SIGNAL INTERACTION TO REGULATE OXYGEN AND SUBSTRATE SUPPLY FOR UTILIZATION BY MUSCLE

Integrative physiology is the field within which CMRC has its strength. The research relates primarily to the function of intact humans in regard to how oxygen and substrates are made available to the muscle, matching supply with demand. Various interventions such as muscle contractions or exercise as well as dietary manipulation and varying the oxygen availability are used to fully explore the mechanisms involved in this regulation. Selected patient groups are also included in our research to elucidate specific control mechanisms providing the mitochondria with an ample supply of substrates. Characteristic for CMRC research has been to combine studies on the system level with the cellular and subcellular levels, both in humans and other species. In the last couple of years major efforts, which will be continued in 1997-98, have been made to adapt and develop new methods and include molecular biology techniques. This will further strengthen our ability to attack a problem on several levels, enhancing our chances to obtain a more complete understanding of the function of humans. The perspectives are therefore good for the CMRC to contribute significantly to the frontier of human physiology in the coming century.

Below follows a description of our visions for the future in regard to research topics and our ability and plans to fulfill our goals. The examples start on the subcellular and cellular level with the muscle and build through organ and systemic responses to end with the central nervous system.

Muscle stimulation causes contraction accompanied by an increase in metabolic rate and recruitment of intramuscular as well as extramuscular energy sources by activation of rate limiting enzymes and plasma membrane substrate transporters. However, answers to fundamental questions are still lacking in regard to the precise character of the stimulus that triggers acute metabolic events or chronic adaptations to muscle stimulation. Illustrating this is the general acceptance that Ca²⁺ release results in ATPase activation and an increase in metabolic rate, but this does not explain how metabolic rate is coupled with workload at a given contraction frequency (and in turn a given Ca²⁺ concentration). Also, it is believed from earlier studies, which may be inadequate because of technical deficiencies and the use of frog muscle which may not behave as mammalian muscle, that glucose transport is solely determined by stimulation frequency and the resulting increase in intracellular Ca²⁺ with no role for work output. This seems unlikely because needs for glucose parallel metabolic rate which varies with workload. The factors, each of which may have an independent and important role as an initial trigger of subsequent responses to muscle stimulation, are membrane depolarization, Ca²⁺ movement, change in energy state (ADP/ATP) or redox state (NAD/NADH), muscle shortening and tension. Demonstrating that "contraction" does not constitute an unambiguous stimulus we have found that high resistance exercise and eccentric exercise cause responses qualitatively different from responses to dynamic concentric exercise, i.e. decrease vs increase in insulin mediated glucose uptake and increase vs no change for insulin mediated protein synthesis in muscle. These findings point to the intriguing possibility that muscle tension during contraction significantly influences acute and chronic adaptations to muscle stimulation by determining the conformational state of enzymes and transporter proteins. We have the methods necessary to control and monitor the mentioned independent, putative trigger factors and to characterize in detail acute and chronic (e.g. myosin synthesis and transformation, enzyme adaptation) responses in isolated rat muscle as well as in human muscle (e.g. healthy subjects, patient groups such as tetraplegics or Duchennes muscular dystrophy). By these means and by parallel basal biochemical studies key scientific questions can be answered and at the same time knowledge can be provided of immediate relevance in prevention and treatment of disease.

• Muscle protein synthesis, growth and fibre type specificity

The activation of a motor unit gives rise to stimuli regulating muscle protein synthesis, and the type of contractions determine, in analogy with the varied metabolic responses described above, which specific proteins are made. The regulation of muscle protein synthesis also involves the endocrine system (anabolic and catabolic hormones) and paracrine factors. We will specifically investigate the role of various cytokines for both growth and differentiation of the muscle cells by using specific activity patterns. The goal is to identify what triggers the on-and-off switch for the genes of the myosin heavy chain isoforms in skeletal muscle of humans and what brings about concomitant changes, such as energy-related enzymes and the coupling to insulin sensitivity. We will also utilize the opportunity to study the regulatory mechanism and the role of stimulation pattern in attempts to use the latissimus dorsi muscle in assisting failing hearts. Moreover, the role of immuno-related cytokines will be studied, especially in regard to the acute phase response. The application of molecular biology methods such as PCR and in situ hybridization will allow us to identify which cells are producing the cytokines and to identify cytokine receptors in skeletal muscles and possible effects of promoter factors like MyoD and Myf5.

