Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 8, 2015). doi:10.1152/ajpregu.00405.2014
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From Tusko to Titin:
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The role for comparative physiology in an era of molecular discovery
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KROGH DISTINGUISHED LECTURE OF THE AMERICAN PHYSIOLOGICAL SOCIETY
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S.L. Lindstedt and K.C. Nishikawa
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Center for Bioengineering Innovation and Department of Biological Sciences,
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Northern Arizona University, Flagstaff AZ, 86011-5640
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Running Page Heading: Comparative Physiology and Modern Biology
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Keywords: Allometry, connectin, muscle contraction, muscle activation, muscular dystrophy
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with myositis (mdm), winding filament hypothesis
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Send correspondence to: Stan L. Lindstedt Department of Biology Northern Arizona University Flagstaff, AZ 86011-5640 FAX: (928) 523-7500 Email: [email protected]
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DISCLOSURES
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No conflicts of interest, financial or otherwise, are declared by the authors.
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1 Copyright © 2015 by the American Physiological Society.
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Abstract
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As we approach the centenary of the term comparative physiology, we re-examine its role in
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modern biology. Finding inspiration in Krogh’s classic 1929 paper, we first look back to some
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timeless contributions to the field. The obvious and fascinating variation among animals is much
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more evident than is their shared physiological unity, which transcends both body size and
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specific adaptations. The “unity in diversity” reveals general patterns and principles of
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physiology that are invisible when examining only one species. Next, we examine selected
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contemporary contributions to comparative physiology, which provides the context in which
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reductionist experiments are best interpreted. We discuss the sometimes surprising insights
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provided by two comparative “athletes,” pronghorn and rattlesnakes, which demonstrate: 1)
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animals are not isolated molecular mechanisms but highly integrated physiological machines; a
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single “rate-limiting” step may be exceptional, and 2) extremes in nature are rarely the result of
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novel mechanisms, but rather employ existing solutions in novel ways. Further, rattlesnake
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tailshaker muscle effectively abolished the conventional view of incompatibility of simultaneous
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sustained anaerobic glycolysis and oxidative ATP production. We end this review by looking
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forward, much as Krogh did, to suggest that a comparative approach may best lend insights in
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unraveling how skeletal muscle stores and recovers mechanical energy when operating
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cyclically. We discuss and speculate on the role of the largest known protein, titin (the third
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muscle filament), as a dynamic spring capable of storing and recovering elastic recoil potential
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energy in skeletal muscle.
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Comparative physiology is most often associated with the interesting and rare adaptations that
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have evolved to match animals to their demanding environments. Indeed, the historic roots of
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comparative physiology included many stories of often fascinating discovery, what Somero (52)
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referred to as “exploratory physiology”. Thus, urine concentration in desert mammals, warm-
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muscles of tunas, and bar-headed geese flying over Everest are among the favorite topics in all
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animal physiology classes.
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These unusual discoveries also have had another impact, that of providing biology with ideal
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model systems for experimental inquiry. In his 1929 paper, the namesake of this lecture, August
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Krogh (30) introduced what we now refer to as the Krogh Principle, namely that ‘For a large
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number of problems there will be some animal of choice or a few such animals on which it can
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be most conveniently studied.’ (30, 33). By selecting the ideal experimental animal, one
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maximizes the ratio of signal to noise, making interpretation of results much simpler. One theme
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of this review is that within the diversity of “comparative physiology” there are numerous
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examples of enhanced signal to noise which simplify and clarify scientific discovery.
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A second theme of this review is a question posed fifty years after Krogh’s review; FE Yates1
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(then editor of AJP) asked the question, “Comparative Physiology: Compared to what?” (58). In
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this essay, we attempt to follow Krogh’s example of examining the contributions of comparative
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physiology looking back and forward, in all instances asking ‘compared to what?’
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1) Emergent principles from disparate data, “Unity in Diversity” (52)
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Disparate data, when amalgamated, may reveal biological insights and emergent patterns that are
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only visible “comparatively.” One striking example unfolds when comparing animals that differ
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in body size. The obvious first observation is that the smallest and largest mammals share nearly
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identical cell structure and cell biochemistry. However body size alone sets the “gas pedal”
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regulating virtually all physiological processes. Krogh’s contemporary, JBS Haldane (13) wrote
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an engaging essay, “On Being the Right Size,” in which he described body size as the single
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most important, but rarely considered, variable among animals.
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LSD and the Elephant
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An enduring example that should never be lost from the collective knowledge of all physiologists
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is the story of LSD and the elephant, a favorite example of Knut Schmidt-Nielsen that we 3
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recount on his behalf. It is difficult to keep male elephants in captivity because each year they go
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through a period of extreme aggression, “musth” which has resulted in injury and even death to
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other zoo animals as well as zoo keepers. For that reason most zoo elephants are females. During
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the early 1960s, perhaps inspired by experiments and notoriety of Timothy Leary, there were an
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unusually large number of “experiments” involving LSD. One of those involved a male elephant,
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Tusko, residing at the time in the Oklahoma City Zoo. The “hypothesis” of this experiment,
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published in Science (57) was to test if administering LSD to Tusko could induce musth.
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(Thankfully, institutions now have Animal Care and Use Committees.) Of course the first task
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was to calculate the LSD dose. As cats had been the subjects of an imprudent volume of LSD
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experimentation, the dose for Tusko was calculated based on the well-established weight-specific
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cat dose; to ensure the safety of the animal, the calculated dose was halved. When the animal
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died shortly after the LSD was administered, the conclusion, as reported in Science, was simply
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that elephants are very sensitive to LSD. Of course drug delivery, metabolism and clearance all
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occur at a rate dictated by an animal’s pace of metabolism, which scales disproportionately with
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body mass. Using this metabolism as the metric, even by conservative calculation, Tusko was
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given about a 10-fold overdose (297 mg vs ~ 32 mg) and there is no evidence that elephants are
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unusually sensitive to LSD (or that it might induce musth!).
