CMRC Highlights 1998

CMRC research activities are in 1998 characterised by successful developments in the ongoing studies to unravel the mechanisms by which muscle blood flow and metabolism are regulated. In addition, a major field study was undertaken in the Andes (Bolivia) from June to August in order to use the adaptation to chronic hypoxia to identify critical factors for circulatory and metabolic regulations in skeletal muscle.

The use of hypoxia in physiological research has a long tradition in Denmark. In 1935 Hohwü-Christensen, then a research assistant to August Krogh, went with an American group to the Andes (Northern Chile). Among the many features of the adaptive process that they studied, the most dramatic phenomenon they described was a marked drop in maximal heart rate and blood lactate during exhaustive exercise. These findings have repeatedly been confirmed, but never explained. With the method developments within the CMRC it was felt that the time had come to approach the problem again, but now to elucidate the regulatory mechanisms bringing about the reduction in heart rate and blood lactate occurring when exposed to severe hypoxia for an extended period of time. The latter phenomenon has been named "the lactate paradox". These were the two central research questions, but as a large group of subjects were to be staying at altitude for a two-month period, other researchers were invited to pursue other research projects. Most of these were linked to the main aims of the study, but in addition brain circulation was studied. In table 1 the various projects and principal investigators are listed. From this list it is also apparent that the international collaboration was extensive.

The practical outcome was most successful, although very severe difficulties were initially encountered. For one thing, a major snowfall blocked for the transportation of equipment to the cosmic research station at Chacaltaya (5.260 metres above sea level; m.a.s.l.) where the subjects and researchers were housed and the experiments performed. Therefore, it took more than three weeks before the laboratory could be established and thus, some of the early studies of the acute response to high altitude had to be deleted from the protocol. Contacts had been made with Bolivian researchers working with natives to the altitude in question. This enabled us to perform collaborative studies on the natives as well, which opened for comparing the degree of adaptation attained in sea level residents exposed for a two-month period to hypoxia with those that had developed their adaptation through evolution.

A first account of our results from the Bolivia study was given at the FASEB meeting in Washington DC in April 1999 where eight abstracts were presented of which four are reproduced below.

1. PARASYMPATHETIC NEURAL TONE INDUCES THE LOWERING OF EXERCISE HEART RATE IN CHRONIC HYPOXIA, BUT BLOCKING OF THE VAGAL NERVE HAS NO INFLUENCE ON O₂ TRANSPORT AND EXERCISE PERFORMANCE

Boushel R, Calbet JAL, Rådegran G, Søndergaard H., Wagner P.D. and Saltin B.

The influence of altitude acclimatization on parasympathetic neural control of heart rate and hemodynamic responses to exercise was assessed in 6 healthy Danish lowlanders (2 females and 4 males; 23.2 years, 76.1 kg) after 8-10 weeks of residence at high altitude (5260 m, Chacaltaya, Bolivia). Heart rate (HR), cardiac output (Q, Indocyanine Green), intra-arterial pressure (MAP, Dialogue 2000) oxygen uptake (VO₂, Parvo Medics) and vascular resistance (TPR) were determined at rest and during upright cycle ergometer exercise in Control (C) and after parasympathetic blockade with Robinul (ROB 0.8 mg) conditions. At rest, ROB increased HR from 73 (^+/-9) to 152 (^+/-10) bpm, but without effect on Q, MAP, TPR or VO₂. During submaximal and maximal exercise, ROB increased HR by 40 bpm, but again had no effect on O₂ transport or performance (Table).  

In summary, it is primarily an elevated vagal tone that lowers HR in chronic hypoxia. When the vagus is blocked it has no influence on O₂ transport or performance as cardiac output is unchanged due to a reduced stroke volume. It is suggested that the lower HR serves to minimize cardiac work and myocardial VO₂ and to optimize the arterial oxygenation.

2. LEG LACTATE PRODUCTION DURING SUB-MAXIMAL EXERCISE UNDER CHRONIC HYPOXIC AND ACUTE NORMOXIC CONDITIONS.

In acute hypoxia blood lactate concentration is elevated during incremental cycle exercise but with chronic exposure a gradual reduction approaching the normoxic level has been described suggesting that lactate production by the active muscle is similar to sea level. The aim of the present study was to describe leg lactate production during sub-maximal cycle exercise at a constant workload (146"7 Watt) after 9 weeks of acclimatization to 5260 m (Chacaltaya, Bolivia) in 7 Danish lowlanders (2 females and 5 males; 24.2, 68.3 kg). Exercise was performed for 20 min breathing ambient air (5260 m) followed by 20 min breathing a normoxic gas mixture (47% oxygen in nitrogen).