The inclusion of monozygotic twins in these studies will allow us to separate the role of genetic and environmental factors. We will also study the patient groups mentioned above, and furthermore, explore the mechanisms underlying decreased protein synthesis in patients with muscle cachexia following severe systemic infections. These groups of patients give us a unique opportunity to study regulatory mechanisms underlying protein synthesis in chronically inactive and active skeletal muscle, and the inclusion of both high cervical injured individuals (quadriplegic) with impaired sympathoadrenergic activity and lower thoracic level spinal cord injured individuals (paraplegic) allow us to investigate the role of the catecholamine response. The role of growth factors such as growth hormone and IGF1 will be investigated by injection of GH and IGF1-stimulating drugs to healthy people and by continuing our studies of GH deficient patients. Patients with Duchennes muscular dystrophy are of special interest as they may have an impaired expression of IGF1 in their muscles. Intervention studies with selected amino acid preparations and growth factors in patients with impaired muscle protein synthesis will provide further information about underlying mechanisms and potential future treatments.

• Muscle mitochondrial substrate choice and respiration

Why carbohydrate oxidation is increased at the expense of lipid oxidation when exercise intensity is increasing remains an intriguing puzzle. Available evidence indicates that the regulation is mainly within the muscle. Focus will be on the role of malonyl-CoA, CPT-1 and acetyl-CoA-carboxylase (ACOAC) in human skeletal muscle. One intervention to be used is endurance training as an increase in lipid oxidation during exercise at a given absolute power output occurs without an increasing supply of fatty acids. By the application of the array of methods we have available a good possibility exists to fully explore the limiting steps in lipid and carbohydrate oxidation during exercise.

At the onset of muscle contraction oxygen extraction is delayed. The cause for this is unknown. It could be a slow unloading of O₂ from the red cells (see below), but it could also be a tardy elevation in mitochondrial respiration. Substrate supply (pyruvate, ADP, NADH) could also be limiting. These different possibilities will be evaluated by in vivo studies and in vitro models such as the perfused rat hindlimb and muscle cells in culture. We will be able to manipulate single factors, e.g. Ca²⁺, ATP and the redox state, which may be important in regulation of PDH. In the studies on humans, we will modulate the PDH activity by dichloroacetate, elevate muscle blood flow with local vasodilators, e.g. adenosine or NO, and vary muscle mass engaged in the exercise, thus creating different conditions for muscle oxygen extraction and availability of substrates to the mitochondria.

Related to these problems is why lactate is formed. There are two situations when the coupling between glycolysis and mitochondrial usage of the formed pyruvate are quite tight. This is observed after repeated intense exercise bouts and exposure (several days) to hypoxia. Both interventions will be used to unravel basic regulatory mechanisms of glycolysis and mitochondrial respiration.

• Intramuscular triglyceride utilization

Muscle contains abundant triglyceride deposits and fat is a major fuel in submaximal exercise. However, the role and regulation of muscle triglyceride breakdown in exercise remains a major unsolved problem. Essential reasons for this are that studies of intramuscular triglycerides are made difficult by the existence of fat cells within muscle. Furthermore, in vivo breakdown of intramuscular triglycerides during exercise may be difficult to detect due to simultaneous synthesis from free fatty acids (FFA). Finally, the enzyme initiating triglyceride breakdown is not known. However, we now have techniques to study single muscle fibres isolated by collagenase from biopsies, we can account for recycling of FFA by use of isotopes, including stable isotopes, and we have demonstrated that HSL (hormone sensitive lipase ) exists in muscle and is a likely candidate as flux generator. Accordingly, we will be able to elucidate the regulation and role of intramuscular triglyceride breakdown during exercise along the same lines that we have followed for muscle glycogen breakdown (effect of exercise intensity, duration, diet, training, hormones and sympathetic activity). A question of key interest will be whether breakdown of intramuscular triglyceride and glycogen, respectively, are always activated in parallel. This would represent significant support for our proposal that feed-forward control is an essential principle in metabolic regulation.