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Metabolism is just one example, but virtually all physiological variables are strongly body size
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dependent. When expressed in the familiar Power Law equation (Y = aXb), body size dependent
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variables fall into quantitative clusters, with similar slope (b) values (42). For example, volumes
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scale linearly (b≅1); blood volume is very nearly 7% of body mass in all mammals and heart
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mass is about 0.5%. Pressures (force/area) are generally independent of size (b≅0) thus blood
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pressure and maximum muscle stress are essentially constant across all mammals. Physiological
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rates (and their reciprocal, times) scale with surprising consistency, -1/4>b>-1/5 (Fig. 1).
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Volume-rates (such as metabolism and the renal or hepatic clearance of drugs, importantly for
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Tusko) scale as (volume × frequency) or about M3/4 (34, 37). This consistent scaling seems to
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confirm AV Hill’s (21) speculation that mammals experience a body size dependent internal
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clock that dictates the pace of life; all time cycles from mitosis to longevity seem to be set to a
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common “physiological clock”. Conforming to this “clock”, all mammals experience roughly the
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same number of heart beats per lifetime, during which they consume roughly the same number of
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calories per gram of body tissue. These emergent patterns should be considered when testing
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models of aging, etc.
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Compared to what: Although the most conspicuous feature of comparative physiology is the
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diversity among animals, consistent “patterns of design” transcend that diversity. These patterns
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become visible only through comparisons. Body size dependent patterns are one example of the
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unity in diversity (52).
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2) Organismal context to interpret reductionist experiments.
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As biologists, we are taught from our earliest graduate classes to design controlled “reductionist”
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experiments when possible isolating one variable for experimentation. This approach is
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particularly valuable in biology dictated by the large number of simultaneous variables.
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Therefore one challenge in experimental design is to isolate and focus as much as possible in
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order to identify cause and effect. The risk, of course, is that experiments can be performed and
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also interpreted in isolation. Ultimately, there must be a whole organism context as the final
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interpretation of our reductionist results.
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Blood flow limitation to maximum oxygen uptake
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One organismal misunderstanding was the consequence of a valuable reductionist technique,
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isolating blood flow to an individual muscle and measuring in vivo work and oxygen uptake.
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This in situ preparation resulted in some valuable insights, but also at least one detour into a cul-
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de-sac. Briefly, by isolating the blood flow to a single muscle cluster (usually dog
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gastrocnemius-plantaris muscle group), the work output could be manipulated and blood flow
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and oxygen uptake all measured in isolation (4). One significant conclusion of these studies, that
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was many times confirmed, is that blood flow was the single limiting factor setting maximum
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muscle metabolism. As the experiments were meticulously done and results consistent and
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unambiguous, it seemed unnecessary to interpret them in a whole animal context. Thus, in 1985
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when Dick Taylor, Bob Armstrong and Harold Laughlin injected microspheres in a dog running
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on the treadmill at the Concord Field Station, muscle blood flows that greatly exceeded the
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maximum measured in situ (and thus thought possible) strongly suggested the presence of a
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methodological error, hence the rejection of their manuscript.
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However, if one were to make one simple calculation, multiply the isolated blood flow and
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oxygen uptake values by the dog’s entire muscle mass (assuming all dog muscles were operating
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at this maximum, which would seem unlikely), rather than overestimating whole animal oxygen
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uptake as expected, these measured values account for only 1/3 of the measured oxygen uptake.
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In fact, these experiments actually demonstrated the obvious: restricting blood flow limits
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oxygen uptake. Eventually, as a result of whole animal experiments, Laughlin introduced the
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concept of “exercise hyperemia” (rather than in situ ischemia!) into the exercise science
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literature (31). It was only by “comparing” isolated muscle values to intact organismal
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measurements that the misinterpretation of blood flow limitation was exposed.
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Pronghorn: the world’s most elite endurance athlete
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Perhaps the ideal Krogh model system for asking if there is a “rate-limiting step” in the uptake,
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transport and utilization of oxygen is an animal with extraordinary oxygen uptake. Pronghorn
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Antilocapra americana, evolved in the barren plains of the western US where strong selective
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pressure from both sprint (cheetah-like cats) and endurance (wolves, coyotes) predators have
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apparently provided strong selective pressure to maximize both speed and endurance. Comparing
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pronghorns with similar sized, but aerobically “typical,” goats, their maximum oxygen uptake is
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five times higher. When we assess the capacities at each step of the oxygen transport cascade,
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from lung to mitochondrial sink, there is no apparent weak link that limits oxygen flow (35) (Fig.
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2). Indeed, rather than a single “rate-limiting” step, these experiments provide evidence of
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“shared” limits, as each link in the oxygen transport cascade seems to have nearly identical
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capacity (56).
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Rattlesnake tailshaker muscle: A stationary athlete
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Western diamondback rattlesnakes, Crotalus atrox, are fairly “typical” reptiles aerobically, with
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little capacity for sustained activity. They possess one surprising attribute; rattlesnakes can
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maintain very high frequency rattling for hours. The tailshaker muscle is highly aerobic, nearly
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one third mitochondria and one fourth sarcoplasmic reticulum (50). Using Magnetic Resonance
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Spectroscopy (MRS) to estimate the energy cost, we found that as only a single twitch is
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necessary to contract the muscle, the cost per contraction is exceptionally low (9, 43). However,
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there was still a mismatch between ATP production and ATP cost. This mismatch was only
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resolved when this observation was revisited on the whole animal level. What Conley and his 6
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colleagues found belied our textbook knowledge; rattlesnakes use sustained anaerobic glycolysis
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to supplement aerobic ATP production on a steady-state basis (fig. 3). The rich blood supply and
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minimal muscle size results in a solution of exporting lactate to other tissues to be oxidized there.
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Putting this high frequency muscle into a whole animal context resulted in a “shake-up” of our
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traditional view of glycolysis and greatly expanded our understanding of muscle energetics (29).
