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Ultrastructural basis of airway smooth muscle contraction Kuo, Kuo-Hsing 2003

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ULTRASTRUCTURAL BASIS OF AIRWAY SMOOTH MUSCLE CONTRACTION By KUO-HSING KUO M.D., National Taiwan University, 1990 M.Sc, National Yang-Ming Medical University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Anatomy and Cell Biology) We accept this thesis as conforming to the required standard 7 THE UNIVERSITY OF BRITISH COLUMBIA July 2003 © Kuo-Hsing Kuo, 2003 Abstract Smooth muscle is ubiquitous and controls vital function in our body. Airway smooth muscle (ASM) is the main effecter that controls the caliber and hence airflow resistance of the airways. Dysfunction of the A S M is a major contributor to the distress of asthma and other obstructive airway diseases. The emphasis of my thesis research is to elucidate the basic mechanism of A S M contraction, especially from the ultrastructural point of view. The general hypothesis, based on previous observations on mechanical behavior of the tissue, is that A S M cells are highly adaptable to their external environment and are able to maintain optimal contractile function over a large length range. Specific hypotheses regarding mechanisms of length adaptation of A S M are 1) the myosin filaments are structurally labile in the relaxed state, and it is this lability that facilitates plastic remodeling of the A S M to accommodate large changes in cell geometry while maintaining optimal contractile function; 2) in A S M cells adapted to long lengths, polymerization produces more myosin filaments to account for the observed increase in muscle power output and shortening velocity; the reverse is true for A S M adapted to short lengths. Three major groups of experiments were carried out in this thesis research. The first group of experiments was carried out to examine inter- and intra-cellular organization of the contractile filaments. We showed electronmicroscopic and functional evidence that contractile filaments in A S M lied parallel to the longitudinal axis of the cell bundle, in contrast to the obliquely arranged filaments depicted in conventional models. The parallel arrangement of the contractile filaments was observed to be maintained despite the fact that individual cells were spindle-shaped. This was accomplished through filament ii attachment to dense plaques on the cell membrane and the plaques were in turn connected to like-structures on neighbouring cells. Intracellularly the parallel arrangement was maintained despite the centrally located nucleus. This was accomplished by attachment of actin filaments to the nuclear envelope and making the nucleus a force transmitting structure. The results suggest that A S M cells form a mechanical syncytium and are able to function properly only as a group. The second group of experiments was carried out to examine myosin filament lability and its relationship to the ability of the muscle to generate force. Specifically we studied the relationship between isometric force generation and myosin thick filament density in cell cross-sections, measured electronmicroscopically, following length oscillations applied to the relaxed porcine trachealis muscle. The results indicate that thick filaments in A S M are labile; depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. The third and final group of experiments examined the mechanisms by which plastic adaptation of A S M to large changes in muscle length is accomplished. In these experiments we showed that isometric force produced by A S M was independent of muscle length over a 2-fold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity, power output and myosin filament density varied similarly to length change. The data can be explained by a model where additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. 111 In summary, results from this thesis research has provided much needed ultrastructural data for constructing a preliminary model that explains some aspects of plastic behavior of A S M . The most important finding of this research is perhaps that the ultrastructure of smooth muscle is not as "permanent" as that of striated muscle; the malleable contractile apparatus of smooth muscle makes it necessary to interpret the ultrastructural data in the context of functional states under which the tissue is fixed for examination. iv Table of Contents Abstract ii Table of Contents v List of Figures ix List of Tables xi List of Abbreviations xii Acknowledgements xiii Chap. 1 General introduction 1 1.1 Airway smooth muscle and obstructive airway disease 1 1.1.1 Airway smooth muscle: an overview 1 1.1.2 Obstructive airway diseases and airway hyperresponsiveness 2 1.1.3 The role of airway smooth muscle in obstructive airway diseases 4 1.2 Plasticity of smooth muscle: Does the striated muscle model apply? 5 1.2.1 The characteristic features of smooth muscle cell 5 1.2.1.1 Lability of the thick filaments 5 1.2.1.2 Large functional length range for smooth muscle 6 1.2.2 Can a model with fixed filament lattice accommodate smooth muscle cell function? 7 1.3 Hypotheses regarding plasticity in smooth muscle 9 Chap.2 Specific aims 13 2.1 Morphological observations on airway smooth muscle 13 2.2 Inter- and intra-cellular force transmission in intact smooth muscle 13 2.3 Thick filament lability induced by mechanical perturbation 14 v 2.4 Ultrastructural basis of adaptation of airway smooth muscle to different lengths 15 Chap.3 Material and method 16 3.1 Measurements of mechanical properties 16 3.2 Preparation for ultrastructural study 17 3.3 Image processing and data analysis 18 Chap.4 Morphological observations of airway smooth muscle 20 4.1 Morphological observations: past and present 20 4.2 Organelles of airway smooth muscle cell 23 4.2.1 Cell membrane 23 4.2.2 Caveolae 23 4.2.3 Sarcoplasmic reticulum 24 4.3 Contractile apparatus 25 4.3.1 Overview of the contractile apparatus 25 4.3.2 Thick filaments 26 4.3.3 Thin filaments 27 4.3.4 Intermediate filaments 27 4.3.5 Dense bodies / plaques 27 Chap.5 Intra- and inter-cellular force transmission in intact airway smooth muscle bundle . 31 5.1 Previous structural models proposed for smooth muscle based on observation of isolated cells 31 5.2 Notes on experimental procedure 33 vi 5.3 Arrangement of contractile filaments observed in intact airway smooth muscle bundle . 33 5.3.1 Parallel arrangement of contractile filaments 33 5.3.2 Association of contractile filaments and nuclei 34 5.4 Nucleus as a force carrier 36 5.4.1 Elasticity of the nuclear envelope . 36 5.4.2 Mechanical integrity of the nuclear envelope 39 5.5 A proposed model of filament architecture in bundle of trachealis - a mechanical syncytium 41 Chap.6 Myosin thick filament lability induced by mechanical strain 52 6.1 Background 52 6.2 Notes on experimental procedure 54 6.3 Isometric force development after mechanical strain 55 6.3.1 Force decrease caused by applied length perturbation 55 6.3.2 Adaptive process of force generation after length perturbation 56 6.3.3 Time course of force development after length perturbation 57 6.4 Decrease in density of thick filaments after mechanical strain 59 6.4.1 Effect of length perturbation on density of thick filaments 59 6.4.2 Lability of thick filaments 60 6.4.3 Reorganization of thick filaments caused by applied length perturbation 62 6.5 Rhythmic length-perturbation of airway smooth muscle as a model of deep inspiration 64 Chap.7 Adaptation of airway smooth muscle to different lengths 72 vii 7.1 Background 72 7.2 Notes on experimental procedure 73 7.3 Mechanical properties at different lengths 74 7.3.1 Length independence of force generation 74 7.3.2 Length dependence of shortening velocity and power output 74 7.3.3 Energetic efficiency of active contractile units at different lengths 75 7.3.4 Possible explanation for muscle adaptation at different lengths 76 7.4 Morphological observations at different muscle lengths 77 7.4.1 Volume conservation of smooth muscle cells at different lengths 77 7.4.2 Density of myosin thick filaments at different lengths 77 7.4.3 Estimated thick filament content in a whole cell at different lengths 78 7.5 A proposed mechanism for smooth muscle adaptation to large length changes 79 7.5.1 A model with variable number of contractile units in series at different cell lengths 79 7.5.2 Plastic adaptation of smooth muscle and its clinical implication 82 Chap.8 Conclusion 93 Reference 95 viii List of Figures Figure 1 The dependence of active tension generation by striated muscle fiber on the sarcomere length. 11 Figure 2 Proposed model for plastic alternation to different lengths 12 Figure 3 Electron micrograph of a transverse section of trachealis cells. 29 Figure 4 Contractile apparatus in trachealis muscle • - 30 Figure 5 Transverse section of trachealis cells within an intact muscle bundle. 44 Figure 6 Longitudinal section of trachealis cells within an intact muscle bundle. 45 Figure 7 Electron micrographs showing a series of consecutive transverse sections of a trachealis cell. 46 Figure 8 Inverse relationship between the number of myosin filaments and the nuclear area. 47 Figure 9 Direct attachment of thin filament to the nuclear envelope. 48 Figure 10 Thin filaments observed in clusters of mitochondria. 49 Figure 11 Change of nuclear length during isometric contraction. 50 Figure 12 A proposed model of filament architecture in a bundle of tracheali. 51 Figure 13 Experimental procedure 67 Figure 14 Time course of changes in tetanic force (closed circles) and myosin thick filament density (open circles) associated with a length oscillation 68 Figure 15 Time course of isometric force development before and immediately after length oscillation 69 Figure 16 Electron micrographs of cross sections of airway smooth muscle before and after length oscillation. 70 ix Figure 17 Change of force generation at different lengths 83 Figure 18 Change of shortening velocity at different lengths 84 Figure 19 Change of power output at different lengths 85 Figure 20 Electron micrographs of cross sections of airway smooth muscle at Lref and 2.0 Lref 86 Figure 21 Change of cell cross-sectional area at different lengths 87 Figure 22 Change of filament density at different lengths 88 Figure 23 Change of estimated thick filament content in whole cell at different lengths 89 Figure 24 Pooled values of isometric force, shortening velocity, power output, myosin filament density and estimated filament content 90 Figure 25 Proposed model with variable contractile units in series at different lengths 91 x List of Tables Table 1 Myosin filament densities and cell cross-sectional areas 71 Table 2 Force, velocity, power and myosin filament density of intact airway smooth bundle at different lengths. 92 x i List of Abbreviations A S M - airway smooth muscle ATP - adenosine triphosphate COPD - chronic obstructive pulmonary disease E M - electronic microscopy Lref - reference length s - second SE - standard error SR - sarcoplasmic reticulum Xll Acknowledgements First and foremost, I would like to extend my appreciation to my advisor, Dr. Chun Y. Seow who inspired my interest and dedication to the discipline of muscle physiology. I thank him for his patience, guidance, persistence and devoted interest in my education and career. I would like to express grateful thanks to my committee members, Dr Bruce Crawford, Dr. Wayne Vogl and Dr. Peter Pare, who provide me with a plenitude of advice and support during my years in the Department of Anatomy and the McDonald Research Laboratories/iCAPTURE Centre in St. Paul Hospital. I would like to thank my colleague and peers who have made this academic and life experience enjoyable and rewarding. And, finally, my deepest thanks to my family who provided with heartfelt support and encouragement throughout my university education. xm Chap.1 General introduction 1.1 Airway smooth muscle and obstructive airway disease 1.1.1 Airway smooth muscle: an overview The airway caliber and hence airway flow resistance is regulated by the contraction and relaxation of airway smooth muscle. To understand how airway smooth muscle controls airway diameter, one needs to know the anatomical location of airway smooth muscle within the airway wall and the constraints that the various components of the airway wall have on the muscle. Beyond the larynx, the respiratory tract continues as the trachea, which is then divided into two primary bronchi. The conducting portion of the respiratory tract consists of -16 generations of branching bronchioles. Beyond the terminal bronchioles, the thin-wall respiratory bronchioles, alveolar ducts and alveoli constitute the respiratory portion of the respiratory tract, where the exchange of oxygen and carbon dioxide with the blood takes place. Histologically, the components of the conduction portion of the respiratory tract include the lining epithelia, fibroblastic connective tissue, airway smooth muscle (ASM) and / or incomplete cartilaginous rings or plates. Smooth muscle exists in almost all the airways. 1 Even though contraction of A S M is the main contributor in narrowing the airways and to increase airway flow resistance, little is known about the function of A S M under physiological conditions. The function of any muscle is that it contracts when activated, and this is also true for A S M . Muscle contraction is usually a normal process occurring under physiological conditions that creates motion, generates force, sets tone or stabilizes shape. In the case of A S M , there is no concrete idea as to what the role A S M performs. There is no known disease entity or appreciable physiological deficit that is associated with loss of A S M contractility. It seems that, when contracting normally, A S M may not have compelling function and, when contracting excessively, A S M only serves to cause problems (Seow and Fredberg, 2001). Ironically research in airway smooth muscle is not driven by the desire to understand its normal physiology, but by its implications in diseases such as asthma and chronic obstructive pulmonary disease (COPD). 