The August Krogh Institute: Capillaries and beyond

Scand J Med Sci Sports 2015: 25 (Suppl. 4): 16 21 doi: 10.1111/sms.12552 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Review Article The August Krogh Institute: Capillaries and beyond G. Sjøgaard Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark Corresponding author: Gisela Sjøgaard, PhD, DrMedSci, Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Campusvej 55, Odense DK-5230, Denmark. Tel: +45 2498 4063, E-mail: gsjogaard@health.sdu.dk Accepted for publication 11 August 2015 Bengt Saltin knew very well the history and work of the giants whose shoulders he was standing upon, such as August Krogh and Johannes Lindhard. He was basically a physiologist interested in physical activity and exercise, particularly in the cardiovascular and muscular responses. Some of his major original contributions were (a) the human muscle model in terms of the one-legged, knee extensor quantifying work by the high-precision Krogh ergometer and, using this, challenging Krogh s proposed autoregulation of capillary blood flow during exercise; (b) the electrolyte fluxes quantification on an intra- and extra-cellular level in human muscle during exercise to reveal such changes as possible fatigue mechanisms; and (c) the evidence presented that underlined the health-enhancing effect of physical exercise training from bedside to workplace. August and Marie Krogh were icons during the golden age of exercise physiology (1940 1970) of the Laboratory for the Theory of Gymnastics, GLAB, University of Copenhagen. Thus, when the laboratories were planned to be moved from the Rockefeller building in Copenhagen to a new building ready in 1970, strong arguments were posed to name it the August Krogh Institute (AKI), in honor of our Nobel Prize winner on 28 October 1920. This is the exact same institute where Bengt Saltin in 1973 started his professorial carrier in Denmark. As a student I had witnessed the physical move of the laboratories in 1971 now I was witnessing a change in research paradigm. I was stunned when I had listened to Bengt Saltin s first lecture and learned that it was actually possible to take muscle samples from healthy people and analyze them for capillary density and fiber types. This was the beginning of very exciting years for myself, the GLAB, and the whole research area of exercise physiology. August Krogh has impacted tremendously on exercise physiology and only small glimpses with immediate relevance for this paper will be touched upon. Understanding transport of oxygen for metabolism requires knowledge of regulatory processes in the capillaries and Krogh s work on transfer of molecules and ions between capillary blood and tissue is a cornerstone of his scientific contributions leading to the Nobel Prize. Krogh s proposal that the oxygen requirement in the tissue and the oxygen supply to the tissue would regulate capillary blood flow in muscle during work was by then generally accepted (Schmidt-Nielsen, 1997). However, Bengt Saltin challenged the concept of such autoregulation by the muscle especially during high-intensity, whole-body work. The beginning of sport science in Denmark August Krogh was known as an ambitious physiologist in the beginning of 20th centennial and was in play when the University of Copenhagen, after a long dispute in 1909, decided to accept new education for teachers in gymnastics. To ensure scientific content of high quality for the theoretical part, the university wanted August Krogh to supervise this; eventually, however, the physician Johannes Lindhard was employed as docent in gymnastics theory in 1909. Nevertheless, August Krogh was a mentor for the younger Danish exercise physiologists at GLAB, Erling Asmussen, Erik Hohwü-Christensen, and Marius Nielsen; he called the younger physiologists the three musketeers. He engaged in helping them all to study abroad using his international network and played an essential role in their scientific successes in human exercise physiology encompassing exposure to altitude, heat, and humidity. August Krogh together with Lindhard profiled the research from the beginning of GLAB to focus on physiological responses to muscular work, and the main focus was on circulation and muscle. From around 1940 the three musketeers took the lead and engaged in additional areas, as can be seen from the publication profile shown in Fig. 1, redrawn from Poulsen (2009). No doubt Bengt was aware of the attractive research milieu, but Hohwü-Christensen was especially instrumental for his move. Hohwü-Christensen had, in 1935, been the leader of an expedition in the 16