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Molecular basis of muscular contraction During the past century, remarkable progress has been made elucidating the molecular mechanisms of muscle contraction (reviewed in 23, 25). While most of this research has been focused at the level of single demembranated muscle fibers (e.g., 11), technical advances have made it possible to explore the process of muscle contraction at ever smaller scales, in single myofibrils (32) or even single sarcomeres (18, 48). These reductionist studies have produced an internally consistent description of the biochemistry and biophysics of cross-bridge cycling in muscle. However, despite this progress, the goal of predicting how muscle force changes during natural movements at the level of the intact organism has remained elusive despite decades of intensive research (7, 49). Muscle models based on the sliding filament theory and used in neuromusculoskeletal simulations (e.g., 59) fail to predict muscle force under physiologically relevant conditions, even during purely isometric contractions (6).
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In summary, as biologists we appreciate and thus embrace physiological principles. The
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seduction of molecular clarity is that little or no context is required for interpretation. No animal
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exists as a mere compilation of molecules, it is only by putting these molecular discoveries back
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into a whole animal context that insights mature into physiological principles. Thus it is useful to
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remain cautious of the “sufficiency of proof” axiom, namely that far less evidence is required to
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establish an idea as “fact” than is required to dislodge that idea once established.
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Compared to what: Reductionist experiments are the basis of scientific discovery and the pace of
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data acquisition has never been greater than in this era of molecular biology. Comparative
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physiology provides a scaffold on which data from reductionist experiments can be built into
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biological insights. 3) Constructing a molecular model from comparative data.
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Identification of cross-species patterns does not provide explanations, but these patterns may be
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the starting point for novel hypotheses. Identified patterns can guide experimental inquiry; we
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end this review with a look forward.
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Most animals use their muscles cyclically in locomotion, whether running, swimming or flying
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(51). In almost all instances, cyclic muscle use involves the storage and recovery of elastic recoil
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potential energy. Just as experiments by AV Hill (20) provided insights into the properties of
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muscle, our understanding of cyclic muscle use has been greatly enhanced by the work loop
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analysis introduced by Robert Josephson (27). Once again, body size is a dominant factor
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influencing this behavior. In all modes of movement, body size dictates frequency of muscle use.
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Here we focus on galloping mammals, but the same principles apply to swimming or flying, as
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well as other cyclical movements.
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It has been 40 years since Heglund et al. (14) identified the strong link between body size and
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stride frequency among galloping mammals; body size alone seems to set stride frequency (s-1) =
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4.5 kg-0.14. So consistent is this scaling of stride frequency that we have developed a student
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biomechanics lab that demonstrates two principles: 1) humans hopping in place do so at the same
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frequency as a galloping mammal of the same size (Fig. 4); and 2) changing the self-selected
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frequency doubles the energy cost per hop, greatly reducing apparent muscle efficiency (34).
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Running mammals select a stride frequency that maximizes the recovery of elastic recoil energy.
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In addition to selecting a “lowest cost” stride frequency, there are other properties of skeletal
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muscle that become apparent only in context. For example, the energy cost of muscle use is not
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just a function of the magnitude of force production, but also depends on muscle movement.
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When stretched, muscle force is enhanced, and when muscle shortens, force is depressed (1). As
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a result, when we compare the identical magnitude and duration of force production, shortening
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is the most energetically costly and lengthening the least costly (46). These properties require a
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“functional supplementation” to the traditional model of the sliding filament theory. Skeletal
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muscle behaves as though it contains an internal spring. Titin: a two-stage molecular muscle spring Sufficient experimental data are now available to re-construct our current model of skeletal muscle to coincide with (comparative) whole animal observations of muscle behavior in vivo.
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The molecular structure of titin unambiguously identifies it as an elastic protein. At a molecular weight of ~4.2 MDa (55), it is the largest known protein; a mole of titin outweighs Tusko by about a ton. Discovered first in the late 1970’s (41), titin was unknown to the Huxleys (24, 26) and thus it could not have been incorporated into in the sliding filament theory. Since its discovery, titin has been thought to contribute to muscle passive tension (39, 40, 54) and sarcomere integrity (22). Titin’s elastic I-band region is composed of two serially linked spring elements: tandem immunoglobulin (Ig) domains and the PEVK segment (10, 39). At relatively short sarcomere lengths, passive stretch straightens the folded tandem Ig domains but with little change in passive tension. Only at longer (often non-physiological) sarcomere lengths, does the elongation of the PEVK segment result in a steep increase in passive tension (Fig. 6). Because the compliant and stiff titin “springs” are in series, titin has been thought to be too compliant to play a role in active muscle stiffness (12), begging the question, why would it contribute to muscle stiffness only at non-physiological muscle lengths? There is now sufficient evidence to suggest that titin is a dynamic spring, the stiffness of which depends on muscle activation. Furthermore, titin stiffness varies with both muscle length and the presence and direction of muscle movement. Whole muscle