1.1.2 Obstructive airway diseases and airway hyperresponsiveness Increased airway narrowing in response to nonspecific stimuli, also known as airway hyperresponsiveness (Woolcock and Peat, 1989), is a characteristic feature of human obstructive airway diseases such as asthma and COPD (Moreno et al., 1986). The underlying mechanisms of airway 2 hyperresponsiveness are still unknown. Several mechanisms have been proposed to explain causes of the airway hyperresponsiveness, including alterations in the neurohumoral control of airway smooth muscle (Boushey et al, 1980; de Jongste et al., 1991), increase sensitivity of A S M (Antonissen et ah, 1979; Jiang et al., 1992; Stephens et al., 1988), increased mucosal permeability ( Persson et al., 1990), increased mucosal secretions ( Jeffery et al., 1992; Lambert et al., 1991), and mechanical factors related to remodeling of the airways ( James et al., 1989; Wiggs et al., 1990). Regardless of the potential contributors of these mechanisms, the reversibility and rapid onset of the airway narrowing indicate that contraction of A S M may play a central role in the airway hyperresponsiveness occurring in obstructive airway diseases. Considerable work has been carried out in A S M research to elucidate the relationship between A S M contractility and airway hyperresponsiveness (Hargreave et al., 1981; Opazo-Saez et al., 2000). The evidence suggests that the primary cause of airway hyperresponsiveness in obstructive airway diseases exists at the level of smooth muscle cells within the airway wall and obstructive airway diseases are associated with an increase in the contractility of A S M (de Jongste et al., 1987; Lamert et al., 1993). 3 1.1.3 The role of airway smooth muscle in obstructive airway diseases The causes of airway hyperresponsiveness in human obstructive airway diseases mainly include inflammatory response and exaggerated A S M contractility. These causes may lead to excessive airway narrowing and increased airway resistance. From a clinical point of view, if exaggerated contractility of airway smooth muscle can be uncoupled from airway inflammation, asthma may no longer be a disease. Therefore, an increased understanding on the contractility of airway smooth muscle is critical and should be taken into consideration when studying human obstructive airway diseases. Recently, it has been reported that A S M shows prominent structural and functional alterations in subjects with asthma or COPD ( Ebina et al., 1990; Wiggs et al., 1992; Thomson et al., 1996; Seow et al, 1998; Opazo-Saez et al., 2000). The structural and functional alternations include increased thickness of A S M layer and increased amount of A S M shortening. Therefore, further understanding of A S M structure and function may provide new insights for mechanisms underlying obstructive airway diseases. 4 1.2 Plasticity of smooth muscle: Does the striated muscle model apply? 1.2.1 The characteristic features of smooth muscle cell The reason that smooth muscle is described as "smooth" is because the muscle lacks striation which is characteristic of skeletal and cardiac muscles. Smooth muscle contains contractile filaments, i.e., myosin thick filaments and actin thin filaments, just as the striated muscles do. In addition, smooth muscle also contains dense bodies and dense plaques that are thought to be equivalent to the Z disks in striated muscles. Despite these similarities, smooth muscle possesses unique features not found in striated muscles, as described below. 1.2.1.1 Lability of the thick filaments Thick filament lability in smooth muscle was firstly suggested by Shoenberg (Shoenberg, 1969) and Rice et al. (Rice et al., 1970). It has been proposed that myosin thick filaments in smooth muscle might form during contraction and dissolve during relaxation (Shoenberg, 1969; Rice et al., 1970). It has been shown that thick filaments are more abundant in smooth muscle fixed in the solutions with low pH or with high divalent cations. Since the feature of activation in smooth muscle involves both a low pH and an 5 increased calcium concentration, it has been suggested that thick filaments undergo dynamic changes during the contraction-relaxation cycle. In airway smooth muscle, an important line of evidence for myosin filament lability is the observation of decreased density of thick filaments after mechanical perturbation applied to relaxed. A S M (Kuo et al., 2001). The results suggest that thick filaments are not stable in the relaxed state and are susceptible to depolymerization by mechanical agitation. Polymerization of the thick filaments during contractile activation has also been observed in A S M (Herrera et al, 2002; Kuo et al, 2003). The results seem to support the model proposed by Shoenberg (Shoenberg, 1969) in that polymerization of myosin monomers were found associated with activation; it disagrees with the Schoenberg model in that only partial dissolution of the thick filaments were observed in the relaxed state; many thick filaments remained intact in the relaxed state and were readily seen with electron microscopy (Kuo et al, 2001; Herrera et al, 2002; Kuo et al, 2003). 1.2.1.2 Large functional length range for smooth muscle It has been recognized for a long time that smooth muscle, compared to striated muscle, can function over a much larger range of length (Uvelius, 6 1976). This feature is required by the large volume changes of some hollow viscera, such as bladder, bowl and uterus. Under normal conditions the volume of a urinary bladder can be reduced about 500-fold within 1 minute. If all dimensions were reduced in the same proportion, the circumference of the bladder wall would decrease by about 8-fold. The change in the circumference may not reflect the change in muscle length, because there are additional tissues, such as loose connective tissue that enclose the muscle tissue: The work by Uvelius (Uvelius, 1976) has shown that a change of ~7-fold in the cell length in smooth muscle of the urinary bladder is associated with the maximal bladder volume change. He suggested that the length range of individual cell in the urinary bladder was indeed as large as the volume changes of the organ. 1.2.2 Can a model with fixed filament lattice accommodate smooth muscle cell function? In striated muscle, there have been many hypotheses for the mechanism of contraction (Huxley, 1980). The most accepted theory involves the so-called independent force generators (Huxley, 1980), which have become synonymous with the crossbridges observed between thick and thin filaments. The theory explains the sliding of filaments by the action of an array of 7 individual elements within the filaments which could generate force in the direction of shortening. The isometric force depends on the number of attached crossbridges and is proportional to the degree of overlap between the thick and thin filaments. With the fixed filament lattice array, isometric force generation is in good agreement with that expected from the degree of overlap between thick and thin filaments and the length-tension relationship shows decline at partial overlap (Figure 1, pagel l)(Gordon et al., 1966). An important feature of the fixed filament array (sarcomeres) is the declined isometric force generation with respect to an increase in length beyond the optimal length where the overlap between thick and thin filaments is maximal, and force will be zero at a long length where thick and thin filaments no longer overlap. At lengths shorter than the optimal, force will also be reduced because filaments undergo double and triple overlap. For a typical sarcomere, a 60% stretch from the optimal length will reduce the force to zero. Hence, it seems very unlikely that a 7-fold range of functional length in smooth muscle could be accommodated by the fixed filament array found in striated muscle. An important question in smooth muscle physiology is therefore, what is the structural basis that allows smooth muscle to function over such a large length range? 8 1.3 Hypotheses regarding plasticity in smooth muscle As mention above, there are two intriguing features in smooth muscle. One is the large functional length range that cannot be explained by the fixed filament array structure found in striated muscle. The other intriguing feature is the lability of smooth muscle myosin thick filaments. The thick filaments of smooth muscle depolymerize, at least partially, during relaxation and repolymerize upon activation. These unique features of smooth muscle have prompted Pratusevich et al. (Pratusevich et ai, 1995) to propose a model for smooth muscle where plastic reorganization of the contractile filament arrays could explain the ability of smooth muscle to adapt to large changes in length. The structural lability could confer plasticity on the filament lattice that would enable the muscle to adapt to large length changes by rearranging the contractile units within the cell. My thesis research has produced substantial amount of evidence in support of the plasticity theory, especially from the ultrastructural point of view. The scientific content of this thesis is mainly based on 3 published papers where I am the lead author (Kuo et al., 2001; Kuo et al., 2003a; Kuo et al., 2003b). The objectives of the thesis are to 1) present an overview on ultrastructural observations in airway smooth muscle, 2) examine the ultrastructural evidences of plastic alternations after mechanical perturbations applied to airway smooth muscle and 3) examine the ultrastructural evidences 9 of plastic alternations in airway smooth muscle adapted to different lengths. Our working hypothesis is that structural lability of the thick filaments confers plasticity on the filament lattice and enables smooth muscle cells to adapt to new cell dimensions after length perturbation by varying the number of contractile units in series (Figure 2 , page 12) . 10 Figure 1 The dependence of active tension generation by striated muscle fiber on the sarcomere length. The positions indicated correspond to sarcomere length of (a) 3.65 mm, (b) 2.2mm, (c) 1.65mm and (d) 1.05mm. (Modified from Gordon et al., 1966) b 10 20 30 Sarcomere lengrh (u.m) m u m M I I I I H i m n m n i l i l l i c n Figure 2 Proposed model for plastic alternation to different lengths Reference Length to Passively Lengthened After Repeated Activation Reference Length 12 Chap.2 Specific aims 2.1 Morphological observations on airway smooth muscle There is an abundance of ultrastructural data in the literature on vascular, visceral and other smooth muscles; such data on A S M , however, are conspicuously missing. In Chapter 4, we present a series of electron micrographs depicting contractile and cytoskeletal elements as well as organelles in porcine trachealis. The objective is to provide an overview of ultrastructural observations on A S M cells. 2.2 Inter- and intra-cellular force transmission in intact smooth muscle Many models of isolated smooth muscle cells depict obliquely arranged contractile filaments. According to our observations on intact A S M bundles, the contractile filaments run parallel to the long axes of the muscle cells. We therefore hypothesize that the arrangement of contractile filaments are parallel 13 to the long axes of the muscle cells and the direction of force transmission, as described in Chapter 5. Little is known about how force is transmitted through or around the relatively large and centrally located nuclei in smooth muscle. We hypothesize that part of the contractile filaments anchor onto the nuclear envelop complex, and the nuclei of smooth muscle cells can carry force during muscle contraction, as described in Chapter 5. Thick filament lability induced by mechanical perturbation To examine ultrastructural changes that may be associated with the plastic alternation of the contractile apparatus, in Chapter 6, we examine the dynamic changes of thick filament density after mechanical perturbation and during the process of force recovery. We hypothesize that thick filaments are labile and can be induced to depolymerize by mechanical perturbation and will repolymerize when the muscle cells are allowed to recover without further mechanical agitation. 14 Ultrastructural basis of adaptation of airway smooth muscle to different lengths In order to carry out its normal physiological function, a smooth muscle cell can be stretched to a length several times of its original. The mechanism underlying the muscle's ability to maintain optimal contractile function over the large length range is still poorly understood. In Chapter 7, we examine ultrastructural basis of plastic alternation of airway smooth muscle at different lengths. We hypothesize that smooth muscle adapts to large length changes by varying the number of contractile units in series. 