Lactate concentration and net leg lactate release was much higher with chronic hypoxia compared to acute normoxia. This may imply that muscle lactate production after acclimatization is similar to exposure to acute hypoxia, and a lactate paradox may not exist.

3. IS ACCLIMATION INDUCED POLYCYTHEMIA AN ADVANTAGE FOR MAXIMAL EXERCISE PERFORMANCE UNDER HYPOXIC CONDITIONS IN HUMANS?

Calbet JAL, Rådegran G, Boushel R, Søndergaard H, Wagner PD, and Saltin B.

The influence of hemoglobin concentration on maximal exercise performance was assessed in 8 healthy Danish lowlanders (3 females and 5 males; 24.2 years, 76.1 kg) after 8-10 weeks of residence at high altitude (5260 m, Chacaltaya, Bolivia). Peak oxygen uptake (VO₂peak), load (Wpeak), cardiac output (Indocyanine Green), femoral venous blood flow (LBFpeak, thermodilution) and heart rate (HRpeak), as well as pO₂, pCO₂, hemoglobin concentration (Hb) and saturation (SO₂, Radiometer) were determined during incremental upright cycle ergometer exercise until exhaustion under three different conditions: Control (C), Isovolemic Anaemia (IA) and Hypervolemic Anaemia (HPA). Despite that Hb concentration was reduced to pre-acclimation values in IA, Wmax and VO₂peak were the same as in C. As shown in the Table, the effect of low Hb was almost compensated for by increasing LBFpeak and, to less extent, by increasing oxygen extraction (O₂Ex). Fatigue always occurred at similar levels of arterial pO₂ (45-47 Torr) and hemoglobin saturation (73-75%).

The similar cardiac output, pO₂ and arterial saturation at exhaustion suggests an O₂ supply limitation to maximal exercise capacity under these conditions.

4. FEMORAL ARTERY EXERCISE BLOOD FLOW RESPONSE TO HYPOXIA IN DANISH LOWLANDERS AND AYMARA NATIVES.

Rådegran G, and Saltin B.

Femoral artery blood flow (F_aBF, ultrasound Doppler) was measured during rest and one-legged knee-extensor exercise (1L-KEE) in sixteen Danish lowlanders (DL) at sea level (SL) and after 7-10 weeks of altitude (ALT, 5260 m above SL, m.a.s.l.), as well as at ALT in six Aymara natives (AN, normally resident at 3600-4200 m.a.s.l.). In DL, (i) [Hb] and Hct increased (p<0.003) at ALT to a level similar (p=ns) as in AN (189 g l^-1 and 53%, respectively). (ii) With acute exposure to hypoxia (11% O₂) in submaximal 1L-KEE, FaBF was elevated with 50%. (iii) In chronic hypoxia F_aBF returned to SL values and was insignificantly different from the F_aBF response in AN at equivalent power outputs. (iiii) Hyperoxia (acute, 55%) in the chronic hypoxia condition brought F_aBF down by 25%; a response which was of a similar magnitude in AN. These data underline the critical role of arterial oxygen content in skeletal muscle blood flow regulation. Thus, it can be concluded that in chronic hypoxia, [Hb] is elevated allowing for the muscle blood flow to return to a normoxic level, which is similar to that observed in AN. The effect on F_aBF of acute hypoxia at SL, and hyperoxia after chronic ALT exposure, further emphasises that limb blood flow in exercise is regulated to match O₂ delivery to the metabolic demand.

From abstract number 2 and from figure B it is apparent that we could not confirm the existence of the so-called "lactate paradox". Indeed, our subjects attained the same lactate production and accumulation in muscle and blood that are observed during acute hypoxia in spite of the fact that they had been chronically exposed to hypoxia for approximately 2 months. Thus, our findings challenge the existing dogma that a "lactate paradox" really exists. At present we have no obvious explanation, but it is noteworthy that our results resemble those obtained in the natives.

