Airway Smooth Muscle in Asthma Perturbed Equilibria of Myosin Binding JEFFREY J. FREDBERG Harvard School of Public Health, Boston, Massachusetts
The classic theory of airway lumen narrowing was developed to explain the determinants of airway narrowing, and why that narrowing can become excessive in asthma. The classic theory emphasizes that muscle length and airway caliber are set by a force balance in which the active force generated by airway smooth muscle statically is in mechanical equilibrium with the passive reaction force developed by the elastic load against which that muscle has shortened. Since both forces depend on muscle length, the muscle accommodates itself to the length at which these opposing forces come into static balance (1, 2). If the external load should change in time, as would occur with lung inflation, for example, then the activated muscle is believed to accommodate itself to a sequence of states lying along its static force–length characteristic (3–6). The isometric forcegenerating capacity of airway smooth muscle is set principally by muscle mass, muscle contractility, and muscle position on its static force–length characteristic (2, 7). Taken together, evidence available in the literature suggests that there is no systematic difference in force-generating capacity between muscle from the normal versus the asthmatic lung, although this evidence is equivocal (8, 9). Moreover, animal studies now suggest that the force-generating capacity of normal airway smooth muscle is, in any event, more than sufficient to close all airways in the lung (10, 11). The passive reaction force against which the muscle shortens is set principally by the elasticity and geometry of the airway wall, tethering of the airway to the lung parenchyma, and the state of lung inflation (5, 11, 12). Each of these factors, in turn, has its own determinants that are known to be modified with chronic airway inflammation (13, 14). This classic framework explained much of what needed to be explained, but our understanding of the causality linking airway inflammation to its ultimate mechanical consequence— excessive airway narrowing—remains fragmentary. For example, for reasons that remain poorly understood the plateau of the dose–response curve is elevated in asthma, or abolished altogether, suggesting that those factors that limit maximal airway narrowing in the normal lung, whatever they are, have somehow been attenuated in the asthmatic lung. And no less important than the mystery of the plateau is the perplexing role of deep inspirations (3, 15–18). Deep inspirations that are attendant to spontaneous breathing may be the most potent of all known bronchodilating agencies, and they comprise the first line of defense against bronchospasm. But in the spontaneous asthmatic attack this potent bronchodilating mechanism fails. Indeed, Fish and colleagues (16) suggested that it may be the failure of this very defense mechanism that is the most telling end effect of the inflammatory cascade and, therefore, the proximal cause of excessive airway narrowing in asthma. Moreover, there is ample evidence from the work of Ingram and
Correspondence and requests for reprints should be addressed to Jeffrey J. Fredberg, M.D., Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail:
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colleagues (17–19) to show that, if anything, in an asthmatic attack deep inspirations only make matters worse. The inability of deep inspirations to relax airway smooth muscle during episodes of spontaneous asthmatic obstruction remains unexplained.
A NEW FRAMEWORK: PERTURBED EQUILIBRIA OF MYOSIN BINDING The effects of tidal stretch on airway smooth muscle were first addressed by Sasaki and Hoppin, and by Gunst and colleagues (20–23). These investigators demonstrated that imposition of tidal changes in muscle length depresses active force. In isolated, maximally activated airway smooth muscle, imposed fluctuations of length about a fixed mean length cause graded depression of muscle force F (averaged over the stretch) and muscle stiffness E, and augmentation of the specific rate of ATP use and the hysteresivity [equivalent to the loss tangent, related to the viscosity, and a rough index of bridge cycling rate (24–26)]. Also, imposed force fluctuations about a fixed mean distending force systematically bias the airway smooth muscle toward lengthening (fluctuation-driven muscle lengthening depicted in Figures 1 and 2). To explain how the tidal action of lung inflations modulates smooth muscle function we have put forward the theory of perturbed myosin binding (24–27). The theory holds that with each breath lung inflation strains airway smooth muscle. These periodic mechanical strains are transmitted to the myosin head and cause it to detach from the actin filament much sooner than it otherwise would have. This premature detachment profoundly reduces the duty cycle of myosin, typically to less than 20% of its value in the isometric steady state, and depresses to a similar extent total numbers of bridges attached and active force. Of the full isometric force-generating capacity of the muscle, therefore, only a rather modest fraction ever comes to bear on the airway narrowing, even when the muscle is activated maximally. At the macroscopic level the fully activated muscle becomes much less stiff and much more viscous, becoming in effect a gooey liquid that is characterized at the molecular level by perturbed binding equilibria (few crosslinks attached at any moment, but cycling rapidly), almost as if the muscle had “melted.” In pathological circumstances, however, the load fluctuations impinging on myosin can become compromised. For example, the chronically inflamed airway and its peribronchial adventitia remodel in a way that is thought to uncouple the muscle from these load fluctuations, and such an uncoupling would permit myosin to approach an unperturbed binding equilibrium, in which case the muscle would shorten, stiffen, and virtually freeze in the latch state (24, 28). In doing so the myosin duty cycle would tend toward 100% of its value in isometric contraction, with levels of force generation believed to be sufficient to close all the airways of the lung (10). The effects of load fluctuations as predicted from first principles of myosin binding dynamics are for the most part indistinguishable from those determined experimentally, both in terms of the threshold levels and the magnitudes of the effects
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Figure 1. Evolution of mechanical properties of a representative muscle during contraction against a steady force of component 0.32Fo on which is superimposed force fluctuations (0.2 Hz) of graded amplitude ␦F. (a) Mean muscle length over each force cycle. (b) Loop stiffness (percentage of maximum isometric value). (c) Hysteresivity (dimensionless). Force fluctuations drive the contractile state away from static equilibrium conditions.