• Glucose and fatty acid transport

A major goal of our research is to elucidate the mechanisms involved in transport of glucose and fatty acids into skeletal muscle. For glucose transport, emphasis will be on the molecular mechanisms activated by insulin and contractions, and also on the mechanisms by which muscle contractions affect the insulin signalling pathway. Much of the ground work will be performed in animal models and cultured muscle cells in which the role of possible modulating agents on glucose transport will be studied using microinjection of antibodies and peptides. Every effort will be made to verify if the results apply to intact human muscle. Extension of our studies to patients with NIDDM may help us gain insight into the pathophysiological mechanisms involved in insulin resistance. Immunoflourescence and immunocytochemical electron microscopy will be used to study the association of GLUT4 containing vesicles with components involved in vesicle translocation. Immunoprecipitation of GLUT4 containing vesicles will allow for characterization and identification of vesicle protein components and enzyme activities, e.g. PI3-kinase activity. Known steps in the insulin signalling cascade will be studied in resting and exercised normal and insulin resistant muscle in an effort to elucidate the extent to which contractions activate/influence the insulin signalling cascade. Our recent results implicate a role of Ca²⁺ in both contraction- and insulin stimulated glucose transport. Ca²⁺-imaging in muscle fibres will be attempted to study spatial and temporal Ca²⁺ movements after stimulation with insulin and/or contractions. A goal of our studies will also be to investigate the extent to which the above mentioned mechanisms and glucose transport might be affected by different contraction patterns (concentric vs. eccentric, loaded vs. unloaded) as well as by physical training/inactivity.

In terms of fatty acid transport our giant vesicle technique also offers unique possibilities to characterize fatty acid transport kinetics at rest and after exercise in human skeletal muscle. Furthermore, studies will elucidate which of the membrane bound fatty acid transporters (FABPpm, FAT, FATP) are present in human sarcolemma, and their physiological role will be characterized by use of antibodies against the various transporters. In addition, we will study the potential role of physical training, inactivity, and varying sarcolemmal membrane composition (induced by dietary interventions) on expression and functional role of these transporter proteins and on intracellular fatty acid binding proteins (FABPc).

• Compounds produced by contracting muscles, endothelial cells, and the interstitium activate sensory nerve endings, induce vasodilatation and affect pH regulation

The electrical and metabolic activity in contracting muscle releases compounds such as lactate, H⁺ and K⁺ to the interstitial space and later to the blood. The temporary accumulation of these compounds in the interstitial space may have several effects on muscle function. One area of interest will be pH regulation both at the cellular level and in the interstitium in association with muscle activity. The changes in interstitial pH may directly influence the activity of the sensory nerve endings involved in muscle reflexes, and may influence fluxes of other compounds, such as the carrier mediated transport of glucose, free fatty acids, and amino acids. For instance, in vivo ingestion of bicarbonate and pharmacological blockers can be used to evaluate the role of interstitial buffer capacity and pH regulating transport. Such in vivo studies will be carried out both with traditional blood sampling and with the microdialysis technique. The latter technique will allow us to study ion transport in a specific area of a contracting muscle under different types of interventions both at systemic level, e.g. infusion of chemical agents in an artery, and locally, e.g. infusion of a chemical agent in the microdialysis probe. For example, the technique will allow us to study the effect of pH and epinephrine (infused through the probe) on transport of potassium in muscle at rest and during exercise. In more long-term perspectives the intention is to evaluate how muscle energy metabolism is coupled to ion transport. These studies will be supplemented with in vitro investigations of the transport systems involved in muscular pH regulation. A new approach we shall take is to identify changes in positively charged ion concentrations in the interstitium of skeletal muscle and by the use of electron probe (x-ray) microanalysis.