stiffness increases ~3-fold upon activation (8) and evidence is building that titin plays a key role (Fig. 5) (2, 3, 19, 38, 53). Titin 1) has a number of Ca2+ binding sites (53); 2) An epitope of T2 titin binds to actin with high affinity in the presence of Ca2+ (28). Recent studies by Leonard and Herzog (32) and Powers et al. (47) demonstrate that titin-based stiffness increases upon Ca2+-activation of myofibrils stretched beyond overlap of the thick and thin filaments. The N2A region of titin is in an ideal position for modulation of titin stiffness through Ca2+-dependent binding to thin filaments. Binding of titin to actin at this location would eliminate low-force straightening of proximal tandem Ig domains in the I-band that normally occurs upon passive stretch of myofibrils at slack length (39). Furthermore, when Ca2+-activated sarcomeres are stretched, only the PEVK segment would elongate, producing a much higher force (Fig. 7). Binding of N2A titin to actin upon muscle activation can account for the increase in titin-based stiffness with activation in myofibrils from skeletal muscle (17, 32). When we re-examine the sliding filament hypothesis, supplementing the cross-bridges with a dynamic role for titin, it
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becomes possible to explain several puzzling aspects of muscle physiology, including the low energy cost and enhancement of force during active stretch (15, 42, 44, 45). The winding filament hypothesis The ability of the traditional sliding filament theory to account for the muscle length-dependent properties is significantly enhanced when the cross bridges partner with a Ca2+-activated titin filament. This mechanism has been described in detail (44). The hypothesis proposes first, that titin binds to actin upon Ca2+-influx (28); and second that titin is wound on the thin filaments by the cross-bridges, because the cross-bridges not only translate but also rotate the thin filaments. The resultant “winding filament” hypothesis explains how titin modifies both force and energetic cost in skeletal muscle. Most animals use their muscles cyclically, storing and recovering elastic recoil energy. Under these conditions, muscles exhibit enhancement of force with stretch (1); depression of force with shortening (1); and a low cost of force production during active stretch (5, 46). Questions regarding the fundamental mechanisms for these properties have persisted since before the advent of the sliding filament theory and remain unresolved to the present day (19). The fact that these fundamental properties of muscle physiology defy explanation suggests that our understanding of the process of muscle contraction is incomplete. Compared to what: Muscles in intact organisms display properties that are not apparent in traditional static force-velocity relationships. In particular, enhanced force and low cost of lengthening contractions suggests the presence of an active muscle spring. Titin is a molecular spring that apparently has the property of changing it stiffness in the presence of Ca2+. In summary, comparative physiology continues to provide valuable contributions to modern physiology. First, many physiological patterns only emerge when making comparisons; these have been valuable in providing testable hypotheses for many physiological processes from cardiovascular function to aging. In addition, a comparative approach can yield insights that may remain obscure in most animals. Both pronghorn and rattlesnakes may be among “nature’s extremes” but in pushing physiological mechanisms they provide a robust signal to noise that allows us to examine our assumptions. Finally, it was specifically by taking a comparative view that resulted in a novel hypotheses of muscle function. While there are still many unanswered
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questions, we feel the winding filament hypothesis best explains many aspects of muscle function. 206 207 208 209 210 211 212
FOOTNOTE 1. FE Yates was the editor of the AJP, apparently selected for his big ideas rather than attention to detail. His name was misspelled on this editorial and thus the author found in the references is “FE Yales”.
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ACKNOWLEDGMENTS
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We thank Novo Nordisk for their support of the Krogh Distinguished lectureship of the
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American Physiological Society.
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GRANTS
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Research on titin has been funded by NSF IOS-1025806 and IIP-1237878 to KCN, and Northern
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Arizona University’s Technology Research Initiative Fund.
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1552, 1997. 50. Schaeffer PJ, Conley KE, Lindstedt SL. Structural correlates of speed and endurance in skeletal muscle: the rattlesnake tailshaker muscle. J Exp Biol 199:351-358, 1996. 51. Schaeffer PJ, Lindstedt SL. How Animals Move: Comparative Lessons on Animal Locomotion. Comprehensive Physiology, Wiley, 2013. 52. Somero, GN. Unity in diversity: a perspective on the methods, contributions, and future of comparative physiology. Annu Rev Physiol 62: 927-937, 2000. 53. Tatsumi R, Maeda K, Hattori A, Takahashi K. Calcium binding to an elastic portion of connectin/titin filaments. J Muscle Res Cell Motil 22:149-162, 2001. 54. Wang K, McCarter R, Wright J, Beverly J, Ramirez-Mitchell R. Regulation of skeletal muscle stiffness and elasticity by titin isoforms: A test of segmental extension model of resting tension. Proc Natl Acad Sci USA 88: 7101-7105, 1991. 55. Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis 24: 1695-1702, 2003. 56. Weibel ER, Taylor CR, Hoppeler H. The concept of symmorphosis: a testable hypothesis of structure-function relationship, Proc Natl Acad Sci USA 88:10357-10361, 1991. 57. West LJ, Pierce CM, Thomas WD. Lysergic Acid Diethylamide: Its Effects on a Male Asiatic Elephant, Science 138:1100-1103, 1962. 58. Yales, FE. Comparative physiology: compared to what? Am J Physiol 237: R1-2, 1979. 59. Zajac FE. Muscle and tendon: properties, models, scaling, and application to biomechanics and motor control. Crit Rev Biomed Engr 4: 359-411, 1989.
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FIGURE CAPTIONS
Fig. 1: Body size scaling among mammals. Most variables scale in quantitatively predictable patterns, expressed as straight lines on log-log plots. In particular because physiological frequencies (e.g., heart and respiratory rates) scale with the same slope, ratios of frequencies are constant, all mammals have about four heartbeats per breath.
Fig 2: Is there a rate-limiting step that constrains oxygen uptake? Maximum aerobic capacity of pronghorn is about 4.5 times that of similar size goats. When each step in the oxygen transport cascade is quantitatively compared, there is an apparently equal increase in capacity in lung, cardiovascular delivery and mitochondrial oxygen capacity; each is increased by a nearly equal magnitude. In what may be nature’s most elite endurance athlete, there is no evidence of a “weak link” in the oxygen transport cascade. Rather these animals provide evidence of shared limitation; natural selection may rarely preserve excess structure. Modified from (35).
Fig 3: The rattlesnake tailshaker muscle ATP supply and demand during rattling. About 60% of the ATP demand (filled bar) is filled though oxidative phosphorylation within the muscle (open bar) but 40% though steady state glycolysis (striped bar). Adapted from Kemper et al., (29).
Fig 4: Stride frequency in galloping mammals is a predictable function of body size. When humans hop in place, the preferred (comfortable) frequency falls exactly on the line for galloping mammals. Adapted from Heglund et al. (14) and Lindstedt et al. (36).