15 Chap.3 Material and method 3.1 Measurements of mechanical properties Porcine tracheal smooth muscle was used for the experiments. The tracheas were obtained from a local abattoir. The tracheas were kept in ice-cold physiological saline immediately after their removal from the animals. Muscle strip (approximately 6 x 1 x 0.3 mm in dimension) was dissected from a trachea and attached to clips made of aluminum foil. The clipped muscle strip was then mounted in a muscle bath; one end of the strip was attached to the force transducer and the other end to the servomotor. The muscle bath contained physiological saline solution at pH 7.4, bubbled with a gas mixture (5% C 0 2 , 95% 0 2) and containing (in mM) NaCl 118, KC1 5, NaH 2 P0 4 1.2, NaHC0 3 22.5, MgS0 4 2, CaCl 2 2 and Dextrose 2g/L. The muscle preparation was equilibrated in the bath for about 1 hour to obtain a maximal, stable isometric force. During the equilibration period, the muscle was stimulated electrically once every 5 min, and a reference length (Lref) for the muscle was determined as well. The length where rest tension was just detectable, approximately the in situ length, defines L^f- The duration of the electrical field stimulation was 12 s, the stimulator frequency was 60 Hz. Muscle force was measured with a photoelectric type of force transducer with a resonant 16 frequency of 1 kHz and a signal-to-noise ratio of >50. Isotonic shortening velocity was measured by quick-releasing the muscle from plateau of an isometric contraction to a pre-set isotonic load. 6-10 such releases were performed on a muscle preparation to obtain force-velocity data used in the curve fitting to generate a force-velocity curve. Preparation for ultrastructural study The muscle strips were fixed at the time points described in experimental procedures, for 15 min while it was still attached to the apparatus. The replacement of physiological saline by fixing solution occurred in less than 1 s. The fixing solution was pre-warmed to 37 °C, the same temperature as the bathing physiological saline. The fixing solution contained 1.5% glutaraldehyde, 1.5% paraformaldehyde in 0.1 M Na-cacodylate buffer. After the initial fixing (15 min), the sample was removed from the apparatus, cut into small blocks (approximately 1 x 0.5 x 0.1 mm in dimension) and placed in the same fixing solution for 2 hours at 4°C on a shaker. This was then followed by a 3 x 10 min wash in 0.1 M Na-cacodylate buffer. The tissue was then fixed with 1% OSO4 in 0.1 M Na-cacodylate buffer for 2 hrs, followed by a 3 x 10 min wash with distilled water. This was followed by en bloc staining with 1% uranyl acetate for 1 hr and a 3 x 10 min wash in distilled water. The tissue was then dehydrated through graded series 17 of ethanols of 50%, 70%, 80%, 90%, 95%, each for 10 min, followed by 100% ethanol (3x10 min) and propylene oxide (3x10 min). The specimen was then ready for embedding in resin (TAAB 812 mixing, medium hardness). The specimen was allowed to "soak" in the resin overnight before embedding in molds and placed in oven at 60 °C for 8 hrs. The blocks were sectioned on microtome using a diamond knife. The thickness of the sections was about 90 nm. The sections were then stained in 1% uranyl acetate for 4 min followed by a 3 min staining in Reynolds lead citrate. A Phillips 300 electron microscope was used for obtaining the images of the cross-sections of the muscle cells. Image processing and data analysis The sampling and analysis were carried out in a "blinded" manner. The sample codes were revealed only after the analysis for each group was complete. Each picture contained one whole cell cross-section. The myosin thick filaments (with a diameter of 15-20 nm) were identified by eye and counted for the whole cell cross-section. The density was obtained by dividing the number of thick filaments by the total area of the cell cross-section minus the areas occupied by nuclei, mitochondria and other cellular organelles. The measurement of length, perimeter and area were aided by the 18 use of a morphometric digital device (Carl Zeiss, Germany.) and a image analysis software (Image Pro Plus). 19 Chap.4 Morphological observations of airway smooth muscle 4.1 Morphological observations: past and present The structural studies of smooth muscle have been carried out for more then one century (Kolliker, 1849), but the knowledge about structural organization of smooth muscle are still far short of being well established, compared to our understanding of striated muscle ( Huxley, 1957). Up to now, smooth muscle cell has only been shown to be spindle-shaped in appearance, to have a central-located and elongated nucleus, and with clustered mitochondria located near the two poles of a nucleus, advanced very little beyond that already been established by the turn of last century ( Heidenhain, 1900; McGill, 1909). The physiology and pathophysiology of airway smooth muscle contraction in terms of structure-function relationship is also poorly understood. Our knowledge about the contractile mechanism in airway smooth muscle has been based largely on the sliding-filament theory of contraction developed for striated muscle (Huxley and Hanson, 1954). The existence of myosin thick filaments and actin thin filaments, as well as the 20 cytosolic dense bodies and membrane-associated dense plaques (that are thought to be equivalent to the Z-disks in striated muscle) in smooth muscle has provided the basic elements necessary for assembling, at least in theory, a contractile unit analogous to a sarcomere (Bagby et al.„ 1983). This suggests that the sliding-filament mechanism could be operative in smooth muscle. Unlike in striated muscle, the structure of a contractile unit in smooth muscle is only vaguely defined. The consensus, nevertheless, is that it consists of a side-polar myosin thick filament interacting with two parallel actin thin filaments that are anchored onto two dense bodies positioned on either side of the thick filament (Hodgkinson et al, 1995). The dimension of a contractile unit has yet to be accurately determined. The recently proposed plasticity hypothesis postulates that contractile units may be added or removed from the contractile machinery of a smooth muscle cell in a process called length adaptation (Pratusevich et al, 1995), and that the actin filaments may not attach to dense plaques until the muscle is activated (Gunst et al, 1995). The dimension of a contractile unit may even change during activation due to lengthening of the thick filaments (Seow et al, 2000; Xu et al, 1997). The dynamic ultrastructure of smooth muscle is in sharp contrast with the static ultrastructure of striated muscle. It has been proposed that a malleable structure allows smooth muscle to accommodate large changes in length while maintaining optimal contractility (Ford et al, 1994). The malleable structure however poses a major challenge to 21 investigators attempting to delineate the structure-function relationship in smooth muscle. The situation is further exacerbated by the fact that different smooth muscles exhibit different degrees of malleability in their ultrastructure (Gillis et al, 1988; Godfraind-De Becker and Gillies, 1988; Xu et al, 1997; Kuo et al, 2001; Herrera et al, 2002). This may explain the slow progress in elucidating structural mechanism of contraction in smooth muscle. The available ultrastructural data on smooth muscle are mostly from studies of vascular and visceral smooth muscles (Bagby et al, 1983). There are limited ultrastructural data on airway smooth muscle in the literature. The assumption that there is no fundamental difference in ultrastructure between airway and other smooth muscles may be valid to some extent; the observed unique properties associated with each muscle however are likely manifestations of the difference in ultrastructure and malleability of the structure. To understand the structure-function relationship in airway smooth muscle, we cannot rely solely on structural data gathered from other smooth muscles. In the thesis, the ultrastructure of contractile and cytoskeletal elements, and the spatial organization of organelles of airway smooth muscle are described in detail. A brief description of the in situ relationship of airway smooth muscle to other components of the airway wall is also included as a reminder of the structural interdependence of airway smooth muscle and the airway wall in regulating airway caliber. It is important to point out that a 22 micrograph is a "snapshot" that shows ultrastructure of a cell which often is in a dynamic state; the data has to be interpreted in the context of cell activation (Xu et al., 1997; Herrera et al., 2002; Qi et al.„ 2002), adaptation or equilibration after a mechanical perturbation (Kuo. et al., 2001; Qi et al., 2002) and other non-equilibrium states. 4.2 Organelles of airway smooth muscle cell 4.2.1 Cell membrane The cell membrane and membrane-associated elements in smooth cells do not differ, in appearance, from that of other cell types (Figure 3, page29). Most noticeably are the electron-dense bands, known as dense plaques, and the flask-shaped invaginations, known as caveloae. The outer layer of the cell membrane is coated by basal lamina. 4.2.2 Caveolae The cell membrane of airway smooth muscle has numerous flask-shaped invaginations (Figure 3, page29), known as caveloae (or caveloae 23 intracellulares, surface vesicles, plasmalemmal vesicles). Caveolae are fairly uniform in size. In our preparations, the dimensions of caveolae are about 90nm in width and 120rtm in length. Caveolae have a characteristic association with sarcoplasmic reticulum. The arrangement is shown to be important in calcium recycling (Lee et al., 2002). Caveolae are not randomly distributed over the cell surface but are grouped in rows with dense plaque intervening between them. 4.2.3 Sarcoplasmic reticulum Smooth muscle cells are well endowed with endoplasmic reticulum (sarcoplasmic reticulum, SR). The SR is often located near the plasma membrane in association with caveolae (Figure 3, page29). There appears to be two general types of SR: superficial SR and deep SR (Figure 3, page29). Three-dimensionally a SR can be visualized as an irregularly shaped pancake with hollow inside. A superficial SR is positioned with its flat face parallel to the plasma membrane; a deep SR is positioned with its flat face perpendicular to the plasma membrane. The deep SR appears to provide a conduit for calcium transport within a cell. It has been speculated that the SR-mitochondrial junctional space is another buffer barrier for calcium diffusion that serves to regulate mitochondrial calcium concentration (Lee et al., 2002). 24 4.3 Contractile apparatus 4.3.1 Overview of the contractile apparatus The cytoplasm of tracheal smooth muscle cells is largely occupied by myofilaments. Figure 4 (page30) shows a cross section of a trachealis cell. The myosin thick filaments have an irregular cross-sectional profile (due to the presence of cross bridges on the filament), with an average diameter of 15-20 nm. The diameter of an actin thin filament is about 6 nm, and at the magnification shown in Figure 4 (page30), it has a circular profile for its cross section. A third type of filament seen in smooth muscle is intermediate filament, so called because it is intermediate in size between thin and thick filaments. Intermediate filaments have a circular cross section with a diameter of 10 nm. Dense bodies are also regular features seen in electron micrographs of tracheal smooth muscle cells. Dense bodies and dense plaques are structures thought to provide anchorage for actin and intermediate filaments. The network of actin and intermediate filaments connected through dense bodies is thought to form the scaffold of cytoskeleton. Microtubules (long hollow tubes with a diameter of 25 nm) can often be seen in the cytoplasm of trachealis cells; they are relatively few in number but are regularly present. These dynamic structures are involved in intracellular motility such as 25 generation of cell processes, transportation of organelles and chromosome movement in the mitotic spindle. They are part of the cytoskeleton, but not considered as part of contractile appratus. 4.3.2 Thick filaments Thick filaments of smooth cells were rarely seen in the early electron microscopic preparations (Devine and Somlyo, 1971). There were mainly two causes that had been blamed for the failure to visualize the thick filaments. These causes included swelling of muscle cells (Jones et al., 1973), inadequate block staining for thick filaments and lack of equilibration for muscle cells (Devin & Somlyo 1971). Indeed, the main reason for the difficulty in visualizing the thick filaments may be caused by their lability, a recently recognized feature. Thick filaments have now been found in all types of smooth muscle examined with proper preparative technique. In airway smooth muscle, diameter of thick filaments is variable, ranging from 12 to 20 nm in our preparation (Figure 4, page30). In transverse section, the thick filaments of smooth muscle have an irregular profile and are distinguishable from those of striated muscle with regular profile. 26 4.3.3 Thin filaments Unlike thick filaments, thin filaments in smooth muscle have been observed since the early work in ultrastructural study. Diameter of a thin filament is about 6nm in our preparation (Figure 4, page30). In transverse section, they have circular profile and form a fairly uniform population. 4.3.4 Intermediate filaments The filaments of the third type measure around 10 nm in diameter and have a clear-cut circular profile in transverse section (Figure 4, page30). Intermediate filaments are well preserved by conventional fixative, compared to thick filaments and thin filaments. 4.3.5 Dense bodies / plaques Dense bodies are electron-dense structures scattered in cytoplasm. They vary in diameter between 30 nm and 200 nm in our preparation. Intermediate filaments are often observed to associate with dense bodies (Figure 4, page30) and show a structural continuity (Cooke, 1976). Some preparations clearly 27 show thin filaments penetrating and merging with dense bodies (Ashton et al.,1975). Dense bodies and dense plaques were suggested to be similar structures and they probably correspond to Z-lines of striated muscle (Pease & Molinari, 1960) and provide the attachment sites of thin filaments. By using immunocytochemical technique, both dense bodies and dense plaques show similar labeling-pattern of alpha-actinin (Schollmeyer et al., 1973). In our preparation, the dimensions of the majority of the dense plaques are about 200nm to 400nm in length and 50nm to 80nm in width (Figure 4, page30). 