinone. Journal of Physiology, 474, 291-302

Westerblad H, Allen DG (1994) Calibration of fluorescent [Ca2+] indicators in muscle. (Letter) Biophysical Journal, 66, 926-927

Duty S, Allen DG (1994) The distribution of intracellular calcium concentration in isolated single fibres of mouse skeletal muscle during fatiguing stimulation. Pflügers Archiv (in press)

Westerblad H, Allen DG (1994) Slowing of relaxation, [Ca2+]i and [Mg2+]i during prolonged tetanic stimulation of intact, single fibres from mouse skeletal muscle. Journal of Physiology (in press)

Turvey SE, Allen DG (1994) Changes in myoplasmic sodium concentration during exposure to lactate in perfused rat hearts Cardiovascular Research (in press)

BOOK EDITED

Lee JA, Allen DG, eds (1993) Modulation of Cardiac Calcium Sensitivity: A New Approach to Increasing the Strength of the Heart. Oxford University Press, Oxford (347 pp)

CHAPTERS IN BOOKS

1990

Allen DG, Smith GL (1990) The mechanism of cellular damage following metabolic blockade in the heart. Imaging Analysis and Simulation of the Cardiac System, Sideman S, Beyar R, eds, Freund Publishing House, London, 505-523

Poronnik P, Cook DI, Allen DG, Young JA (1990) Diphenylamine-2-carboxylate (DPC) blocks calcium influx in cultured mouse mandibular cells. Exocrine Secretion II, Wong PDY, Young JA, eds, ISES, Hong Kong, 113- 114 *

Wegman EA, Poronnik P, Cook DI, Allen DG, Young JA (1990) Mechanism of inhibition of bethanecol evoked secretion from the sheep parotid gland by tetraethylammonium. Exocrine Secretion II, Wong PDY, Young JA, eds, ISES, Hong Kong, 129-132 *

Allen DG, Westerblad H, Lee JA (1990). Intracellular mechanisms of fatigue in skeletal muscle. Fatigue in Sport and Exercise, Hargreaves M, ed, Victoria Institute of Technology, Footscray, 16-20

1992

Allen DG (1992) The interval-strength relationship of cardiac muscle: Past, present and future. The Interval- Force Relationship of the Heart; Bowditch Revisited, Noble, MIM, Seed WA, eds, Cambridge University Press, Cambridge, 43-65

1993

Lee JA, Allen DG (1993) Altering the strength of the heart: Basic mechanisms. Modulation of Cardiac Calcium Sensitivity: A New Approach to Increasing the Strength of the Heart, Lee JA, Allen DG, eds, Oxford University Press, Oxford, 1-36

Orchard CH, Allen DG, Boyett MR, Elliott AC, Kirby MS (1993) The effects of acidosis on intact cardiac muscle. Modulation of Cardiac Calcium Sensitivity: A New Approach to Increasing the Strength of the Heart, Lee JA, Allen DG, eds, Oxford University Press, Oxford, 89-114

Allen DG, Lee JA, Elliott AC (1993) Effects of anoxia and ischaemia on cardiac muscle. Modulation of Cardiac Calcium Sensitivity: A New Approach to Increasing the Strength of the Heart, Lee, JA, Allen DG, eds, Oxford University Press, Oxford, 283-303

Lee JA, Allen DG (1993) Future directions. Modulation of Cardiac Calcium Sensitivity: A New Approach to Increasing the Strength of the Heart, Lee JA, Allen DG, eds, Oxford University Press, Oxford, 339-342

Lännergren J, Westerblad H, Allen DG (1993) Mechanisms of fatigue as studied in single muscle fibres. Neuromuscular Fatigue, Sargeant AJ, Kernell D, eds, Royal Netherlands Academy of Arts and Sciences, North-Holland, Amsterdam, 3-9

Lee JA, Allen DG (1993) Intracellular calcium in cardiac ischaemia. Atherosclerosis Reviews, Leaf A, Weber PC, eds, Raven Press, 25, 151-161 Journal?

Allen DG, Cairns SP, Turvey SE, Lee JA (1993) Intracellular calcium and myocardial function during ischaemia. Interactive Phenomena in the Cardiac System, Advances in Experimental Medicine and Biology, 346, Sideman S, Beyar R, eds, Plenum Publishing Corporation, New York, 19-29

Lännergren J, Westerblad H, Allen DG (1994) Fatigue of striated muscle: metabolic aspects. In The Thorax, Roussos C, ed (in press)

CONFERENCE ABSTRACTS AND PRESENTATIONS in 1993

Allen DG Duty S, Westerblad H (1993) Metabolic changes in muscle during exercise; their effects on muscle function. Proceedings of the Australian Physiological and Pharmacological Society, 24, 65-75

Allen DG (1993) Cellular mechanisms of fatigue in mammalian skeletal muscle: components due to metabolites and excitation-contraction coupling. Proceedings of the Australian Physiological and Pharmacological Society, 24, 109P Melbourne (Feb) (invited speaker)

Allen DG (1993) Ischemia and intracellular calcium handling. Name of Meeting? Journal of Molecular and Cellular Cardiology, 25 (suppl II), S1. Oita, Japan (May) (invited speaker)

Westerblad H, Allen DG (1993) What determines the relaxation speed in skeletal muscle? International Union of Physiological Sciences, abstract 116.1. Glasgow, UK (Jun)

Lee JA, Allen DG (1993) Reperfusion-induced changes in intracellular [Ca2+] following ischaemia of variable duration in isolated mammalian cardiac muscle.International Union of Physiological Sciences, abstract 139.11. Glasgow, UK (Jun)

Allen DG, Duty S (1993) Muscle fatigue - is the failure of calcium release spatially homogeneous within the cell? Proceedings of the Australian Society for Biochemistry and Molecular BiologyI, 25, 69, Adelaide (July) (invited speaker)

Allen DG (1993) Intracellular calcium during myocardial ischaemia. Baker Symposium, Cellular Signalling, Melbourne (Dec) (invited speaker)

?was this published etc?/details?

Ansselin AD, Davey DF, Allen DG (1993/4?) ATP evokes a rise in intracellular calcium in cultured adult rat Schwann cells. International Journal of Neuroscience, (submitted) (see D. Davey's report)

1993 PHYSIOLOGY REPORT - ALLEN