Fig 5: Schematic diagram of a skeletal muscle half-sarcomere illustrating the layout of titin and the winding filament hypothesis. Top) Each titin molecule is bound to the thin filaments (blue) in the Iband, and to the thick filaments (purple) in the A-band. The N2A segment (red) is located between the proximal tandem Ig segments (orange) and the PEVK segment (green). Middle) Upon Ca2+ influx, N2A titin (red) binds to thin filaments (blue). Bottom) Cross bridges (purple) wind PEVK titin (orange) on thin filaments in active muscle. From Nishikawa et al. (44). Fig. 6: Schematic diagram of passive and active stretch in skeletal muscle sarcomeres. A) Passive stretch of muscle sarcomeres. Slack sarcomere (top), in which the proximal tandem Ig domains (orange) and PEVK segment (green) are short and folded. Middle) As the sarcomere is stretched beyond its slack length, the proximal tandem Ig segments unfold approximately to their contour length. Bottom) After the proximal tandem Ig segments have reached their contour length, further stretching extends the PEVK segment. Adapted from Granzier and Labeit (17). B) Active stretch of muscle sarcomeres. Upon activation (top) N2A titin binds to actin. Only the PEVK segment (green) extends when active muscle is stretched (bottom), due to binding of N2A to thin filaments.
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From Tusko to Titin:
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The role for comparative physiology in an era of molecular discovery
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KROGH DISTINGUISHED LECTURE OF THE AMERICAN PHYSIOLOGICAL SOCIETY
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S.L. Lindstedt and K.C. Nishikawa
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Center for Bioengineering Innovation and Department of Biological Sciences,
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Northern Arizona University, Flagstaff AZ, 86011-5640
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Running Page Heading: Comparative Physiology and Modern Biology
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Keywords: Allometry, connectin, muscle contraction, muscle activation, muscular dystrophy
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with myositis (mdm), winding filament hypothesis
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Send correspondence to: Stan L. Lindstedt Department of Biology Northern Arizona University Flagstaff, AZ 86011-5640 FAX: (928) 523-7500 Email: [email protected]
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DISCLOSURES
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No conflicts of interest, financial or otherwise, are declared by the authors.
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1 Copyright © 2015 by the American Physiological Society.
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Abstract
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As we approach the centenary of the term comparative physiology, we re-examine its role in
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modern biology. Finding inspiration in Krogh’s classic 1929 paper, we first look back to some
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timeless contributions to the field. The obvious and fascinating variation among animals is much
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more evident than is their shared physiological unity, which transcends both body size and
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specific adaptations. The “unity in diversity” reveals general patterns and principles of
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physiology that are invisible when examining only one species. Next, we examine selected
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contemporary contributions to comparative physiology, which provides the context in which
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reductionist experiments are best interpreted. We discuss the sometimes surprising insights
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provided by two comparative “athletes,” pronghorn and rattlesnakes, which demonstrate: 1)
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animals are not isolated molecular mechanisms but highly integrated physiological machines; a
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single “rate-limiting” step may be exceptional, and 2) extremes in nature are rarely the result of
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novel mechanisms, but rather employ existing solutions in novel ways. Further, rattlesnake
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tailshaker muscle effectively abolished the conventional view of incompatibility of simultaneous
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sustained anaerobic glycolysis and oxidative ATP production. We end this review by looking
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forward, much as Krogh did, to suggest that a comparative approach may best lend insights in
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unraveling how skeletal muscle stores and recovers mechanical energy when operating
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cyclically. We discuss and speculate on the role of the largest known protein, titin (the third
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muscle filament), as a dynamic spring capable of storing and recovering elastic recoil potential
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energy in skeletal muscle.
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Comparative physiology is most often associated with the interesting and rare adaptations that
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have evolved to match animals to their demanding environments. Indeed, the historic roots of
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comparative physiology included many stories of often fascinating discovery, what Somero (52)
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referred to as “exploratory physiology”. Thus, urine concentration in desert mammals, warm-
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muscles of tunas, and bar-headed geese flying over Everest are among the favorite topics in all
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animal physiology classes.
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These unusual discoveries also have had another impact, that of providing biology with ideal
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model systems for experimental inquiry. In his 1929 paper, the namesake of this lecture, August
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Krogh (30) introduced what we now refer to as the Krogh Principle, namely that ‘For a large
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number of problems there will be some animal of choice or a few such animals on which it can
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be most conveniently studied.’ (30, 33). By selecting the ideal experimental animal, one
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maximizes the ratio of signal to noise, making interpretation of results much simpler. One theme
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of this review is that within the diversity of “comparative physiology” there are numerous
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examples of enhanced signal to noise which simplify and clarify scientific discovery.
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A second theme of this review is a question posed fifty years after Krogh’s review; FE Yates1
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(then editor of AJP) asked the question, “Comparative Physiology: Compared to what?” (58). In
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this essay, we attempt to follow Krogh’s example of examining the contributions of comparative
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physiology looking back and forward, in all instances asking ‘compared to what?’
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1) Emergent principles from disparate data, “Unity in Diversity” (52)
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Disparate data, when amalgamated, may reveal biological insights and emergent patterns that are
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only visible “comparatively.” One striking example unfolds when comparing animals that differ
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in body size. The obvious first observation is that the smallest and largest mammals share nearly
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identical cell structure and cell biochemistry. However body size alone sets the “gas pedal”
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regulating virtually all physiological processes. Krogh’s contemporary, JBS Haldane (13) wrote
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an engaging essay, “On Being the Right Size,” in which he described body size as the single
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most important, but rarely considered, variable among animals.
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LSD and the Elephant
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An enduring example that should never be lost from the collective knowledge of all physiologists
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is the story of LSD and the elephant, a favorite example of Knut Schmidt-Nielsen that we 3
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recount on his behalf. It is difficult to keep male elephants in captivity because each year they go
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through a period of extreme aggression, “musth” which has resulted in injury and even death to
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other zoo animals as well as zoo keepers. For that reason most zoo elephants are females. During
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the early 1960s, perhaps inspired by experiments and notoriety of Timothy Leary, there were an
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unusually large number of “experiments” involving LSD. One of those involved a male elephant,
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Tusko, residing at the time in the Oklahoma City Zoo. The “hypothesis” of this experiment,
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published in Science (57) was to test if administering LSD to Tusko could induce musth.