The August Krogh Institute studying man in a stressful environment that Bengt addressed in so many studies since (Saltin et al. 1987). The present review will focus on three topics where Bengt Saltin has given pioneering scientific contributions: the knee extension model, muscle electrolyte dynamics, and physical activity for health. The knee extension model Fig. 1. Percentage of peer-reviewed original papers at GLAB. Publication profile of peer-reviewed papers from the Laboratory for the Theory of Gymnastics during the two 30-year periods: 1909 1939 and 1940 1970, redrawn from Poulsen (2009). Andes together with other world famous physiologists studying human cardiorespiratory responses to high altitude (Christensen & Frobes, 1937). During the Second World War he came to Stockholm and was a teacher of Bengt Saltin at Gymnastik och Idrotts Högskolan, Stockholm, Sweden, and later a main mediator for Bengt Saltin s decision to move to Denmark and accept a professorship at the AKI in Copenhagen. The new AKI had been equipped with very advanced facilities such as a temperature-adjustable climate chamber with the original Krogh precision balance installed to assess sweat loss while the subject was seated on the bicycle, a swimming flume, a hypobaric chamber, and, of course, a treadmill on which even racing bicycle experiments were performed to study human physiological responses during exercise in numerous ambient conditions. Now, Bengt Saltin very effectively introduced new research methodologies biopsies were taken and analyzed for capillary density in human muscle divided into the numbers surrounding type I and type II fibers (Sjøgaard & Saltin, 1980; Sjøgaard, 1982). Inspired by his teacher, some of the first experiments Bengt was running at the AKI in 1973 were in the hypobaric chamber, taking subjects to about 3000 m in altitude to reduce their maximal oxygen capacity to 80% of that at sea level. Biopsies were taken in relation to intensive prolonged bicycle work that I was involved in as a master student. Many technical problems were encountered but eventually the detailed analysis of fiber type-specific glycogen depletion during prolonged high-intensity work resulted in a gold medal award from the University of Copenhagen (Sjøgaard, 1975). Together with many other researchers, Bengt Saltin later continued the high-altitude research from the first period at the AKI in real mountains, for example, Mount Everest (Savard et al., 1995). Likewise, he very soon took advantage of the advanced climate chamber and the high standard of research within heat stress at the AKI (Nielsen et al., 1990), adding to his engagement of Basic human physiology was the overall interest of Bengt Saltin and he advocated the department to be termed Human Physiology and not Theory of Gymnastics because he understood the importance of studying generic underlying mechanisms of human physiological responses to exercise. He was successful in 1989 when the official change was accepted. More importantly, he had also been successful in studying basic mechanism of physiological responses in exercising humans. Various animal models are available; however, humans are unique (e.g., in their muscle fiber type distribution, anatomy, or disease patterns) and therefore it was pertinent to identify the best possible human muscle model for studying basic human exercise physiology. The biceps brachii had proven well suited for biomechanical and neuromuscular studies but was a less fruitful approach for cardiovascular and metabolic studies. This is because it is difficult to obtain venous effluent blood from that muscle because of the complex vascular supply in the shoulder region and because the muscle is rather small for repeated biopsy sampling. Using the knee extensors in a setup where the workload was quantified with precision by the Krogh bicycle was promising (Andersen et al., 1985a). Nice linear relationships were demonstrated for HR, VO 2, and VE. There was only one disturbing aspect. Resting values were quite different from 0 W knee extension work resulting in a jump from rest to 0 W. Much later detailed biomechanical modeling presented evidence that knee extension at 60 rpm requested an internal work rate of 10 W for lifting the lower leg by the knee extensors (Sjøgaard et al., 2002). Based on this the total work rate of the knee extensors was estimated as the sum of the external work rate measured by the Krogh ergometer and the internal work rate calculated using the biomechanical model. Redrawing the original figure now showed a continuous slope for HR and VO 2 relative to the total work rate during knee extension (Fig. 2). The knee extension model has been used extensively as it was first published and was a prerequisite for Bengt Saltin being able to demonstrate that during maximal exercise by a small fraction of the body s muscle mass (i.e., one leg s knee extensor of around 2.5 kg), the exercising muscle could accommodate much larger blood flow of 2 2.5 L/kg/min than ever reported previously (Andersen & Saltin, 1985b). This was a major breakthrough because this demonstrated that the concept proposed by Krogh of 17

Sjøgaard 0 10 20 30 40 50 60 70 Internal Total work rate, Wa s Work rate ~10 Wa s Fig. 2. Knee extension model. Oxygen uptake vs total power during one-legged knee extension calculated as the sum of external work rate determined by the Krogh bicycle ergometer and the internal work rate of the knee extensors for lifting the lower leg and foot (for more details of the model for internal work rate, see Sjøgaard et al., 2002). The original figure presenting the relationship between oxygen uptake and external power only with a discontinuous jump from rest to 0 W is from Andersen et al. (1985a). blood flow autoregulation by the muscle could not suffice to explain the distribution of blood flow during whole-body maximal work. Assuming a total muscle mass of 30 kg exercising at high intensity, a maximal perfusion of all muscle would request a cardiac output of 60 75 L/min, which is far beyond values of around 40 L/ min measured in even the most well-trained athletes. Muscle electrolyte dynamics