28 Figure 3 Electron micrograph of a transverse section of trachealis cells. (A) Electron micrograph of a transverse section of trachealis cells. Calibration bar: 1 pm. (B) - (E) Enlarged areas from Panel A. (B) Showing a group of mitochondria (indicated by "M"), Golgi apparatus (indicated by "G") and rough endoplasmic reticulum. Calibration bar: 200nm. (C) Showing dense plaques, (circle) SR (arrowheads) and caveolae (arrows). (D) Showing a superficial SR (arrowheads) and caveolae (arrows). E) Showing a deep SR (arrowheads) and caveolae (arrows). Panel B - E have the same magnification. 29 Figure 4 Contractile apparatus in trachealis muscle The bulk of the cytosol is occupied by myofibrils. The insect shows enlargement of some myofibrils on the panel (black square). Myosin filaments (indicated by arrow) are surrounded by numerous actin filaments. Intermediate filaments are often observed to associate with dense body (circle). Calibration bar: 1pm. 30 Chap.5 Intra- and inter-cellular force transmission in intact airway smooth muscle bundle 5.1 Previous structural models proposed for smooth muscle based on observation of isolated cells Force generation in smooth muscle is achieved through cyclic interaction of myosin cross bridges with actin filaments similar to that found in striated muscle (Guilford et al., 1998). How the force is transmitted within and between smooth muscle cells however is still poorly understood. The lack of precise knowledge about the contractile filament architecture in smooth muscle has hindered our efforts to understand the mechanism and structure of force transmission. It has been recognized long ago that most smooth muscle cells in vivo function electrically as a syncytium in a so called "effector muscle bundle" (Burnstock and Prosser, 1960; Burnstock, 1970). Whether a mechanical syncytium exists in the same effector bundle of smooth muscle is not known. Many models of contractile filament structure in smooth muscle are based on observations made in isolated cells (Fay and Delise, 1973; Fisher and Bagby, 1977; Small, 1977) whose mechanical couplings with adjacent cells have been severed during the process of isolation. The models are mostly that of a single cell with obliquely orientated contractile filaments 31 relative to the longitudinal axis of the cell. When an isolated cell contracts, corkscrew-like shortening of the cell is often observed (Bagby, 1983; Warshaw et al., 1983), with an exception of observation by Small (Small, 1985) where antivinculin staining of isolated guinea pig vas deferens cells showed filament attachment sites parallel to the longitudinal axis of the cell, even in the shortened state. A contentious point still being debated is whether the corkscrew-like shortening observed in isolated cells could happen in an intact muscle bundle or is it a result of decoupling of mechanical connections of the cell with its neighboring cells and extracellular matrix. If smooth muscle cells do not work as individuals but as a group in an effector muscle bundle, the architectural design of the contractile filaments may be optimized for the group but not for individual cells. In other words, through dense plaques the contractile filaments may be organized into a trans-cellular mechanical syncytium where force can be generated and transmitted in contractile filaments lie parallel to the longitudinal axis of the muscle bundle and the coincidental axis of force transmission, even at the tapered ends of the cells. In that regard force transmission within the constituent cells may not be uniform at all cross sections and when isolated from its neighbors an individual cell may be dysfunctional. Studies of filament organization in smooth muscle cells therefore may be more appropriately carried out in a bundle of cells where intercellular connections are intact. 32 5.2 Notes on experimental procedure For this portion of the thesis research, small bundles of swine tracheal smooth muscle were used in the assessment of mechanical properties and fixation for electron microscopy. Cells in these tissues had their longitudinal axes aligned to the axis of force transmission during fixation. This allowed us to examine the orientation of contractile filaments with respect to the direction of force transmission. The multi-cellular preparations also allowed us to examine the mechanical connections between cells and how these connections influenced intracellular organization of contractile filaments. 5.3 Arrangement of contractile filaments observed in intact airway smooth muscle bundle 5.3.1 Parallel arrangement of contractile filaments Smooth muscle cells normally aggregate into small bundles within which the cells lie parallel (with respect to their longitudinal axes) to one another (Bagby, 1983); this is also true for trachealis cells. Mechanical connections among the cells are provided by the cell-matrix adhaerents that contain two opposed dense plaques from adjacent cells. Figure 5 (page44) shows a transverse section of trachealis cells within a muscle bundle. Mechanical couplings between cells are evident (examples of cell-matrix 33 adhaerents are circled). Contractile filaments (actin and myosin filaments) seen in the section are perpendicular to the cell cross-section (see inset). To see the filament orientation with respect to the cell's long axis, longitudinal sections were obtained; an example is shown in Figure 6 (page46). Dense bodies and filaments are seen lined up with the axis of force transmission, which is also the longitudinal axis of the muscle bundle. The filaments lie parallel to one another; the parallelism is not interrupted by cell boundaries or nuclei. When filaments run into cell membrane they attach to dense plaques; the plaques are located all over the cell surface and not just in the region of tapered ends (Figure 5 and Figure 6). Continuity of force transmission across a cell boundary is accomplished through coupling provided by two opposed adjacent dense plaques located on neighboring cells; an example is shown in Figure 6 (oval) where contractile filaments in both cells attach to the inner aspects of the dense plaques that in turn are connected extracellularly by amorphous electron-dense material. The prevailing filament orientation is not altered by the intervening cell boundary. When filaments run into a nucleus, there is no evidence of circumvention (Figure 6, inset). There is evidence that contractile filaments attach directly to nuclei, as shown below. 5.3.2 Association of contractile filaments and nuclei 34 Figure 7 (page46) shows 3 transverse sections (out of an eleven-consecutive series of slices with an average thickness of 100 nm per slice) of a trachealis cell. We counted the number of myosin thick filaments in the whole cross-section for each of the 11 serial sections and correlated the filament number to the fraction of area occupied by the nucleus. We found an inverse relationship between the number of myosin filaments and the nuclear area (Figure 8, page47). The number of myosin filaments decreases when the area of nucleus increases. In muscle, force is mainly carried by myosin filaments. In order to transmit force smoothly, the number of myosin filaments has to be constant in per cross-section. The decrease of the number of myosin filaments in cross-section of different segments of a cell is not a constant, suggesting that another force-carrier rather then contractile filaments may exists. The inverse relationship between the number of myosin filaments and the nuclear area suggests that the contractile filament arrays ended where the nucleus started and the nucleus may carry part of force. High magnification electron micrograph of a region near the nuclear pole of an actively shortened trachealis cell shows the association of thin filaments and the nuclear envelope during isotonic contraction (Figure 9, page48). The association-sites on the envelope are patchy; pulling by the actin filaments appears to be responsible for the "folds" seen on the nuclear envelope. 35 Near the two poles (ends) of a nucleus, clusters of mitochondria and other membranous organelles are often found. Although myosin filaments are not seen within the clusters, actin filaments are often observed to go through the clusters and likely to associate to the nuclear envelope (Figure 10,page49). 5.4 Nucleus as a force carrier 5.4.1 Elasticity of the nuclear envelope To determine whether the force generated by the contractile filaments exerted on the nucleus, we examined the longitudinal lengths of nuclei in two groups of trachealis strips: relaxed and contracted. Each pair of the strips (approximately 6 mm long, 2 mm wide and 0.3 mm thick) was obtained from bisecting a rectangular piece of trachealis to ensure that both strips had the same length. One strip was fixed for electron microscopy in the relaxed state while the other strip was fixed in an isometrically (constant length) contracted' state. The average width of cells were 1.30±0.076 pm and 1.32±0.083 pm for the relaxed and activated groups, respectively. Therefore the volume of the cells is conserved during isometric contraction. The final muscle contraction was induced by addition to the muscle bath 0.1 mM of Acetylcholine to ensure continued activation during fixation. Muscle strips were fixed at the plateau of contraction (120 s after stimulation) while they were still attached to the experimental apparatus (Herrera et al., 2002; Qi et al., 2002) and force 36 generated by the muscle was monitored throughout the period of fixation (15 min). In the contracted muscles, there was a small decline in force (<5% maximal force) during fixation. Longitudinal lengths of the nuclei from the two groups (relaxed and contracted) were measured from electron micrographs (examples shown in Figure 11, page50). The averaged values from 5 pairs of muscle preparations from 5 tracheas were 9.24±0.12 pm and 11.36±0.12 pm for the relaxed and activated groups, respectively. The ratio of the activated over the relaxed group indicated a 23.4%±1.7 (SE) increase in the longitudinal lengths of nuclei, suggesting that activated contractile filaments were exerting tension on the nucleus. The strain of the nucleus under isometric force was therefore 0.234±0.017. To calculate the axial tensile strength of the nuclear envelope, we first determined the stress in the activated muscles by dividing the active isometric force (88.3±11.0 mN, n=5) by the cross-sectional area occupied by the muscle cells. In these trachealis preparations, muscle cells on average occupied 76%±9.6 of the total cross-sectional area, the balance of the area was occupied by connective tissue. The averaged active stress in the muscle cells was calculated to be 196+10 kPa (n=5). If we assume that the tensile stress is uniform in a cell cross-section, then the average stress on the nucleus is the same as that on the rest of the cell, i.e., 196 kPa. The elasticity of the nucleus under isometric stress can be calculated as 196±10 kPa/0.234±0.017, which gives a value of 837.6±74.4 kPa. The stress, however, may not be evenly distributed in a cell cross-section, as suggested by Fig.3(page29). The fraction 37 of nuclear area increased from zero to -60% of the total area, the total number of myosin filaments per cell cross-section decreased only by -47%. If force is proportional to the number of myosin filaments, this means that force transmitted through the cytoplasmic area surrounding the nucleus in this particular cell is 53% of the total force generated by the cell, which leaves 47% of force going through the nucleus. The disparity between the percentage area occupied by the nucleus (60%) and the force transmitted through it (47% of total force) means that the stress on the nucleus is about 78% (i.e., 0.47/0.6 « 0.78) that of the averaged stress in this particular example. We examined the fractional areas occupied by the nuclei in cell cross-sections morphometrically in the 5 trachealis strips fixed in the contracted state. In 40 cell cross-sections showing full nuclei (8 cells per strip), we found that the fraction of total cell area occupied by the nucleus was 0.549±0.026. The number of myosin filaments in these sections expressed as a fraction of the number of myosin filaments in a separate group (but from the same tracheas) of 40 cell cross-sections without nuclei, was 0.551±0.061. This means that although the nuclear area on average is slightly greater than 50% of the total cell area, the number of myosin filaments attaching to the nucleus is on average slightly less than 50%. The stress on the nucleus is therefore less than the average stress calculated for the whole cell. Assuming that isometric force is proportional to the number of myosin filaments in a cross-section, a correction factor can be obtained: (0.449±0.061) / (0.549±0.026) = 0.818+0.118. With this correction, we obtained a corrected stress on the 38 nucleus: 160.3±24.5 kPa, and a stiffness of 685±116 kPa for the nuclear envelope. This stiffness value is very close to the modulus of elasticity of elastin fibers, -600 kPa (Fung, 1993). 