The concentrations of adenosine and NO also increase in the interstitium during contractions. We will determine what role adenosine and NO play in muscle blood flow regulation and muscle function. Two primary aims are to reveal the mechanisms underlying adenosine and NO formation by muscle and vascular cells, and to study the interaction between muscle and vascular cells with regard to adenosine and NO formation. Our main approach to examine these issues will continue to be the use of primary rat skeletal muscle and smooth muscle and endothelial cell culture models. These models allow the investigation of adenosine and NO formation and metabolism at rest and during electrical stimulation of muscle in presence and absence of vascular cells. The intention for the future is to develop skeletal muscle cell cultures and muscle microvascular endothelial cell cultures of human origin.

• Compounds released by contracting muscle to the blood stream modulate heart rate response, vascular tone, glucose and fatty acid release from extramuscular stores

Our research has shown that many events elicited by contractions cannot be satisfactorily explained by known mechanisms, e.g. known neuroendocrine mechanisms cannot fully account for hepatic glucose production, lipolysis, splanchnic vasoconstriction, or the ventilation and heart rate increase in exercise. Quite recently it has become clear that fat cells secrete a variety of peptide messengers with a broad spectrum of actions throughout the body. Even muscle has been shown to possess the machinery necessary for protein secretion. We have provided evidence that contracting muscle produces a glycogenolytic hormone and that a blood-borne factor from muscle may be necessary for a normal increase in heart rate in exercise. These facts point at a new research area with an enormous potential: We want to use our expertise with incubation, perfusion and culture of muscle and with chemical separation to define the substances secreted by resting and contracting muscle. Furthermore, we will measure the response of any discovered substance to in vivo exercise and characterize its effects and physiological role in our isolated muscle, liver and adipose tissue systems as well as in intact rat and humans.

• Arterial oxygen content, skeletal muscle blood flow, and off-loading of O₂ from the Hb molecule in skeletal muscle

The search for a universal oxygen sensor has focused mainly on in vitro mechanisms operating at the cellular and molecular levels. At the CMRC we are in the unique position of being able to examine oxygen sensing in intact humans and to experimentally probe the responsible cellular and molecular mechanisms. We have recently shown that haemoglobin, and more specifically the arterial oxygen content, influences limb blood flow, and that arterial oxygen tension has only a small regulatory effect. This observation leads to several testable hypotheses about what property or properties of the red blood cell signals that changes in arterial oxygen content and how and where this signal is sensed.

We will answer four questions that may explain how arterial oxygen content is sensed to regulate limb blood flow and oxygen delivery. First, ATP release from erythrocytes as they pass through peripheral vascular beds, proportional to the number of unoccupied O₂ binding sites on the Hb molecules, may be the signal that is sensed to regulate limb blood flow. ATP may induce vasodilatation through an ATP receptor triggering formation of nitric oxide (NO) or a prostaglandin causing the relaxation. An alternative could be that the vasodilatation is caused by adenosine with the ATP serving as a substrate. Second, we will evaluate the idea that the haemoglobin molecule functions as a scavenger of NO in the peripheral vascular bed with low [Hb] causing more NO to be available to induce vasodilatation. Third, a Po₂ sensitive cytochrome P450 enzyme has recently been identified that forms HETE-20 which is a potent vasoconstrictor in all animal systems tested (rat renal, cerebral and striated muscle beds). Almost no HETE-20 is formed at a Po₂ of 40 Torr with a linear increase in formation as Po₂ increases. A fourth important aspect of oxygen sensing in the intact organism is the possibility that unloading of oxygen from the red blood cell to the exercising muscle may be enhanced by signals originating in the exercising muscle. Recent evidence from our laboratory suggests that off-loading of O₂ from the haemoglobin molecule may be a limiting factor for oxygen uptake by muscle with pH playing a critical role in determining the rate of off-loading. We will explore these mechanisms for oxygen sensing and control of oxygen delivery in healthy human subjects during submaximal and maximal knee extension exercise with varied levels of arterial oxygen content and tension. Furthermore, manipulations that alter red blood cell acid-base status (e.g. by ameloride), and NO, ATP and prostaglandin metabolism will be employed to determine the contribution of these factors to oxygen sensing and delivery in the intact exercising human muscle.