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(Thankfully, institutions now have Animal Care and Use Committees.) Of course the first task
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was to calculate the LSD dose. As cats had been the subjects of an imprudent volume of LSD
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experimentation, the dose for Tusko was calculated based on the well-established weight-specific
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cat dose; to ensure the safety of the animal, the calculated dose was halved. When the animal
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died shortly after the LSD was administered, the conclusion, as reported in Science, was simply
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that elephants are very sensitive to LSD. Of course drug delivery, metabolism and clearance all
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occur at a rate dictated by an animal’s pace of metabolism, which scales disproportionately with
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body mass. Using this metabolism as the metric, even by conservative calculation, Tusko was
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given about a 10-fold overdose (297 mg vs ~ 32 mg) and there is no evidence that elephants are
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unusually sensitive to LSD (or that it might induce musth!).
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Metabolism is just one example, but virtually all physiological variables are strongly body size
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dependent. When expressed in the familiar Power Law equation (Y = aXb), body size dependent
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variables fall into quantitative clusters, with similar slope (b) values (42). For example, volumes
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scale linearly (b≅1); blood volume is very nearly 7% of body mass in all mammals and heart
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mass is about 0.5%. Pressures (force/area) are generally independent of size (b≅0) thus blood
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pressure and maximum muscle stress are essentially constant across all mammals. Physiological
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rates (and their reciprocal, times) scale with surprising consistency, -1/4>b>-1/5 (Fig. 1).
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Volume-rates (such as metabolism and the renal or hepatic clearance of drugs, importantly for
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Tusko) scale as (volume × frequency) or about M3/4 (34, 37). This consistent scaling seems to
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confirm AV Hill’s (21) speculation that mammals experience a body size dependent internal
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clock that dictates the pace of life; all time cycles from mitosis to longevity seem to be set to a
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common “physiological clock”. Conforming to this “clock”, all mammals experience roughly the
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same number of heart beats per lifetime, during which they consume roughly the same number of
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calories per gram of body tissue. These emergent patterns should be considered when testing
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models of aging, etc.
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Compared to what: Although the most conspicuous feature of comparative physiology is the
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diversity among animals, consistent “patterns of design” transcend that diversity. These patterns
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become visible only through comparisons. Body size dependent patterns are one example of the
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unity in diversity (52).
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2) Organismal context to interpret reductionist experiments.
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As biologists, we are taught from our earliest graduate classes to design controlled “reductionist”
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experiments when possible isolating one variable for experimentation. This approach is
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particularly valuable in biology dictated by the large number of simultaneous variables.
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Therefore one challenge in experimental design is to isolate and focus as much as possible in
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order to identify cause and effect. The risk, of course, is that experiments can be performed and
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also interpreted in isolation. Ultimately, there must be a whole organism context as the final
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interpretation of our reductionist results.
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Blood flow limitation to maximum oxygen uptake
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One organismal misunderstanding was the consequence of a valuable reductionist technique,
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isolating blood flow to an individual muscle and measuring in vivo work and oxygen uptake.
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This in situ preparation resulted in some valuable insights, but also at least one detour into a cul-
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de-sac. Briefly, by isolating the blood flow to a single muscle cluster (usually dog
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gastrocnemius-plantaris muscle group), the work output could be manipulated and blood flow
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and oxygen uptake all measured in isolation (4). One significant conclusion of these studies, that
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was many times confirmed, is that blood flow was the single limiting factor setting maximum
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muscle metabolism. As the experiments were meticulously done and results consistent and
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unambiguous, it seemed unnecessary to interpret them in a whole animal context. Thus, in 1985
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when Dick Taylor, Bob Armstrong and Harold Laughlin injected microspheres in a dog running
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on the treadmill at the Concord Field Station, muscle blood flows that greatly exceeded the
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maximum measured in situ (and thus thought possible) strongly suggested the presence of a
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methodological error, hence the rejection of their manuscript.
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However, if one were to make one simple calculation, multiply the isolated blood flow and
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oxygen uptake values by the dog’s entire muscle mass (assuming all dog muscles were operating
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at this maximum, which would seem unlikely), rather than overestimating whole animal oxygen
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uptake as expected, these measured values account for only 1/3 of the measured oxygen uptake.
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In fact, these experiments actually demonstrated the obvious: restricting blood flow limits
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oxygen uptake. Eventually, as a result of whole animal experiments, Laughlin introduced the
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concept of “exercise hyperemia” (rather than in situ ischemia!) into the exercise science
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literature (31). It was only by “comparing” isolated muscle values to intact organismal
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measurements that the misinterpretation of blood flow limitation was exposed.
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Pronghorn: the world’s most elite endurance athlete
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Perhaps the ideal Krogh model system for asking if there is a “rate-limiting step” in the uptake,
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transport and utilization of oxygen is an animal with extraordinary oxygen uptake. Pronghorn
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Antilocapra americana, evolved in the barren plains of the western US where strong selective
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pressure from both sprint (cheetah-like cats) and endurance (wolves, coyotes) predators have
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apparently provided strong selective pressure to maximize both speed and endurance. Comparing
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pronghorns with similar sized, but aerobically “typical,” goats, their maximum oxygen uptake is
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five times higher. When we assess the capacities at each step of the oxygen transport cascade,
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from lung to mitochondrial sink, there is no apparent weak link that limits oxygen flow (35) (Fig.
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2). Indeed, rather than a single “rate-limiting” step, these experiments provide evidence of
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“shared” limits, as each link in the oxygen transport cascade seems to have nearly identical
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capacity (56).