5.4.2 Mechanical integrity of the nuclear envelope The nuclear envelope of a eukaryotic cell consists of the nuclear membrane and the underlying fibrous layer called nuclear lamina (Moir et al., 1995). Cytoskeletal proteins and intermediate filaments form an extensive network that connects the nuclear envelope to the cortical cytoskeleton underlying the plasma membrane (Djabali, 1999). Unlike that in skeletal muscle, the relatively large and centrally located nucleus of smooth muscle interrupts the array of contractile filaments spanning from one end of the cell to the other. Mechanical equilibrium requires that force transmitted by the contractile filaments along the cell length be continuous and uniform. This means that the contractile filaments either have to go around the nucleus, or anchor onto the nuclear envelope and use the envelope as a passive structure to transmit force. Evidence from the present study suggests that the latter may be the case. This implies that physical integrity of the nuclear envelope is important for smooth muscle's mechanical function. A compliant envelope will result in substantial internal movement when the muscle is activated and lead to low mechanical efficiency. The elongation of the envelope in airway 39 smooth muscle under isometric tension is about 2 um (see Results). In a 100-200 um long cell, this represents a 1-2% change in cell length. Stretching a relaxed trachealis strip also caused an increase in the average length of the nuclei (results not shown). Although one can get an estimate of the stiffness of the nuclear envelope by measuring the force of stretch and the amount of nuclear elongation, there are factors that complicate the analysis. The nucleus is connected to the rest of the cell in all directions through a network of intermediate filaments and cytoskeletal proteins (Bagby, 1983); stretching a cell longitudinally will result in forces acting axially on the nuclear poles as well as a lateral compression of the nucleus, both contribute to the elongation of the nucleus. Without knowing the exact architecture and mechanical properties of the cytoskeletal network, it is impossible to separate the contributions to nuclear strain by the axial and lateral forces and render the estimate of the axial stiffness inaccurate. The use of force generated by the contractile filaments in an isometric (constant length) contraction simplifies the analysis because the cytoskeleton is not stretched and therefore there is no lateral force involved. The ultrastructure of nuclear "ghosts" or nuclear envelopes without the membrane (Riley and Keller, 1978) has been examined and it appears that the structure is essentially the same in a great variety of cells derived from mammals, birds and plants. Currently the mechanical integrity of a defective nuclear envelope can only be assessed morphologically by observing changes in the shape of the envelope, for example, formation of blebs or herniations 40 (Vigouroux et al., 2001). The present study demonstrates a simple and straightforward way of accurately quantifying axial stiffness of the nuclear envelope under physiological strain. Mutations in genes that encode different envelope proteins can now be quantitatively studied to gain insights into the roles of the proteins in conferring integrity to the envelope. Cyclic mechanical strains applied to cultured smooth muscle cells are known to enhance production of certain proteins by the cells (Smith et al., 2000; Lee et al., 2001). The strain-initiated signal transduction pathway in these cells is not clear. Although "stretch receptors" on the plasma membrane may be involved, our finding indicates that strains applied to smooth muscle cells can be transmitted directly to the nucleus, and the transmission is more effective if the muscle is activated. Cyclic isometric contraction and relaxation of smooth muscle cells can also lead to mechanical perturbation of the. nuclei and may have a modulatory function on specific protein transcriptions. A proposed model of filament architecture in bundle of trachealis - a mechanical syncytium Morphological evidence gathered in this part of thesis research supports a notion that individual cells in a smooth muscle bundle may not be a "self-contained" functional unit of force generator. Non-uniformity in terms of the 41 number of myosin filaments per cell cross-section in individual cells is prevalent. This has raised a question of how does smooth muscle as a tissue maintain mechanical equilibrium (i.e., uniform force generation and transmission) along its entire length. An answer to the above question is shown in Figure 12. To achieve uniform force transmission along the length of a muscle bundle, same amount of force has to be transmitted across each cross-section (perpendicular to the direction of force transmission) of the bundle. The force is actively generated and carried by the contractile filaments and passively borne by the nuclei. This arrangement allows an aggregate of "non-uniform" cells to form a functional unit, a syncytium, where force can be generated and transmitted uniformly. If smooth muscle cells function as a mechanical syncytium, mechanical equilibrium of force transmission within a single cell is no longer required. This removes a crucial constraint on the modeling of filament architecture in a single cell. For instance, force generated in the tapered region of a cell can be much less than the force generated in the mid-region. In Figure 5 the thick and thin filament numbers in the tapered ends (small cell cross-sections on the left) are much less compared to those seen in the cross-section near the mid-segment (large cell cross-section on the right, Figure 5). This is consistent with the model proposed in Figure 12 and no special filament architecture is required to account for the apparent disparity created by the difference in the 42 number of contractile filaments found in different cell cross-sections, if the individual cells belong to a mechanical syncytium. Another implication of the proposed model of mechanical syncytium is that isolated single cells of smooth muscle may not be a good model for studying mechanical function, because they are not designed to function as individuals. However, under certain conditions, for example, when an isolated smooth muscle cell is long and the tapered ends are not included in the mechanical study, reliable measurements can still be obtained (Warshaw and Fay, 1983). 43 Figure 5 Transverse section of trachealis cells within an intact muscle bundle. Examples of cell-matrix adherent are circled. Cross-sectional profiles of myosin and actin filaments are visible in the magnified area (square). Arrows point to myosin filaments surrounded by actin filaments. Arrowheads point to dense bodies. Calibration bar: 1 pm. 44 Figure 6 Longitudinal section of trachealis cells within an intact muscle bundle. The general orientation of contractile filaments and dense bodies (arrowheads) aligns with the longitudinal exes of the cells. A n example of intercellular coupling of dense plaques is highlighted (oval). The inset shows myosin filaments (arrows) near a nucleus (N). Calibration bar: 1 urn. 45 Figure 7 Electron micrographs showing a series of consecutive transverse sections of a trachealis cell. (A) A transverse section near a nuclear pole with a cluster of centrally located mitochondria and other organelles. (B) A transverse section cutting across the tip of a cigar-shaped nucleus. (C) A transverse section showing full cross-section of a nucleus. (D) An enlarged picture of A showing myosin thick filaments (arrows) surrounded by actin thin filaments (inset). All cross-sections come from a series of 11 consecutive sections (-100 nm in thickness for each section). Calibration bar: 1 pm. 46 Figure 8 Inverse relationship between the number of myosin filaments and the nuclear area. A plot of cell cross-sectional area occupied by the nucleus and the number of myosin thick filaments found in the cytoplasmic area in the 11 serial sections. 47 Figure 9 Direct attachment of thin filament to the nuclear envelope. Electron micrograph showing actin filaments attaching to nucleus in an isotonically shortened trachealis cell. Calibration bar: 1 pm. . 48 Figure 10 Thin filaments observed in clusters of mitochondria. Electron micrograph of a transverse section of trachealis cell showing a cluster of mitochondria and other organelles. The enlarged area shows microtubules (hollow, 25 nm in diameter) and actin thin filaments (solid, 6 nm in diameter) going through the cluster. Calibration bar: 1 urn. 49 Figure 11 Change of nuclear length during isometric contraction. Longitudinal sections of trachealis cells fixed in the relaxed (top) and contracted (bottom) state. The average lengths of the nuclei measured from 40 such pictures for each condition were 9.22um±0.12(SE) and 11.36um±0.12 respectively for relaxed and contracted cells. Calibration bar: 2 pm. 50 Figure 12 A proposed model of filament architecture in a bundle of tracheali. 51 Chap.6 Myosin thick filament lability induced by mechanical strain 6.1 Background Functional studies indicate that adaptation of airway smooth muscle to a large length change occurs in two steps. First, there is a decrease in the ability of the muscle to generate force immediately after the length change. Second, the decrease in force generation is followed by a period of force recovery when the muscle is allowed to adapt at a fixed length without further mechanical perturbation (Pratusevich et al., 1995; Wang et al., 2000). Structural studies of skeletal muscle adaptation to chronic stimulation at short lengths (Jakubiec-Puka and Carraro, 1991) have also revealed two steps in the process of muscle remodeling. The first phase of skeletal muscle remodeling is associated with disorganization of the structure of sarcomere, which appears during the initial hours of stimulation. The alternation in the structure of sarcomere is followed by a period of recovery during which the muscle regains its normal appearance of sarcomere. Remodelling of the contractile apparatus in a chronically shortened skeletal muscle is accomplished by deletion of sarcomeres in series so that optimal filament overlap is reestablished (Farkas and Roussos, 1983; Jakubiec-Puka and Carraro, 1991). 52 The time required for structural remodeling in skeletal muscle (from hours to days) is much longer than that required for adaptation of airway smooth muscle to length change, which takes only minutes (Farkas and Roussos, 1983; Jakubiec-Puka and Carraro, 1991; PratusevicheJ al., 1995 and Wang et al., 2000). The fundamental mechanism, however, could be the same. Disorganization of the contractile apparatus in airway smooth muscle following a large length change could be the first step in the remodeling process that leads to reorganization of the contractile apparatus that in turn would lead to a full adaptation of the muscle to a new length with optimal overlap of the contractile filaments. Characterization of structural changes that accompanies functional changes during the process of smooth muscle adaptation has never been done and is the focus of this chapter. A stepwise change in length is not the only way to initiate the process of adaptation in smooth muscle. It has been shown that by applying a brief period of length oscillation to a relaxed muscle one can achieve the same purpose (Wang et al., 2000). The advantage of using length oscillation instead of a stepwise length change is that after the oscillation the muscle returns to its original length, and that allows measurements of functional and structural changes associated with the reorganization of the contractile apparatus during the adaptation period to be made at the same muscle length. In this chapter, we used electron microscopy to examine the density of myosin thick filament in cross-sections of airway smooth muscle fixed at various stages during the adaptation process initiated by length oscillation. 53 Notes on experimental procedure Four tracheas were used for the experiments in this part of the thesis research. Five strips of muscle were isolated from each trachea. A reference length (Lref) was obtained for the muscle preparation during the period of equilibration. Lref was associated with a resting tension that was between 1-2% of maximal isometric tension. These muscle strips were fixed at five different time points during the process of length oscillation and adaptation, as illustrated in (Figure 13, page67). Time 1 (pre-oscillation) was the time point when muscle strip was equilibrated and the force generation nearly maintained constantly. Time 2 (post-oscillation) was the time point immediately after length oscillations applied. The amplitude of the sinusoidal length oscillation used was 60% of Lref (peak-to-peak). The oscillation therefore caused a 30% Lref stretch of the muscle. The maximal passive force measured during the oscillation was about % of maximal isometric force. The frequency of the oscillation was 0.5 Hz. This frequency is close to the respiration rate of pigs. Time 3 was the time point when 1 tetanus was produced by 12 second electrical field stimulation at a 5-minute interval after length oscillation. Time 4 was the time point when 2 tetani was produced by 12 second electrical field stimulation at a 5-minute interval after length oscillation. Time 5 was the time point when the force generation completely recovered to the value of force generation at time 1. 54 6.3 Isometric force development after mechanical strain 6.3.1 Force decrease caused by applied length perturbation Isometric force was obtained from averaging digitized isometric force traces (Figure 14,page68). Data at each time point represent results averaged for four strips from four different animals. The maximal force generation immediately after length oscillation (time 2) is depressed about 25% compared to the maximal force generation before length oscillation (time 1). The decrease of the maximal force after length oscillation is unlike to be due to disruption of cross-bridge dynamics because the length oscillations are applied before the muscle is stimulated. Therefore a non-cross bridge mechanism appear to play a role in the effect of length oscillation on smooth muscle contractility (Shen et ai, 1997, Wang et al, 2000 ). Possible mechanisms involved in the effect of length oscillation on smooth muscle contractility has been suggested to be due to reorganization of contractile filaments or cytoskeleton (Pratusevich et al., 1995, Gunst et al., 1995). However, it is still not conclusive without structural evidence on the effect of length oscillation. 