• Intraabdominal adipose tissue, insulin sensitivity and lipid storage

Until recently it has in intact organisms only been possible to study the integrated lipolytic response of all adipose tissues. Microdialysis and abdominal vein catheterization have now made studies of selected subcutaneous fat depots possible. However, we have found that responses of these fat depots only poorly reflect physiological,"whole body fat" responses. In vitro, rat intraabdominal adipocytes are more metabolically active than subcutaneous adipocytes, and we have confirmed this finding in microdialysis studies in intact rats. From such findings and estimations of intraabdominal fat mass in various conditions it is generally speculated that intraabdominal adipose tissues play a major role in physiology and pathophysiology (e.g. in the insulin resistance syndrome). Thus, studies of the previously inaccessible human intraabdominal adipose tissue are of major interest. For that reason we are developing techniques for ultrasound guided microdialysis of perirenal fat in humans. Furthermore, we expect to be able to carry out transjugular (transhepatic) catheterization of the portal and mesenteric veins draining intraperitoneal fat stores. With these techniques we will have unique possibilities for studies of the effects of exercise and training on intraabdominal adipose tissue in regard to insulin sensitivity and FFA mobilization. If combined with infusion of isotopically labelled FFA and catheterization of muscle and subcutaneous fat, it will be possible to give a complete account of FFA metabolism in exercise, including intra- and intercellular futile cycling.

• Functional mapping of "Central Command"

Findings of marked increases in heart rate and blood pressure during attempted exercise with partial neuromuscular blockade or regional anaesthesia of the exercising limb(s) have been the basis for formulation of the hypothesis that cardiovascular regulation is subserved by specific brain structures: the "central command". However, little is known about the localization and interrelations between the brain regions involved in such influence on the cardiovascular system. Further, little is known about the functional relationship between "central command" and well known functional brain systems like the motor/pre-motor regions and regions involved in somatosensory function. In previous, less sensitive single photon computerized emission tomography (SPECT) studies we found focal cerebral activation both in regions assumed related to "central command" as well as in foci assumed to reflect somatosensory feedback from the cardiovascular system induced by post-exercise muscle ischemia.

With the use of high resolution positron emission tomography (PET), morphological magnetic resonance imaging (MRI) and fast echo planar functional MRI (fMRI) we will further pursue the understanding of the functional organization and anatomical localization of the "central command" in the human brain. During repeated brain PET ¹⁵O-H₂O and fMRI blood oxygen level dependent (BOLD) examinations we will determine the regional cerebral blood flow (rCBF) changes caused by activation of the sympathetic and parasympathetic nervous systems, as well as the effects of relevant blockade of the systems by substances like atropine, glycopyrron, beta- and alpha adrenergic blockers and by regional anaesthesia of the relevant sympathetic feedback from the exercising and resting limb(s). A methodological evaluation of the BOLD signal obtained with fMRI and quantified PET rCBF will be performed, and we will attempt to optimize the methods for evaluation of delicate structures in the medulla and spinal cord; areas not previously studied with these methods.

With the PET technique we will also seek further insight into the neurotransmitter systems involved in "central command" by imaging and quantification of the binding of specific radio-labelled receptor ligands: Dopamine-D₁ and -D₂, 5HT and, if possible, alpha- and beta-adrenergic receptor ligands. Further, there are plans for radio-labelling of vasoactive peptides. Other measurements can be obtained in conjunction with the PET and fMRI studies, e.g. retrograde jugular vein sampling of neuropeptides, e.g., epinephrine, norepinephrine, calcitonin gene related peptide, NPY, substance P and VIP. Non-invasive near infrared spectroscopy and transcranial Doppler techniques can also be applied.

The above methods will allow us to study the anatomical localization and functional relationships of "central command" and to provide opportunities to describe how the cerebral circulation adapts to exercise and to manipulation of baroreceptors. Finally, the methods also provide possibilities to quantify peripheral parameters such as muscular blood flow and glucose consumption (PET), ATP turnover (MR spectroscopy), determination of activated muscle volumes (MRI) and regional muscular water diffusion (fMRI).