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Rattlesnake tailshaker muscle: A stationary athlete
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Western diamondback rattlesnakes, Crotalus atrox, are fairly “typical” reptiles aerobically, with
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little capacity for sustained activity. They possess one surprising attribute; rattlesnakes can
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maintain very high frequency rattling for hours. The tailshaker muscle is highly aerobic, nearly
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one third mitochondria and one fourth sarcoplasmic reticulum (50). Using Magnetic Resonance
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Spectroscopy (MRS) to estimate the energy cost, we found that as only a single twitch is
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necessary to contract the muscle, the cost per contraction is exceptionally low (9, 43). However,
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there was still a mismatch between ATP production and ATP cost. This mismatch was only
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resolved when this observation was revisited on the whole animal level. What Conley and his 6
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colleagues found belied our textbook knowledge; rattlesnakes use sustained anaerobic glycolysis
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to supplement aerobic ATP production on a steady-state basis (fig. 3). The rich blood supply and
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minimal muscle size results in a solution of exporting lactate to other tissues to be oxidized there.
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Putting this high frequency muscle into a whole animal context resulted in a “shake-up” of our
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traditional view of glycolysis and greatly expanded our understanding of muscle energetics (29).
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Molecular basis of muscular contraction During the past century, remarkable progress has been made elucidating the molecular mechanisms of muscle contraction (reviewed in 23, 25). While most of this research has been focused at the level of single demembranated muscle fibers (e.g., 11), technical advances have made it possible to explore the process of muscle contraction at ever smaller scales, in single myofibrils (32) or even single sarcomeres (18, 48). These reductionist studies have produced an internally consistent description of the biochemistry and biophysics of cross-bridge cycling in muscle. However, despite this progress, the goal of predicting how muscle force changes during natural movements at the level of the intact organism has remained elusive despite decades of intensive research (7, 49). Muscle models based on the sliding filament theory and used in neuromusculoskeletal simulations (e.g., 59) fail to predict muscle force under physiologically relevant conditions, even during purely isometric contractions (6).
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In summary, as biologists we appreciate and thus embrace physiological principles. The
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seduction of molecular clarity is that little or no context is required for interpretation. No animal
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exists as a mere compilation of molecules, it is only by putting these molecular discoveries back
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into a whole animal context that insights mature into physiological principles. Thus it is useful to
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remain cautious of the “sufficiency of proof” axiom, namely that far less evidence is required to
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establish an idea as “fact” than is required to dislodge that idea once established.
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Compared to what: Reductionist experiments are the basis of scientific discovery and the pace of
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data acquisition has never been greater than in this era of molecular biology. Comparative
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physiology provides a scaffold on which data from reductionist experiments can be built into
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biological insights. 3) Constructing a molecular model from comparative data.
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Identification of cross-species patterns does not provide explanations, but these patterns may be
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the starting point for novel hypotheses. Identified patterns can guide experimental inquiry; we
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end this review with a look forward.
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Most animals use their muscles cyclically in locomotion, whether running, swimming or flying
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(51). In almost all instances, cyclic muscle use involves the storage and recovery of elastic recoil
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potential energy. Just as experiments by AV Hill (20) provided insights into the properties of
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muscle, our understanding of cyclic muscle use has been greatly enhanced by the work loop
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analysis introduced by Robert Josephson (27). Once again, body size is a dominant factor
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influencing this behavior. In all modes of movement, body size dictates frequency of muscle use.
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Here we focus on galloping mammals, but the same principles apply to swimming or flying, as
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well as other cyclical movements.
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It has been 40 years since Heglund et al. (14) identified the strong link between body size and
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stride frequency among galloping mammals; body size alone seems to set stride frequency (s-1) =
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4.5 kg-0.14. So consistent is this scaling of stride frequency that we have developed a student
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biomechanics lab that demonstrates two principles: 1) humans hopping in place do so at the same
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frequency as a galloping mammal of the same size (Fig. 4); and 2) changing the self-selected
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frequency doubles the energy cost per hop, greatly reducing apparent muscle efficiency (34).
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Running mammals select a stride frequency that maximizes the recovery of elastic recoil energy.
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In addition to selecting a “lowest cost” stride frequency, there are other properties of skeletal
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muscle that become apparent only in context. For example, the energy cost of muscle use is not
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just a function of the magnitude of force production, but also depends on muscle movement.
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When stretched, muscle force is enhanced, and when muscle shortens, force is depressed (1). As
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a result, when we compare the identical magnitude and duration of force production, shortening
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is the most energetically costly and lengthening the least costly (46). These properties require a
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“functional supplementation” to the traditional model of the sliding filament theory. Skeletal
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muscle behaves as though it contains an internal spring. Titin: a two-stage molecular muscle spring Sufficient experimental data are now available to re-construct our current model of skeletal muscle to coincide with (comparative) whole animal observations of muscle behavior in vivo.