55 6.3.2 Adaptive process of force generation after length perturbation We observed that immediately after the length oscillation, the maximal force was depressed. Then, the maximal force gradually recovered to the level of pre-oscillation after a series of isometric contractions over a period of 30minut.es (Figure 14,page68). It has been shown that large-amplitude oscillation causes a relatively long-term depression of force (Wang L. et al, 2000). There are factors other than disruption or cross-bridge dynamics to explain the relatively long-term effect caused by large-amplitude oscillation. A possible explanation is that the perturbation results in rearrangement of contractile machinery in muscle cells. The time course of force recovery shows an exponential process (Figure 14), perhaps reflecting that the recovery process appear to involve receptors binding and signal transduction. Hence, the exponential function of force recovery suggests that the recovery process may be a biochemical process and it includes rearrangement of contractile filaments, polymerization of myosin filaments and/or re-anchoring of actin filaments to attachment sites. 56 6.3.3 Time course of force development after length perturbation The time course of isometric force development probably reflects, to a certain extent, the kinetic properties of the crossbridges and the arrangement of the contractile units. Figure 15 shows the averaged isometric force (n=4) in the rising phase of force development elicited by electrical field stimulation. The closed circles are obtained from averaging isometric force traces before the length oscillation (corresponding to the contraction just before Time 1 in Figure 13); the open circles are obtained from averaging force traces after length oscillation (corresponding to the contraction just after Time 2 in Figure 13). The force traces are normalized to their respective maximal values. The onset of isometric force development is observed to be delayed by 0.20±0.01 (s) in the post-oscillation contraction, compared to that in the pre-oscillation contraction. The -0.2 s delay could be due to an oscillation-induced detachment of the "weakly attached" crossbridges that are normally present in the relaxed state (Seow and Stephens, 1987). The delay could also be due to an oscillation-induced disorganization of the contractile and/or cytoskeletal filaments, or even the extracellular matrix, which could be manifested as a decrease in the stiffness of the series elastic element of the muscle preparation. Besides the delay in the onset of force development, the maximal rate of force development also shows difference between pre-oscillation and post-57 oscillation (Figure 15). The maximal rate of force development (dP/dt) occurs early in contraction. For the pre-oscillation contraction (n = 4), dP/dt = 0.265±0.031 (P0/s), and for the post-oscillation contraction (n = 4), dP/dt = 0.324±0.043 (TVs), where P 0 is the maximal isometric force for each of the contractions. The maximal rates of force development for the pre- and post-oscillation contractions are significantly different (p<0.05). It is interesting that the rate of force development is increased after the length oscillation (Figure 15). The rate increase may be due to an increase in the cycling rate of the crossbridges, which may in turn be due to an increase in myosin light chain phosphorylation. Another explanation for the rate increase is that the number of contractile units in series is increased after the oscillation. This is because the rate of force development is partially determined by the rate of shortening of the contractile element in series with the series elastic element. The shortening velocity of the contractile element is proportional to the number of contractile units in series. If length oscillation results in de-polymerization and de-fragmentation of the thick filaments, it is possible to have shorter but more contractile units spanning the cell length, which would increase shortening velocity of the cells. In a study by Wang et al. (Wang et al., 2002), they have found that the shortening velocity is increased after a tracheal muscle has been subjected to a period of length oscillation in the relaxed state. 58 Despite a large force decrease caused by the applied length oscillation, the change in the time course of force development is relatively small. This implies that the kinetics of cross-bridge had not been altered drastically by the length oscillation. 6.4 Decrease in density of thick filaments after mechanical strain 6.4.1 Effect of length perturbation on density of thick filaments To examine the effect of length oscillation on myosin filament structural lability, muscle strips were fixed at different time points before and after a brief period of sinusoidal length oscillation and also during the time course of force recovery, as illustrated in Figure 13(page67). Figure 16(page70) shows examples of electron micrographs of cross-sections of relaxed airway smooth muscle cells fixed at Time 1 (pre-oscillation) and Time 2 (post-oscillation). The absolute densities of myosin thick filament (in the unit of filament number per pm2) for the 5 conditions are listed in Table l(page71). The thick filament density is markedly reduced after the mechanical perturbation (the density in Time 1 and Time 2 is 46.8±1.6pm" 2 and 33.8±1.6pm" 2, respectively). There is 59 no obvious change in the density of thin filaments, although accurate measurement of the thin filament density was not carried out. To examine the ultrastructural change that accompanies force change due to length oscillation, the averaged (n=4) thick filament densities for the samples fixed before and after the length oscillation (Time 1 and 2 in Figure 13,page67) and during the post-oscillation force recovery (Time 3, 4 and 5 in Figure 13) are plotted in Figure 14(page68), along with the measurements of isometric force. A close agreement between the density and force is observed. The absolute densities of myosin thick filament (pm") for the 5 conditions are listed in Table l(page71). The cross-sectional areas of the muscle cells fixed under the 5 conditions are also listed in Table 1. One-way A N O V A shows that there is no significant difference among the areas (p = 0.26). 6.4.2 Lability of thick filaments Lability of myosin thick filament in smooth muscle has been a contentious issue ever since the structure of the tissue was first investigated. As mentioned earlier, it was postulated by Shoenberg (Shoenberg, 1969) and Rice et al. (Rice et al., 1970) that myosin thick filaments of smooth muscle dissolved during relaxation and formed upon activation. Their hypothesis was based on the observation that thick filaments were more abundant when the muscle was fixed for electron microscopy either at low pH (Kelly and Rice, 1968) or in high-divalent cation concentration (Shoenberg, 1969), conditions 60 that were associated with muscle activation. In addition, thick filaments were often difficult to find when the muscles were fixed at rest (Shoenberg and Needham, 1976). However later studies clearly demonstrated the existence of thick filaments in smooth muscle in the relaxed, non-phosphorylated state (see review by Somlyo and Somlyo, 1992). More recently, structural studies on rat anococcygeus smooth muscle have provided evidence that the myosin filaments may increase in length and number during activation (Gills et al., 1988; Godfraind-De et al, 1988; Watanabe et al, 1993 and Xu et al, 1997). In these studies it was found that the myosin lability is present only in anococcygeus smooth muscle and not in guinea pig taenia coli. This tissue specificity may provide an explanation for the contradictory results in the early studies. Based on the more recent structural studies, it can be concluded that, myosin filaments are present in relaxed smooth muscle; however, in some smooth muscle (e.g., rat anococcygeus) the length and/or number of the thick filaments may increase upon stimulation of the muscle, and in others (e.g., taenia coli) the thick filaments may be stable. 61 6.4.3 Reorganization of thick filaments caused by applied length perturbation The results from this part of the thesis research show that mechanical perturbation is another factor that could affect the appearance of myosin thick filament in smooth muscle. If a smooth muscle sample is fixed immediately after dissection, mechanical manipulation associated with the dissection may cause a drastic decrease in the number of thick filaments, or even a total disappearance of the filaments. The length oscillation applied to the muscle tissue in the present study is comparable to that applied in vivo by a deep inspiration, and is relatively small compared to the stretches a tissue might be subjected to during the process of dissection. These results may explain the discrepancies found in some of the early studies where physical and physiological states of the tissues were not taken into consideration during tissue fixation. The thick filament density in a cell cross-section is proportional to the average length and also the number of the thick filaments. The change in the density observed in the present study therefore can be interpreted as a change in thick filament length, or number, or both. A contractile unit in smooth muscle consists of a thick filament plus the adjacent thin filaments. The change in the thick filament length or number therefore has a direct consequence in terms of the muscle's mechanical performance. 62 Functional evidence suggests that airway smooth muscle is able to undergo plastic rearrangement of its contractile apparatus to achieve optimal filament overlap over a large length range (Gunst et al., 1995; Pratusevich et al., 1995). It was speculated that myosin lability might facilitate this plastic restructuring (Ford et al., 1994). Our recent finding of functional evidence suggesting thick filament lengthening during isometric contraction also points to the possibility of myosin lability in trachealis (Seow et al., 2000). The present finding of an inducible myosin thick filament density change by mechanical strain provides strong support for a model of smooth muscle based on filament restructuring as the primary mechanism rather than filament sliding for accommodating large length changes. The side-polar structure of smooth muscle myosin filament described by Xu et al. (Xu et al., 1996) may represent a less stable organization (compared to the bi-polar structure of filaments in striated muscle) that confers lability on smooth muscle thick filaments. Based on the evidence we have shown (Figure 14), it is conceivable that adaptation of airway smooth muscle starts with disassembling of the contractile apparatus, which involves thick filament depolymerization and defragmentation induced by a change in cell geometry. This is then followed by reorganization of the filaments according to the new cell dimension. Repolymerization of the thick filaments then completes the adaptation process. The structural remodelling therefore allows contractile units to be added or 63 subtracted from an existing contractile apparatus so that the optimal overlap between thick and thin filaments can be maintained. This is very similar to the structural remodelling observed in skeletal muscle (Jakubiec-Puka and Carraro, 1991) although the time course of recovery in smooth muscle is much accelerated. The recovery of mechanical performance in skeletal muscle during the process of remodelling has never been characterized. Rhythmic length-perturbation of airway smooth muscle as a model of deep inspiration In healthy subjects, the frequency and depth of spontaneous deep inspiration will increase after inhalation of bronchoconstriction substances such as histamine, and these deep inspirations will also cause a prompt dilating effect in the airways (Orehek et ah, 1980). Deep inspiration has been shown to be able to attenuate bronchospasm in healthy subjects (Fredberg et al, 1997; Moore et al., 1997). The effects induced by deep inspiration involve bronchoprotection and bronchodilation. The bronchoprotective effect of deep inspiration is the reduction in bronchoconstriction resulting from the act of deep inspiration before A S M has been stimulated. The bronchodilating effect is the reduction in bronchoconstriction resulting from the act of deep inspiration after A S M has been stimulated. It has also been shown that an 64 inability of deep inspiration to decrease contractility of A S M is an important feature in asthmatic patients (Fish et al, 1981; Skloot G. et al, 1995; Moore et al, 1997). A numbers of investigators have confirmed that deep inspiration will induce both bronchoprotective and bronchodilating effects on airway (Kapsali T. et al, 2000; Scichilone N. et al, 2000). Even though the mechanism underlying deep inspiration-induced bronchoprotective effect and bronchodilating effect are still unknown, the stretch of A S M may play an important role in these effects. The theory of perturbed equilibrium of myosin binding has been proposed to explain that the bronchodilating effect of deep inspiration is related to disruption of cross-bridge dynamics (Fredberg. et al, 1999). Nevertheless, the bronchoprotective effect of deep inspiration cannot be explained by the disruption of cross-bridge dynamics because the length perturbations are imposed before A S M is stimulated (Shen X. et al, 1997; Wang L. et al, 2000). As mentioned before, the lability of myosin thick filaments is a cardinal feature of smooth muscle cells. Evidence has also shown that the organization of contractile filaments in smooth muscle cells will rearrange in response to changes in muscle lengths and affect the contractility of smooth muscle (Gunst S. J. et al, 1995; Pratusevich V. R. et al, 1995). Non-cross-bridge mechanisms have been suggested to play an role in the reduction of contractility in smooth muscle cells after length perturbations (Shen X. et al, 65 1997). In this thesis, I provide morphological evidence on the lability of thick filaments in A S M cells after length perturbations (Kuo et al, 2001). We suggest that the thick-filament lability may also play a role in the bronchoprotective effect induced by deep inspiration. 