Our main focus is and will be integrated human physiology. However, our future research plans imply a continued interaction between studies in humans and other species and on less integrated systems (e.g. isolated organs, cell cultures and organelles (e.g. plasma membrane, vesicles , mitochondria and nuclei)). The studies on lower levels of organization evidently cannot predict events in intact humans, but they offer more precisely controlled experimental conditions and interpretations on the molecular level. Furthermore, these studies regularly give rise to ideas and development of new techniques, which, in turn, can be applied in studies on humans. Our approach may appear complex but the fact is that the close collaboration between CMRC scientists with mutual interests working on intact humans or on less integrated systems creates a stimulating dynamic milieu with exceptional potential for problem solving. Because establishment of the various groups and of new techniques have taken time the full profits of the centre model are now becoming apparent.

Methods and techniques: We want to emphasize that our human studies allow simultaneous evaluation of the regulation and interplay of several major physiological functions (e.g. cardiovascular, ventilatory, endocrine and substrate delivering systems). This naturally leads to collaboration between CMRC scientists with different specific research interests. Positron emission tomography (PET) makes visualization of the brain nuclei involved in regulation of exercise responses possible. For studies of ventilation and circulation (cardiac output, arterial blood pressure and regional blood flows) we have at our disposal all relevant invasive and non-invasive techniques. In particular, it should be mentioned that we have developed Doppler ultrasound techniques for continuous recording of brain and leg blood flow. Sympathetic nerve activity is directly recorded by superficial microneurography.

Regarding metabolic studies in humans it should be noted that during the spring of 1997 we are adding mass spectrometer facilities to our broad spectrum of physiological and biochemical techniques. This will allow us to use stable isotopes and without health hazards simultaneously measure the kinetics of several substrates in blood and muscle. Nuclear magnetic resonance (NMR) spectrometry makes noninvasive measurements of important metabolic variables possible (e.g. glycogen, pH, ADP/ATP) and by combination with NMR imaging it may be possible to locate metabolic events in muscle more precisely. The muscle biopsy technique will remain a central tool allowing application of the newest biochemical, molecular biology and histochemical techniques.

During CMRC´s first three years we have further developed and evaluated the microdialysis technique for application in muscle and subcutaneous fat tissue. To evaluate metabolic events and signals stimulating afferent nerve activity and local vasodilation the microdialysis technique will be refined to allow measurement of more substrates and potential signalling molecules than now possible. When combined with fluorescent probes it should be possible to obtain continuous measurements of interstitial compounds and pH. The electrode technique will be adapted for continuous monitoring of interstitial ions. Of particular interest is that we will probably be able to open a new important research avenue by direct studies of the metabolism of intraabdominal adipose tissues in humans. One realistic approach will be to develop ultrasound guided microdialysis of perirenal fat. Another will be to carry out transjugular (transhepatic) catheterization of the portal and mesenteric veins draining intraperitoneal fat stores. The latter technique is used in the treatment of patients with cirrhosis of the liver. It is to be expected that within the next few years we will be allowed to use the technique also in more healthy, including obese and diabetic, humans.

Populations to be studied: Our ability to perform extensive studies in humans is based on a long Danish tradition which has lead to a general acceptance of and interest in such research which attracts many foreign researchers to collaborate with the CMRC. For our studies we recruit healthy subjects in various age groups as well as patients selected specifically according to the scientific problem in question. A new approach is the inclusion of monozygotic twins. Patients with specific deficiencies are studied because this will throw light on the role of the missing function or mechanism in normal physiology. At the same time information of importance for the clinical treatment of the patients is obtained. Illustrating our possibilities we have within the past three years studied patients with heart failure, splenectomia, HIV infection, non-insulin dependent diabetes, growth hormone deficiency, obesity, a liver transplant (accordingly lacking liver innervation ), a kidney transplant, adrenalectomy, tetraplegia (disuse atrophy, lack of voluntary "central command" motor control as well as sensory nervous feedback from muscle) and various muscle disorders (glycogen phosphorylase deficiency, phosphofructokinase deficiency, carnitine palmitoyltransferase deficiency and mitochondrial insufficiency).