[Figure 2; the solid lines correspond to predictions from fourstate latch regulation incorporated into Huxley’s sliding filament model (24–29)]. Thus, a seemingly complicated constellation of mechanical, biochemical, and metabolic effects of load fluctuations may be largely accounted for by a single mechanism: myosin dynamics and the way that these dynamics become perturbed by imposed load fluctuations. In addition, this single molecular mechanism tenatively explains and unifies cardinal phenotypic characteristics of asthma that previously had been explained separately and inadequately, and that did not fit the classic understanding of the determinants of airway lumen narrowing. These include the plateau response in healthy individuals (i.e., the previously unidentified factor limiting airway narrowing) and why that narrowing can become excessive in asthma, the multifactorial origins of airway hyperresponsiveness, how allergen sensitization leads to airway hyperresponsiveness, how hyperresponsiveness can persist long after airway inflammation is resolved, and the inability in asthma of deep inspirations to relax airway smooth muscle. It also leads to the first plausible mechanism by which the rate of bridge cycling and its regulation (8, 9, 11, 21, 23) may be reasonably thought to bear on the prevalence of airway hyperresponsiveness in childhood and its changes with lung maturation and allergic status (24, 27, 29).
PERTURBED EQUILIBRIA OR MYOSIN BINDING: NECESSARY BUT NOT SUFFICIENT While perturbed equilibria explain fairly well the fluctuationdriven muscle lengthening and associated events that transpire as load fluctuation amplitude is increased, they fail to account for muscle reshortening that occurs when load fluctuation amplitude is decreased. When the amplitude of the tidal force fluctuation is reduced from 32% back to 8% of optimal force (Fo), the muscle reshortens to a new length, and a new biophysical state, that is substantially different from the prior length under identical loading conditions (Figure 2, point A versus point C). Therefore, the force fluctuation amplitude necessary to keep the muscle at a dynamically equilibrated length (Figure 2, point C) is substantially smaller than that required to break through initially and attain that length (Figure 2,
Figure 2. Pooled observations (filled circles, n ⫽ 6; error bars denote SD between muscle strips, drawn in only one direction for clarity) and expected (solid lines predicted from theory of muscle contraction [24]) values for (a) dynamic equilibrium muscle length versus force fluctuation amplitude. (b) Loop stiffness (percentage of maximum isometric value). (c) Hysteresivity (dimensionless). (d) Tidal length change; is ⌬L/Lo expressed as a percentage.
point B), and under normal circumstances this effect of history seems to be the dominant event. Since airway resistance (Raw) scales roughly with the inverse fourth power of muscle length, these two states (Figure 2, points A and C) would correspond to substantially different levels of airway function; Raw at point C would be much less than at point A (Figure 2). Thus, with the same muscle, with the same stimulus, and with the same current loading, we find dramatically different contractile states brought about only by differences in loading path history. These dependencies on the history cannot be accounted for by bridge dynamics, and may be related to the mechanisms of plasticity in airway smooth muscle discussed by Gunst and coworkers (22).
ISOMETRIC CONTRACTION, UNLOADED SHORTENING, AND OTHER UNNATURAL ACTS Isometric contraction and unloaded shortening of airway smooth muscle have taught us a great deal about the biophysics of airway smooth muscle, but these are unnatural acts that are restricted mainly to the laboratory. In contrast, the perturbed equilibria at issue here are believed to be the very states that govern airway responsiveness. The conformity between predictions from bridge dynamics and existing data is most remarkable and leads us to believe that we have at least a crude understanding of some of the important events at the level of the naked myosin–actin interaction. But the implications ought not to be pressed too far, because these perturbed states re-
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main largely uncharacterized biochemically, metabolically, and structurally.
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IMPORTANT QUESTIONS • Increasing evidence now suggests that normoresponsiveness of the airway corresponds to perturbed equilibria of myosin binding (the “melted”state) secondary to dynamic fluctuations in muscle load. But do airway hyperresponsiveness, airway obstruction, and symptoms correspond to static equilibrium of myosin binding (muscle frozen in latch) secondary to dynamic unloading? • How do the frozen and melted states relate to shortening velocity and its determinants such as myosin isoforms, caldesmon, calponin, and myosin phosphorylation and its determinants? • What mechanisms account for sustained (plastic) effects of tidal loading history?
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