66 Figure 13 Experimental procedure Force records were made during 7 tetani produced by 12 s electrical stimulation at 5 m intervals. Averaged records from 4 experiments are plotted. The muscles were subjected to length oscillations between the first two contractions, and 5 muscle bundles from each of the 4 tracheas were fixed for electron microscopy at the 5 times indicated by the numbered arrows. Oscillation 67 Figure 14 Time course of changes in tetanic force (closed circles) and myosin thick filament density (open circles) associated with a length oscillation The reference values for force and filament density are the values obtained prior to the oscillation (Time 1 in Figure 13) and zero time is the time of onset of the first tetanus after the oscillation. Force recovery is fitted with a mono-exponential function having a rate constant of 0.234 s"1. Force and filament density points are the means of 4 values for 4 separate muscle bundles obtained from each of the tracheas. Error bars indicate S.E.M 5 i i i i i i 0 5 10 15 20 25 Time after onset of first stimulation (min) 68 Figure 15 Time course of isometric force development before and immediately after length oscillation The plots are signal averages o f four traces, one from each trachea, normalized to tetanic force at the end of stimulation. The averaged isometric force (n=4) in the rising phase of force development elicited by electrical field stimulation. The closed circles are obtained from averaging digitized isometric force traces before the length oscillation (corresponding to the contraction just before Time 1 in Figure 13); the open circles are obtained from averaging force traces after length oscillation (corresponding to the contraction just after Time 2 in Figure 13). The force traces are normalized to their respective maximal values. 0 2 4 6 8 10 12 14 Time after Stimulation (s) 69 Figure 16 Electron micrographs of cross sections of airway smooth muscle before and after length oscillation. (A) shows a preparation fixed before a length oscillation (Time 1 in Figure 13). The inset shows the area in rectangle at higher magnification. Arrows indicate myosin thick filament. A thick filament surrounded by thin filaments can be observed. (B) shows a preparation fixed immediately after a length oscillation (Time 2 in Figure 13). 70 Table 1 Myosin filament densities and cell cross-sectional areas Time 1 Time 2 Time3 Time 4 Time 5 Filament Density (filament number/um2) 46.8±1.6 32.8+1.6 36.7±1.7 43.0±2.4 46.112.2 Cross-sectional Area (pm2) 7.4±0.3 7.2±0.2 7.6±0.3 6.9±0.2 7.0+0.3 Note: The 5 time points (or conditions) at which the muscles were fixed for morphometric examination are indicated in Figure 13. The variation in filament density is significant (p<0.05, one-way ANOVA); the variation in cell cross-sectional area is not significant (p>0.05, one-way ANOVA). The tabulated values are averages from 4 muscle preparations from 4 tracheas; for each muscle preparation, 15 morphometric measurements (from 15 micrographs) were made. Means and standard errors are listed. 71 Chap.7 Adaptation of airway smooth muscle to different lengths 7.1 Background Recent studies have demonstrated that the cycle of contraction and relaxation in airway smooth muscle involves polymerization and depolymerization of actin (Jones et al., 1999, Mehta and Gunst, 1999) and myosin (Herrera et al., 2002) filaments. This lability of contractile filaments during muscle activation resembles that of non-muscle motile cells that rely on impromptu assembly of contractile apparatus for motility. Oscillatory strains applied to single airway smooth muscle cells have revealed plastic deformation similar to that observed in soft glassy materials (Fabry et al., 2001). Functional studies of airway smooth muscle indicate that the cells maintain maximal isometric force production (optimal contractile filament overlap) over a 3-fold length change by varying the number of contractile units in series (Pratusevich et al., 1995). This newly emerged "plasticity" model of smooth muscle contraction is incompatible with the concept of contraction based on the relatively restrictive model of striated muscle that possesses filament arrays in fixed lattice, which cannot accommodate large 72 changes in cell length without compromising force production (Gordon et al., 1966). The feasibility of the plasticity model can be tested experimentally based on the model predictions: the recruitment of additional contractile units in muscles adapted to longer lengths should result in an increase in thick filament content in individual muscle cells. In the chapter, we tested the plasticity hypothesis through quantitative analysis of mechanical properties and ultrastructure in airway smooth muscles adapted to different lengths. Notes on experimental procedure Swine tracheal smooth muscle was dissected into a rectangular sheet and all muscle strips used for a single experiment were dissected from one rectangular sheet; all muscle strips from one animal had the same resting length. Two muscle strips were used for each experiment. One strip was studied at the reference length ( L^f ) , which is close to the in situ length, and the other strip was studied at twice the reference length (2.0 Lref)- After at least lh of adaptation at a set length, the muscle strips were then stimulated to produce a ~120s contraction by the addition of 0.1 mM acetylcholine and fixed in the contracted state for E M examination. Acetylcholine was used for the final stimulation to ensure continued activation during fixation. 73 7.3 Mechanical properties a t differen t lengths 7.3.1 Length independence of force generation The values of isometeric force at Lref and 2.0 Lref are 161±12.3 (SE) kPa and 175±23.1 (SE) kPa, respectively (Table 2). Isometric force at 2.0 Lref was not significantly different from the value at Lref (P>0.1, Paired T test) (Figure 17). The finding is parallel to previous studies (Pretusevich et al., 1995; Wang et al., 2000; Kuo et al., 2001). 7.3.2 Length dependence of shortening velocity and power output The values of shortening velocity, normalized by Lref> at L r e f and 2.0 Lref are 0.26±0.03 (SE) L r e f / s and 0.44±0.06 (SE) Lref/s, respectively. The values of power output at L r e f and 2.0 L^f are 7.8±0.6 (SE) mW/g and 13.8±1.0 (SE) mW/g, respectively (Table 2,page92). Shortening velocity (Figure 18,page84) and power output (Figure 19,page85) at 2.0 Lref, in contrast to isometric force, increased significantly compared to the corresponding value at Lref (P<0.05, Paired T test). The result indicates that the shortening velocity is increased by about 69% for a 100% increase in muscle length. The result is similar to that in the previous studies (Pretusevich et al., 1995). Since muscle power output 74 is the product of force and velocity, the results suggest that the power output also have the same length dependence as the shortening velocity. 7.3.3 Energetic efficiency of active contractile units at different lengths What is the mechanism for the length-dependent increase in the mechanical power output of muscle? One possibility is that the increase of mechanical power output at longer length may be accomplished by increasing mechanical efficiency of active contractile units in muscles at longer length. However, data from our previous study on chemically permeabilized A S M preparation have shown that the length dependence of mechanical power output is unlikely due to a change in the rate of ATP utilization of muscle at longer lengths (Kuo et al, 2003). The rate of ATP utilization in the isometrically contracting muscle increased by 35% when the muscle length was increased by 50%. Assuming a linear relationship, a 100% increase in muscle length would result in a 70% increase in the rate of ATP utilization. The ~76%±7.2 (SE) increased mechanical power output for every 100% increase in muscle length found in this thesis research is nearly proportion to the 35% increase in ATP utilization for a 50% increase in muscle length (assuming a linear relationship). The increased power output at longer length of muscle is therefore accompanied by a matched increase in energetic 75 ' utilization. Based on these results, we suggest that the energetic efficiency of the muscle is unchanged at different lengths. 7.3.4 Possible explanation for muscle adaptation at different lengths Another possible explanation for the length-dependent increase in the shortening velocity and mechanical power output is that it is a result of variable number of contractile filaments being placed in series in muscles at different lengths (Pratusevich et al., 1995). The proposed model with variable number of contractile filaments at different cell lengths assumes that the length of thick filaments themselves is the same at different muscle lengths. The increase in thick filament content at a longer muscle length is due to more thick filaments added in series and not to the existing thick filaments getting longer. This model accounts for the present finding that isometric force is length independent because the force generation is proportional to contractile filaments in parallel and the shortening velocity is proportional to those in series. Therefore, we suggest that the number of contractile units in series increase -70% for length doubling, based on the -70% increase in shortening velocity. 76 7.4 Morphological observations at different muscle lengths 7.4.1 Volume conservation of smooth muscle cells at different lengths Electron micrographs from tissue samples are taken for image analysis (Figure 20,page86), including cell cross-sectional area and myosin filament density. The values of cell cross-sectional area at L ^ f and 2.0 L r e f are 8.5±0.7 (SE) pm 2 and 4.4±0.2 (SE) pm 2, respectively (Table 2,page92). Cell cross-sectional area at 2.0 Lref decreased -49% compared to the corresponding value at Lref (PO.05, Paired T test) (Figure 21,page87). The cell volume is the product of cell cross-sectional area and cell length. Therefore, the cell volume is nearly perfectly conserved at different lengths. 7.4.2 Density of myosin thick filaments at different lengths The values of myosin filament density at L r e f and 2.0 L r e f are 83.6±5.8 (SE) pm"2 and 142.4±10.2 (SE) pm"2, respectively (Table 2,page92). The filament density found in this chapter ( ~84 pm"2 ) is much higher then that found in Chapter 6 (-46 pm"2, see Table 1 in page71). This is because the density values listed in the previous chapter were determined in the relaxed muscles, whereas the density reported here was determined in the activated muscles. Myosin filament density at 2.0 L r e f increased significantly compared 77 to the corresponding value at L ^ f (P<0.05, Paired T test) (Figure 22,page88). The result revealed that the myosin filament density is increased by about 70% for a 100% increase in muscle length. 7.4.3 Estimated thick filament content in a whole cell at different lengths If the thick filament length is much less than the length of a cell, and also if the thick filaments are randomly distributed within the whole cell regardless of the cell length, then the total thick filament content can be estimated by the product of thick filament density, cell cross-sectional area and cell length, neglecting the tapered-ends of the muscle cell. Estimated thick filament content (the product of thick filament density, cell cross-sectional area and cell length) at 2.0 Lref increased -76% compared to the corresponding value at L r e f (P<0.05, Paired T test) (Figure 23,page89). The increased value of estimated thick filament content suggests that the thick filament number increases in a lengthened muscle, nearly identical to the increase value of power output (-76%) (Figure 24,page90). It should be noted that the increase in the thick filament number in a lengthened muscle is not associated with an increased in filaments in parallel, because of the length independence of force. Therefore, the observed increase in muscle power 78 output and shortening velocity with muscle length appear to be due to synthesis of new thick filaments and to placement of these newly formed thick filaments in series. 