Studies of patients with enzymatic muscle diseases give important information about metabolic regulation in exercise. In addition, in several patient groups, the chemical milieu in the working muscles is markedly disturbed and depends on the specific disease, a fact which allows conclusions concerning the putative messengers stimulating afferent nerve activity and local vasodilatation. The access to cell cultures (endothelial, skeletal muscle) provides techniques to evaluate which cells produce these substances.

We are expanding our personnel and skills within molecular biology, e.g. we are establishing the vaccinia technique for transfection studies and quantitative in situ PCR technique for specific location and quantification of tiny amounts of mRNA (e.g. in muscle biopsies). Molecular biology techniques (e.g. transgene animals and transfection) will be important in the detailed molecular characterization of contraction induced events, e.g. in elucidation of signal transduction involved in acute glucose transporter recruitment and of regulation of gene transcription and translation involved in glucose transporter and myosin adaptation to altered activity levels. In order to strengthen this field we expect to engage a full-time molecular biologist at the CMRC in the spring of 1997.

For visualization (e.g. of events related to glucose transporter recruitment) we master light microscopy immunofluorescence and immunocytochemistry on the electron microscopy level. We have applied these techniques in animal studies and in coming years we expect to apply them also on human muscle and on transfected or microinjected muscle cell cultures. Finally, we will use fluorescence microscopy and digital video imaging for estimation of intramuscular Ca²⁺ movements. In the future we will use laser scanning confocal microscopy for a more precise localization of the Ca²⁺.

The CMRC researchers work at three locations, the August Krogh Institute, the Panum Institute and the Rigshospitalet (University Hospital). This will continue in the coming period, but it should be emphasized that it would be beneficial from many aspects if more of the CMRC research could be located at one site. Furthermore, with more of our research emphasizing molecular biology, access to appropriate facilities for such work is mandatory in part because of the need to share investment in and maintenance of sophisticated equipment. The CMRC researchers at the August Krogh Institute in the past, i.e. Erik A. Richter, Carsten Juel, Bente Kiens, Jens Bangsbo, and Ylva Hellsten, will continue. Erik A. Richter has become a full professor, and Jens Bangsbo has now a tenured position. The group at the Rigshospital, consisting of Bengt Saltin, Niels H. Secher, Susanne Vissing, and Bente Klarlund Pedersen will also continue in CMRC. In addition, John Vissing who is in charge of the patients with muscle metabolic disorders will join the group. At the Panum Institute, Henrik Galbo, now full professor, Michael Kjær, and Thorkil Ploug will continue to be part of the CMRC, whereas Bjørn Quistorff at the Panum NMR-centre no longer formally will be working within the CMRC, although the collaboration will continue. Bengt Saltin and Henrik Galbo will continue to serve as director and co-director, respectively.

During the first half of this century skeletal muscle metabolism and physiology attracted attention by major groups of excellent researchers in Denmark, most of Europe (especially UK), and in North America (Harvard Fatigue Laboratory). After a period of less research on skeletal muscle this tissue is again more in focus. To this may have contributed the fact that it is now realized that the functional status of skeletal muscle can play a major role for the development of diseases such as type II diabetes and then for cardiovascular disorders. Moreover, with an even larger fraction of the world's population growing older, maintenance of optimal muscle metabolism and function is critical for the individual and society. Although the CMRC research is not primarily focused on either diabetes, cardiovascular diseases or ageing muscles, our research will significantly contribute to the basic understanding of these areas. In other parts of the world major research efforts are also devoted to these problems. Unique for CMRC is the broadness both in regard to experience and access to techniques. These qualities allow comprehensive and penetrating studies of relevant problems; a fact giving the CMRC a role as the one the Scandinavian and English schools of human physiology as well as the Harvard Fatigue Laboratory had in the past.

CMRC researchers are also called upon by many national and international scientific organizations where they contribute by establishment of scientific societies, as board members of scientific societies, as editors of scientific journals, and as organizers of scientific congresses, symposia and educational courses for younger scientists. Thus, by publishing and lecturing and by accepting and being guest researchers, CMRC scientists directly and indirectly have a major influence on the scientific world.