Overexpression of GLUT4 in skeletal muscle by e.g. transgenic techniques or exercise training results in improved glycemic control, which is reflected by a lower postprandial blood glucose concentration and fasting insulin concentration. Under most circumstances the amount of expressed protein is determined by the factors that regulate the rate of transcription of the GLUT4 gene. The promotor region of the GLUT4 gene contains two E-boxes and one MEF2-box, which potentially binds transcription factors of the MyoD and Myocyte Enhancer 2 families, respectively. At the mRNA level we have now by means of Northern blot technique studied the co-expression of 7 different myogenic transcription factors and GLUT4 in different muscles from adult rat and in vitro cultured myotubes, which express variable amounts of GLUT4 protein. A very close positive correlation was found between expression of GLUT4 and the transcription factor MRF4, which indicates a possible involvement of MRF4 in regulation of GLUT4 gene expression.

Another transcription factor is myogenin, which has a function in the neonatal development of muscle differentiation. Lately it has also been proposed that myogenin may be a regulator of the expression of mitochondria in adult muscle. We have found support for this notion in the human muscle. In electrically stimulated muscle of spinal cord patients myogenin is increased with a concomitant proliferation of mitochondria. Cytokines are not transcription factors, but some of them may be inducers of transcription in skeletal muscle via various signal transduction pathways. IL6 is expressed in skeletal muscle with exercise, especially in eccentric exercise and could play a role in remodelling the muscle after damage.

The general view is that contraction-induced muscle glucose transport only depends on stimulation frequency and not on workload. Incubated soleus muscles were electrically stimulated at a given pattern for 5 min. Resting length was adjusted to achieve either no force (0 % P), 50 % (50 % P), or maximum (100 % P) force. Glucose transport (2-deoxy-glucose uptake) increased directly with force development. Glycogen decreased at 0 %P but did not change further with force development. Lactate, AMP and IMP concentrations were higher and ATP concentrations lower when force was produced than when it was not. 5’AMP-activated protein kinase (AMPK) activity increased directly with force. Thus, contraction-induced muscle glucose transport varies directly with force development and is not solely determined by stimulation frequency. AMPK activity is probably an essential determinant of contraction induced glucose transport.

Insulin action in skeletal muscle is known to be influenced by various factors such as activity/inactivity, denervation, diet and recently also by muscle glycogen concentration. We have investigated the mechanism behind the glycogen effect on insulin action. Insulin-stimulated rat muscle glucose transport varied inversely with muscle glycogen concentration in fast twitch but not in slow twitch muscle. In fast twitch muscle, insulin stimulated IRTK (Insulin Receptor Tyrosine Kinase) activity was unaffected by glycogen levels whereas akt/PKB activity varied inversely with muscle glycogen concentration and correlated closely with glucose transport. This is the first demonstration of insulin signalling in skeletal muscle being dependent on muscle glycogen concentration. It also shows that glucose transport in the different muscle fibre types is very differently affected by changes in muscle glycogen concentration. This is in parallel to our recent findings regarding contraction induced muscle glucose transport, which also varies inversely with glycogen concentration in fast twitch but not in slow twitch muscle. Collectively these findings show an important effect of glycogen concentration on muscle glucose transport in fast twitch muscle.

The adenosine receptors in human skeletal muscle have for the first time been directly localised and identified with an immunohistochemical technique. Our findings demonstrate that the three receptor subtypes A1, A2A and A2B are all present in human skeletal muscle. Of these three subtypes only the A2A and A2B receptors are found in the skeletal muscle cells whereas all three subtypes are present in the vascular endothelial and smooth muscle cells. Another observation of interest is that it appears that these adenosine receptor subtypes are only present in endothelial cells of blood vessels that do not contain smooth muscle cells, i.e. capillaries.

In the search for exercise-induced local metabolic factors associated with vasodilation in skeletal muscle, adenosine can now be determined with the microdialysis technique and its production site in the interstitium has been confirmed. In addition, K⁺ can also be determined with the microdialysis technique and possibly also NO. The latter substance plays a major role in maintaining the resting blood flow, but in experiments using L-NMMA or L-NAME to block the n-NOS-activity the exercise hyperaemia is unaffected. Indeed, at present adenosine appears to be the best vasodilator candidate in contracting muscle. This can occur without acting via an enhanced release of K⁺ from contracting skeletal muscle fibres (opening of ATP sensitive K⁺ channels).