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The molecular structure of titin unambiguously identifies it as an elastic protein. At a molecular weight of ~4.2 MDa (55), it is the largest known protein; a mole of titin outweighs Tusko by about a ton. Discovered first in the late 1970’s (41), titin was unknown to the Huxleys (24, 26) and thus it could not have been incorporated into in the sliding filament theory. Since its discovery, titin has been thought to contribute to muscle passive tension (39, 40, 54) and sarcomere integrity (22). Titin’s elastic I-band region is composed of two serially linked spring elements: tandem immunoglobulin (Ig) domains and the PEVK segment (10, 39). At relatively short sarcomere lengths, passive stretch straightens the folded tandem Ig domains but with little change in passive tension. Only at longer (often non-physiological) sarcomere lengths, does the elongation of the PEVK segment result in a steep increase in passive tension (Fig. 6). Because the compliant and stiff titin “springs” are in series, titin has been thought to be too compliant to play a role in active muscle stiffness (12), begging the question, why would it contribute to muscle stiffness only at non-physiological muscle lengths? There is now sufficient evidence to suggest that titin is a dynamic spring, the stiffness of which depends on muscle activation. Furthermore, titin stiffness varies with both muscle length and the presence and direction of muscle movement. Whole muscle stiffness increases ~3-fold upon activation (8) and evidence is building that titin plays a key role (Fig. 5) (2, 3, 19, 38, 53). Titin 1) has a number of Ca2+ binding sites (53); 2) An epitope of T2 titin binds to actin with high affinity in the presence of Ca2+ (28). Recent studies by Leonard and Herzog (32) and Powers et al. (47) demonstrate that titin-based stiffness increases upon Ca2+-activation of myofibrils stretched beyond overlap of the thick and thin filaments. The N2A region of titin is in an ideal position for modulation of titin stiffness through Ca2+-dependent binding to thin filaments. Binding of titin to actin at this location would eliminate low-force straightening of proximal tandem Ig domains in the I-band that normally occurs upon passive stretch of myofibrils at slack length (39). Furthermore, when Ca2+-activated sarcomeres are stretched, only the PEVK segment would elongate, producing a much higher force (Fig. 7). Binding of N2A titin to actin upon muscle activation can account for the increase in titin-based stiffness with activation in myofibrils from skeletal muscle (17, 32). When we re-examine the sliding filament hypothesis, supplementing the cross-bridges with a dynamic role for titin, it
9
becomes possible to explain several puzzling aspects of muscle physiology, including the low energy cost and enhancement of force during active stretch (15, 42, 44, 45). The winding filament hypothesis The ability of the traditional sliding filament theory to account for the muscle length-dependent properties is significantly enhanced when the cross bridges partner with a Ca2+-activated titin filament. This mechanism has been described in detail (44). The hypothesis proposes first, that titin binds to actin upon Ca2+-influx (28); and second that titin is wound on the thin filaments by the cross-bridges, because the cross-bridges not only translate but also rotate the thin filaments. The resultant “winding filament” hypothesis explains how titin modifies both force and energetic cost in skeletal muscle. Most animals use their muscles cyclically, storing and recovering elastic recoil energy. Under these conditions, muscles exhibit enhancement of force with stretch (1); depression of force with shortening (1); and a low cost of force production during active stretch (5, 46). Questions regarding the fundamental mechanisms for these properties have persisted since before the advent of the sliding filament theory and remain unresolved to the present day (19). The fact that these fundamental properties of muscle physiology defy explanation suggests that our understanding of the process of muscle contraction is incomplete. Compared to what: Muscles in intact organisms display properties that are not apparent in traditional static force-velocity relationships. In particular, enhanced force and low cost of lengthening contractions suggests the presence of an active muscle spring. Titin is a molecular spring that apparently has the property of changing it stiffness in the presence of Ca2+. In summary, comparative physiology continues to provide valuable contributions to modern physiology. First, many physiological patterns only emerge when making comparisons; these have been valuable in providing testable hypotheses for many physiological processes from cardiovascular function to aging. In addition, a comparative approach can yield insights that may remain obscure in most animals. Both pronghorn and rattlesnakes may be among “nature’s extremes” but in pushing physiological mechanisms they provide a robust signal to noise that allows us to examine our assumptions. Finally, it was specifically by taking a comparative view that resulted in a novel hypotheses of muscle function. While there are still many unanswered
10
questions, we feel the winding filament hypothesis best explains many aspects of muscle function. 206 207 208 209 210 211 212
FOOTNOTE 1. FE Yates was the editor of the AJP, apparently selected for his big ideas rather than attention to detail. His name was misspelled on this editorial and thus the author found in the references is “FE Yales”.
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ACKNOWLEDGMENTS
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We thank Novo Nordisk for their support of the Krogh Distinguished lectureship of the
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American Physiological Society.
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GRANTS
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Research on titin has been funded by NSF IOS-1025806 and IIP-1237878 to KCN, and Northern
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Arizona University’s Technology Research Initiative Fund.
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FIGURE CAPTIONS
Fig. 1: Body size scaling among mammals. Most variables scale in quantitatively predictable patterns, expressed as straight lines on log-log plots. In particular because physiological frequencies (e.g., heart and respiratory rates) scale with the same slope, ratios of frequencies are constant, all mammals have about four heartbeats per breath.
Fig 2: Is there a rate-limiting step that constrains oxygen uptake? Maximum aerobic capacity of pronghorn is about 4.5 times that of similar size goats. When each step in the oxygen transport cascade is quantitatively compared, there is an apparently equal increase in capacity in lung, cardiovascular delivery and mitochondrial oxygen capacity; each is increased by a nearly equal magnitude. In what may be nature’s most elite endurance athlete, there is no evidence of a “weak link” in the oxygen transport cascade. Rather these animals provide evidence of shared limitation; natural selection may rarely preserve excess structure. Modified from (35).
Fig 3: The rattlesnake tailshaker muscle ATP supply and demand during rattling. About 60% of the ATP demand (filled bar) is filled though oxidative phosphorylation within the muscle (open bar) but 40% though steady state glycolysis (striped bar). Adapted from Kemper et al., (29).
Fig 4: Stride frequency in galloping mammals is a predictable function of body size. When humans hop in place, the preferred (comfortable) frequency falls exactly on the line for galloping mammals. Adapted from Heglund et al. (14) and Lindstedt et al. (36).
Fig 5: Schematic diagram of a skeletal muscle half-sarcomere illustrating the layout of titin and the winding filament hypothesis. Top) Each titin molecule is bound to the thin filaments (blue) in the Iband, and to the thick filaments (purple) in the A-band. The N2A segment (red) is located between the proximal tandem Ig segments (orange) and the PEVK segment (green). Middle) Upon Ca2+ influx, N2A titin (red) binds to thin filaments (blue). Bottom) Cross bridges (purple) wind PEVK titin (orange) on thin filaments in active muscle. From Nishikawa et al. (44). Fig. 6: Schematic diagram of passive and active stretch in skeletal muscle sarcomeres. A) Passive stretch of muscle sarcomeres. Slack sarcomere (top), in which the proximal tandem Ig domains (orange) and PEVK segment (green) are short and folded. Middle) As the sarcomere is stretched beyond its slack length, the proximal tandem Ig segments unfold approximately to their contour length. Bottom) After the proximal tandem Ig segments have reached their contour length, further stretching extends the PEVK segment. Adapted from Granzier and Labeit (17). B) Active stretch of muscle sarcomeres. Upon activation (top) N2A titin binds to actin. Only the PEVK segment (green) extends when active muscle is stretched (bottom), due to binding of N2A to thin filaments.
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