Just before August Krogh s retirement from his professorship in 1945 he had finalized the thesis, The active and passive exchanges of inorganic ions through the surface of living cells and through living membranes, which was published in Proceedings of the Royal Society (Krogh, 1946). He had studied the diffusion of potassium out of the cell and quantified the rate of uptake using 42 K produced in a cyclotron at the Niels Bohr Institute. Later, in 1997, another Danish Nobel Prize winner, Jens Chr. Skov, had in 1957 convincingly demonstrated that the active uptake of potassium was mediated by the Na/K pump (Skou, 1957). All experiments were performed on plant and animal tissue, and as for the human muscle model, Bengt Saltin insisted to find methods to analyze such fluxes in humans to study possible limitations of physical performance. Two methodological steps were taken: (a) the fraction of extracellular muscle water space of the total water content assessed by the H 3 inulin method was introduced in humans and (b) K and Na were analyzed in single slow- and fast-twitch fiber-type fragments from human muscle samples (Sjøgaard & Saltin, 1982; Sjøgaard, 1983). A large challenge was to take all steps necessary to convincingly estimate the extracellular water space in human muscle samples; the steps were (a) choice of marker with sufficient sensitivity to be detected in very small volumes, pointing toward a radioactive tracer; (b) determination of isotope concentrations not exceeding acceptable radiation levels for healthy humans; and (c) verification of the marker s radioactive tracer not entering the intracellular space of muscle fibers. Eventually, all obstacles were tackled using several rats for the validation, and a unique paper was published one of its kind and cited many times every year for the last more than 30 years, as it was taken as a reference standard not only for estimating the relative distributions of water space in humans at rest and during exercise (Sjøgaard & Saltin, 1982) but also for estimating drug pharmacokinetics, modeling in angiogenesis research of the vascular endothelial growth factor distribution, and in magnetic resonance imaging validations. For the electrolyte analyses, the methodological development was more straightforward although demanding high patience (Sjøgaard, 1983) and the paper had lasting impact on muscle fatigue research (Cairns & Lindinger, 2008; Clausen, 2008). The combined analyses of muscle water spaces and electrolyte concentrations in muscle and blood samples allowed for estimating muscle membrane potential (Fig. 3) by the Goldman Hodgkin Katz equation (Hodgkin & 18

The August Krogh Institute Fig. 3. Membrane potential. Extracellular potassium concentration, K e, was calculated from plasma K and intracellular potassium concentration, K i, was calculated based on K e, total muscle K content, and intracellular and extracellular water content in muscle biopsies. Similarly, Na concentrations were calculated. The membrane potential was estimated from the equation E m = 61.5 log 10 [(K e + 0.01Na e) (K i + 0.01/Na i)]; for more details see Sjøgaard (1986). Horowicz, 1959). This showed that the net loss of K during exercise could account for a 5 mv decrease and the increase in extracellular K could account for another 10 mv decrease in membrane potential (for more details, see Sjøgaard, 1990; Sejersted & Sjøgaard, 2000). Such changes could affect muscle membrane excitability and impact on contractility. The use of microelectrodes or the microdialysis technique even revealed that extracellular K concentrations assessed from effluent venous blood would be underestimating interstitial K concentration (Sjøgaard et al., 1988; Juel et al., 2000; Rosendal et al., 2004). These methodological developments advanced our detailed knowledge tremendously from the research on muscle fatigue that Erling Asmussen so excellently had summarized as the basis for this endeavor (Asmussen, 1979). Physical activity for health When establishing the education in gymnastics at the University of Copenhagen, there was some dispute whether this should be associated to the faculty of medicine, philosophy, or mathematics/natural science. Strong arguments regarding the health benefits of gymnastics probably resulted in its initial allocation to the medical faculty, but after only a couple of years it was transferred to the faculty of mathematics/natural science. There were some administrative reasons for this, as this education was not part of the medical candidate exams but only a minor for high school teachers and their major was more common at other faculties. However, a serious fight regarding the lack of scientific evidence of the health-enhancing mechanisms of gymnastics was also ongoing. The practitioners believed gymnastics was health enhancing, but professor Lindhard insisted on evidence for such statement and for this, only objective and precise measurements would be accepted and such evidence was not available at that time. Interestingly, professor Asmussen, although being a capable gymnast and enjoying bicycling, also never agreed on gymnastics having any health benefits; rather, he found gymnastics to be a bit risky. These professorial viewpoints were waved away when Bengt arrived. Before coming to the AKI he had, for example, in Svenska Läkartidning 1970, dealt with the topic Physical training: Practical and medical viewpoints and studied health benefits in former athletes (Saltin & Grimby, 1968). He later engaged heavily in the area of exercise and health (Pedersen & Saltin, 2006). Bengt Saltin had a very broad view on where in society physical activity could promote health, and importantly, in line with Lindhard, he insisted on scientific evidence in terms of physiological responses to physical activity. He had worked together with professor Åsa Kilbom from the National Institute of Occupational Health in Sweden and demonstrated that exercise training was health enhancing for sedentary middle-aged men (Kilbom et al., 1969). Åsa Kilbom played an important role for occupational health in Sweden and Bengt Saltin now wanted me to fill that role in Denmark. Eventually, I was employed at the National Institute of Occupational Health in Demark in 1983 as the first exercise physiologist in an occupational setting and established a department of work physiology and ergonomics. I was enrolled in Åsa Kilbom s international network, and research paradigms were developed with other lead researchers in the field (Armstrong et al., 1993). Musculo-skeletal disorders constitute the major part of work-related disorders and are the major causes of sickness absence. International research efforts elucidated occupational exposures that cause such disorders and proposals on how to minimize those (Armstrong et al., 1993). However, not until the 21st century did it become legitimate to conduct research of physical exercise training of workers for 19

Sjøgaard improving physical capacity as a means of decreasing relative muscle load and pain. The first randomized controled trial in Denmark in an occupational setting was conducted in 2005 after careful planning and negotiation at the workplace to allow workers with sedentary work tasks to train 3 20 min a week within working hours. The results were convincing: muscle strength increased, muscle pain decreased, and cardiorespiratory risk factors decreased (Blangsted et al., 2008; Pedersen et al., 2009). The next step of evidence we wanted to present was the underlying mechanisms in the muscle for such positive findings. Naturally, this involved Bengt Saltin, who was very enthusiastic to join a project that had a setup with a large team jointly between the National Institute of Occupational Health and the University of Copenhagen. The prime finding was again that training could reduce workrelated muscle pain (Andersen et al., 2008a). In particular, strength training of the painful muscle was effective in the long term and multiple muscle adaptations occurred. Muscle mechanics and metabolism improved as evidenced based on analysis of muscle biopsies from the myalgic trapezius muscle. Compared with healthy control trapezius muscle, the myalgic trapezius muscle had the same mean number of capillaries around fibers (Nielsen et al., 2010; Gerdle et al., 2014). However, more detailed morphological analysis showed a number of morphological impairments (Mackey et al., 2010), particularly an excess number of mega fibers with a 40% decrease in capillaries around the fiber area (Andersen et al., 2008b) and impaired oxidative metabolism at rest and with repetitive work (Sjøgaard et al., 2010). Importantly, with exercise training functional capacity improved (Andersen et al., 2014), metabolic capacity increased (Søgaard et al., 2012), as demonstrated even at the gene level (Sjøgaard et al., 2013). Morphological recovery was also documented using advanced immuno-histochemical stainings; for example, satellite cells (Mackey et al., 2011) and neuronal nitric oxide synthase (Jensen et al., 2015). These novel findings can be envisaged to impact tremendously on future treatment and prevention. Perspective Bengt Saltin was always striving toward the highest level to reach novel understanding within human physiology and was also impacting on university structure and education. He was standing on the shoulders of giants and he reached further up. His inspiration has been immense in a steady continuum of more than 50 years of scientific curiosity. It has been like a never-ending endeavor with worldwide research collaboration across fields, from the subcellular muscle level to professional sports and reaching out to physical activity in a health perspective, scrutinizing underlying physiological mechanisms. Bengt Saltin has been like a mountain of knowledge and like a shepherd guiding us along successful trials. Numerous scientists around the world continue his research that will impact enormously on future scientific insight. With inspiration from August Krogh s life and death (Schmidt-Nielsen, 1995) the epilogue says, The enormity of the event has changed our lives a bright and shining light has gone out. Key words: Muscle perfusion, muscular performance and fatigue, exercise as medicine. References Andersen LL, Andersen CH, Skotte JH, Suetta C, Sogaard K, Saltin B, Sjøgaard G. High-intensity strength training improves function of chronically painful muscles: case-control and RCT studies. Biomed Res Int 2014: 2014: 187324. Andersen LL, Kjaer M, Søgaard K, Hansen L, Kryger A, Sjøgaard G. Effect of two contrasting types of physical exercises on chronic neck muscle pain. Arthritis Rheum 2008a: 59: 84 91. Andersen LL, Suetta C, Andersen JL, Kjær M, Sjøgaard G. 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