The last issue to be highlighted is our attempts to link blood flow regulation and oxygen availability to the rate of mitochondrial respiration. We have come far enough to conclude that the delay in elevating mitochondrial respiration at onset of muscle work (which is much shorter than earlier described, i.e. 4-6 s instead of 12-15 s) is not due to lack of O_2. Rather the delay is due the fact that ADP may be lacking and an increased amount of pyruvate (and NADH?) appears to be available in the cytosol within a second. In this work energy transfer of the various processes in the ATP-resynthesis was studied by measuring total heat production at constant power. The amount of heat increased rapidly for 20-30 s and continued thereafter to become further elevated, but at a much slower rate. These data conform to the proposed two-fold higher energy transfer when PCr breakdown provides the energy for ATP-resynthesis, and glycolysis some 20% more than when oxidation provides the energy.

Two major developments occurred in regard to laboratory facilities at Rigshospitalet. The molecular biology group could move into their new facilities at Juliane Maries Vej where the 1^st floor is shared with molecular biologists from the hospital’s cardiology and neurology units. Our centre has now the potential to address questions in the muscle metabolic field which can only by answered by means of these new possibilities. The laboratory will serve as a core facility supporting various CMRC researchers. In addition, the present two post doctoral fellows and the two students working in the laboratory will run their own projects. A laboratory technician will soon be employed.

The other facility is one we have had from the start of the CMRC, but now it has finally found its fully developed stage. It is the laboratory in the main building of Rigshospitalet where Niels Secher performs his experiments and others too, when their studies are technically difficult and highly invasive. In addition to Niels Secher’s office, we have two well-equipped rooms for experiments and access to a third room where various blood analyses can be performed.

During 1998 the CMRC stable isotope laboratory became fully equipped (2 GC-MS, one isotope ratio, and one GC apparatus). The method developments were almost finalised by the end of the year and a number of previously planned metabolic studies were conducted in the fall. The laboratory is staffed with one engineer, one biochemist (researcher) and three laboratory technicians.

During 1998, five of the CMRC active Ph.D.-students and one doctoral student defended their theses.

The CMRC researchers have an extensive network of Danish and foreign research collaborators. Below are listed the research partners who have collaborated on CMRC projects that resulted in a CMRC publication in 1998.

• Jens Bülow, overlæge, Dept. of Clinical Physiology, Bispebjerg Hospital
• Torben Clausen, professor, Dept. of Medical Physiology, Aarhus University

• Arend Bonen, professor, Department of Kinesiology, University of Waterloo, Canada.
• Roberto Bottinelli, associate professor, Institute of Human Physiology, University of Pavia, Italy.
• Samuel W. Cushman, professor, Metab. & Nutrition Section, NIH, Bethesda, USA.
• Salvatore DiMauro, Dept. of Neurology, University of Columbia, New York, USA.
• Jan F. Glatz, assoc. prof., Dept. of Physiol. & Motion Science, Univ. Limburg, the Netherlands.
• Laurie J. Goodyear, assoc. professor, Joslin Diabetes Center, Harvard Medical School, USA
• Terry Graham, professor, Department of Human Biology, University of Guelph, Canada
• A.P. Halestrap, professor, University of Bristol, UK.
• Ronald Haller, professor, Institute for Exercise and Environmental Medicine, Presbyterian Hospital of Dallas, USA.
• Takafumi Hamaoka, professor, Dept. of Preventive Med. and Public Health, Tokyo Medical School, Japan
• Stephen D.R. Harridge, lecturer, the Royal Free Hospital, Dept. of Geriatrics, London, UK.
• Peter Hespel, professor, Catholic University of Leuven, Belgium.
• Harinder S. Hundal, senior lecturer, Dept. of Anatomy and Physiol., University of Dundee, UK.
• Johannes v. Lieshout, assoc. professor, Academic Hospital, University of Amsterdam, the Netherlands.
• Dave A. MacLean, assist. prof., Division of Cardiology, Hershey Medical Center, USA.
• Evelyn Ralston, assoc. professor, Laboratory of Neurobiology, NIH, Bethesda, USA
• Carlo Reggiani, associate professor, Institute of Human Physiology, University of Pavia, Italy.
• Leonard H. Storlien, professor, Dept. of Biomedical Science, University of Wollongong, Australia.
• Lorraine Turcotte, assist. professor, University of Southern California, Los Angeles, USA.
• Ger J.v.d.Vusse, prof., Dept. of Physiol. & Motion Science, Univ. of Limburg, the Nether-lands.

A number of CMRC senior and junior researchers were invited to lecture at numerous congresses, conferences, and symposia, both nationally and internationally, during 1998. The major international congresses/conferences amount to 11. The CMRC did not offer any Ph.D.-courses during 1998, but 3 researchers were invited as teachers at Ph.D.-courses in Denmark.