7.5 A proposed mechanism for smooth muscle adaptation to large length changes 7.5.1 A model with variable number of contractile units in series at different cell lengths After length adaptation, we have shown that isometric force is independent to muscle length and shortening velocity and power output are increased at longer lengths. We interpret the data as suggesting that the number of contractile units in series increases by -76% for length doubling. This interpretation is illustrated in Figure 25 (page91). In the highly simplified case, an increase in the number of contractile units from 3 to 5 as a result of length doubling should cause a -70% increase in shortening velocity and power output but no change isometric force, because the increased contractile units are added in series with the existing ones (Figure 25). The model also predicts the content of myosin thick filaments increases with adapted muscle length. Currently, the only reliable measurement available to 79 us that indirectly estimates myosin filament content is the thick filament density measured in a transverse section of a cell. The indirect measurement of myosin filament content agrees with the prediction of the model (Figure 24). If the thick filament length is much less than the length of a cell, and also if they are randomly distributed within the cell regardless of the cell length, the thick filament density in a cell cross-section should remain constant at different cell lengths provided the filament content (or mass) is length independent (i.e., no filament polymerization or depolymerization with length change). This is analogous to a bag of black and white marbles; as long as the balls are evenly mixed, the number of black marbles per unit area (density) found in any imaginary planes cutting across the bag will be the same whether the bag is stretched or squashed. The present finding that the thick filament density increased in muscles adapted at longer length suggests filamentogenesis. Furthermore, the filament density in a cross-section should reflect filament content of a cell provided that the cell volume is conserved. (Use the same analogy above, the number of black marbles found per unit area in a cross-section is directly correlated to the total number (or content) of black marbles in the bag). The inverse relationship between cell cross-sectional area and cell length (Figure 24 and Table 2) indicates that the cell volume was the same within the lengths studied. The -70% increase in thick 80 filament density found at 100% length increase therefore closely fits the model prediction (Figure 25). The present study therefore provides the first ever evidence that myosin filament content in airway smooth muscle is regulated by muscle length. How is it regulated is unknown. It could be speculated that cytoskeletal deformation may lead to activation of integrin-linked kinase that in turn promotes myosin light chain phosphorylation (Deng et al., 2001) and thick filament formation (Qi et al., 2002). The observation that there is a large increase in the thick filament density (in a matter of seconds) when airway smooth muscle is activated (Herrera et al, 2002) suggests that there is a large pool of monomeric myosin in the trachealis cells. The model shown in Figure 25 assumes that the length of thick filaments is the same at different cell lengths, and the increase in thick filament content at longer muscle length is due to more thick filaments added in series, and not due to the existing thick filaments getting longer, to account for the finding that isometric force is length independent. Another model that predicts the same amount of increase in filament content and length-independence of isometric force allows the thick filament length to increase in inverse proportion to the number of thick filaments in parallel (to keep isometric force constant). This model also has to allow the number of thick filaments in series to vary with muscle length in order to account for the length-dependent shortening velocity. Results presented in this thesis do not allow us to favor 81 one model over the other. The model presented in Fig. 26 however is simpler in the sense that it does not rely on a potentially complicated mechanism that adjusts the thick filament length and the number of filaments in parallel precisely according to the cell length. 7.5.2 Plastic adaptation of smooth muscle and its clinical implication The need for plastic adaptation is quite obvious in smooth muscles lining the wall of hollow organs that undergo large volume changes. It is not clear why such plasticity exists in airway smooth muscle. It is clear, however, that there are a variety of in vivo conditions under which airway smooth muscle could be adapted to pathologically short lengths, especially when inflammation of the airways is involved: frequent and prolonged stimulation . of the muscle, adventitial edema of the airway wall that decreases resting muscle length, reduced elastic tethering of lung parenchyma on the airway wall (due to chronic inflammation) that diminishes resting muscle length, to name just a few (King et al., 1999). Elucidating the mechanism of airway smooth muscle plasticity therefore will, in addition to gaining insights into the basic mechanism of contraction, shed light on the pathophysiology of exaggerated airway narrowing seen in diseases such as asthma and emphysema. 82 Figure 17 Change of force generation at different lengths 1.4 r 1.2 -5 1 -g 0.8 -o 0.6 -0.2 h 0 1 1 1 1 1 Lref 2.0 Lref 83 Figure 18 Change of shortening velocity at different lengths Lref 2.0 Lref 84 Figure 19 Change of power output at different lengths I o Q_ a> o a. Lref 2.0 Lref 85 Figure 20 Electron micrographs of cross sections of airway smooth muscle at L r e f and 2.0 Lref-Examples of electron micrographs from tissue samples fixed under 2 experimental conditions: (A) Contracted at L^f. (B) Contracted at 2.0 L^f. Arrows indicate myosin thick filaments. Calibration bar: 1 pm. 86 Figure 21 Change of cell cross-sectional area at different lengths CO co 2 o "55 O jj s @ < s 1.2 c o 0.8 0.6 u 0) CO o co £ •2 0.4 CO 2 o "55 o CO 0.2 0 Lref 2.0 Lref 87 Figure 22 Change of filament density at different lengths Lref 2.0 Lref 88 Figure 23 Change of estimated thick filament content in whole cell at different lengths 2.5 a) re E .» % 2 LU 1.5 g @ c c o £ « c c ° E c 1 ns <D £ E •o JS i> «= 0.5 ns E W LU 0 Lref 2.0 Lref 89 Figure 24 Pooled values of isometric force, shortening velocity, power output, myosin filament density and estimated filament content Isometric force (F), shortening velocity (V), power output (P), myosin filament density (D) and estimated filament content (C) measured at 2.OxLref normalized by the corresponding values at Lref. Asterisks denote statistical difference from reference (p<0.05, paired T-test). 2.5 3 ns > 2 _ i o c\i © 3 > 1.5 0.5 0 V D 90 Figure 25 Proposed model with variable contractile units in series at different lengths 91 Table 2 Force, velocity, power and myosin filament density of intact airway smooth bundle at different lengths. Isometric Maximum Maximum Muscle Cell Myosin Filament Force Velocity Power Cross-sectional Density (kPa) (Lref/S) (mW/g) Area (filament number (pm2) /pm2) Lref 161±12.3 0.26±0.03 7.8±0.6 8.5±0.7 83.6±5.8 2.0 175±23.1 0.44±0.06* 13.8±1.0* 4.4±0.2* 142.4±10.2* Note: The muscle cell cross-sectional area listed is the total area and not the cytoplasmic area used to calculate the filament density. Means and standard error are listed. Asterisks (*) denote statistically significant difference form appropriate reference values (Paired T-test, p<0.05). 92 Chap.8 Conclusion Results from this thesis research have allowed us to construct a model for airway smooth muscle that explains how the tissue adapts to its external environment and maintains optimal contractile function. Morphological data suggest that airway smooth muscle cells function as a mechanical syncytium (Chapter 5). The parallel arrangement of the contractile filaments is maintained in a bundle of cells that are spindle-shaped and possess centrally-located nuclei; this is possible due to attachment of contractile filaments to dense plaques on the plasma membrane and the relay of force to the adjacent dense plaques on neighboring cells. Direct attachment of contractile filaments to the nuclear envelop has also contributed to the maintenance of parallel arrangement of the contractile filaments in the cell. Functional and morphological data demonstrated the labile nature of the myosin filaments in airway smooth muscle (Chapter 6). This lability is believed to be one of the normal properties of myosin thick filaments of airway smooth muscle and is thought to be the key that allows the muscle to plastically restructure its contractile apparatus according to the demands imposed by the external forces. The mechanical-strain-induced thick filament lability and the consequent reduction in force generation are interpreted as a key mechanism underlying the deep-inspiration associated Bronchodilation. The length-dependent behavior of airway smooth muscle is explained by a model where the number 93 of contractile units varies with the muscle length (Chapter 7). This model relies on key assumptions that the thick filaments are labile and that the contractile apparatus of airway smooth muscle is malleable. Functional and morphological data presented in Chapter 7 strongly supported these assumptions. The notion that airway smooth muscle responds to contractile stimulation with an impromptu assembly of contractile apparatus represents a drastic deviation from the theory that describes striated muscle contraction. The new perspective on how airway smooth muscle contracts that stems from this thesis research hopefully will inspire more studies that will lead to a new level of understanding of smooth muscle in general, airway smooth muscle in particular. 94 Reference Antonissen L A , Mitchell RW, Kroeger EA, Kepron W, Tse KS, Stephens N L (1979) Mechanical alterations of airway smooth muscle in a canine asthmatic model. J Appl Physiol 46: 681-687 Arner A, Hellstrand P (1983) Activation of contraction and ATPase activity in intact and chemically skinned smooth muscle of rat portal vein. Dependence on Ca++ and muscle length. CircRes 53: 695-702 Ashton FT, Somlyo A V , Somlyo AP (1975) The contractile apparatus of vascular smooth muscle: intermediate high voltage stereo electron microscopy. J Mol Biol 98: 17-29 Bagby R M , Young A M , Dotson RS, Fisher BA, McKinnon K (1971) Contraction of single smooth muscle cells from Bufo marinus stomach. Nature 234: 351-352 Bagby R M (1983) Organization of contractile/cytoskeletal elements. Biochemistery of Smooth Muscle, Edited by NL Stephens CRC Press Inc ,Boca Raton.Florida 1-84 Bohr DF, Sitrin M (1970) Regulation of vascular smooth muscle contraction. Changes in experimental hypertension. CircRes 27: Suppl-90 Boushey HA, Holtzman MJ, Sheller JR, Nadel JA (1980) Bronchial hyperreactivity. Am RevRespirDis 121: 389-413 95 Brown RH, Georgakopoulos J, Mitzner W (1998) Individual canine airways responsiveness to aerosol histamine and methacholine in vivo. Am JRespir Crit Care Med 157:491-497 Brown RH, Mitzner W (1998) The myth of maximal airway responsiveness in vivo. J Appl Physiol 85: 2012-2017 Brunstock G, Prosser C L (1960) Conduction in smooth muscle: comparative electrical properties. Am J Physiol 199: 553-559 Brunstock G (1970) Structure of smooth muscle and its innervation. Smooth Muscle, Edited by E Bulbring, A F Brading, A W Jones and T Tomita, Edward Arnold Ltd, London pp. 1-69 Burke B, Mounkes L C , Stewart C L (2001) The nuclear envelope in muscular dystrophy and cardiovascular diseases. Traffic 2: 675-683 Burke B, Stewart C L (2002) Life at the edge: the nuclear envelope and human disease. Nat Rev Mol Cell Biol 3: 575-585 Cooke P (1976) A filamentous cytoskeleton in vertebrate smooth muscle fibers. J Cell Biol 68: 539-556 Cooke PH, Fay FS (1972) Correlation between fiber length, ultrastructure, and the length-tension relationship of mammalian smooth muscle. J Cell Biol 52: 105-116 Cooke PH, Fay FS (1972) Thick myofilaments in contracted and relaxed mammalian smooth muscle cells. Exp Cell Res 71: 265-272 96 De Jongste JC, Mons H, Block R, Bonta IL, Frederiksz AP, Kerrebijn KF (1987) Increased in vitro histamine responses in human small airways smooth muscle from patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 135: 549-553 De Jongste JC, Jongejan RC, Kerrebijn KF (1991) Control of airway caliber by autonomic nerves in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 143: 1421-1426 De Jongste JC (1996) Impact of treatment on bronchial hyperresponsiveness. Pediatr Allergy Immunol 7: 18-24 Deng JT, Van Lierop JE, Sutherland C, Walsh MP (2001) Ca2+-independent smooth muscle contraction, a novel function for integrin-linked kinase. J Biol Chem 276: 16365-16373 Devine CE, Somlyo AP (1971) Thick filaments in vascular smooth muscle. J Cell Biol 49: 636-649 Djabali K (1999) Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery. Histol Histopathol 14: 501-509 Ebina M , Yaegashi H, Chiba R, Takahashi T, Motomiya M , Tanemura M (1990) Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 141: 1327-1332 Fabry B, Maksym GN, Butler JP, Glogauer M,fNavajas.D, Fredberg JJ (2001) Scaling the microrheology of living cells. Phys Rev Lett 87: 148102 Farkas GA, Roussos C (1983) Diaphragm in emphysematous hamsters: sarcomere adaptability. J Appl Physiol 54: 1635-1640 97 Fay FS, Delise C M (1973) Contraction of isolated smooth-muscle cells—structural changes. Proc Natl Acad Sci USA 70: 641-645 Fish JE, Ankin M G , Kelly JF, Peterman VI (1981) Regulation of bronchomotor tone by lung inflation in asthmatic and nonasthmatic subjects. J Appl Physiol 50: 1079-1086 Fisher BA, Bagby R M (1977) Reorientation of myofilaments during contraction of a vertebrate smooth muscle. Am J Physiol 232: C5-14 Flier JS (2000) Pushing the envelope on lipodystrophy. Nat Genet 24: 103-104 Ford L E , Seow CY, Pratusevich V R (1994) Plasticity in smooth muscle, a hypothesis. Can J Physiol Pharmacol 72: 1320-1324 Fredberg JJ, Inouye D, Miller B, Nathan M , Jafari S, Raboudi SH, Butler JP, Shore SA (1997) Airway smooth muscle, tidal stretches, and dynamically determined contractile states. Am J Respir Crit Care Med 156: 1752-1759 Fredberg JJ, Inouye DS, Mijailovich SM, Butler JP (1999) Perturbed equilibrium of myosin binding in airway smooth muscle and its implications in bronchospasm. Am J Respir Crit Care Med 159: 959-967 Fung Y C (1993) Biomechanics:Mechanical properties fo liveing tissues. Second Edition. Springer,New York Gillis JM, CaoML, Godfraind-De Becker A (1988) Density of myosin filaments in the rat anococcygeus muscle, at rest and in contraction. II. J Muscle Res Cell Motil 9: 18-29 98 Godfraind-De Becker A, Gillis JM (1988) Analysis of the birefringence of the smooth muscle anococcygeus of the rat, at rest and in contraction. I. J Muscle Res Cell Motil 9: 9-17 Gordon A M , Huxley AF, Julian FJ (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol 184: 170-192 Guilford WH, Warshaw D M (1998) The molecular mechanics of smooth muscle myosin. Comp Biochem Physiol B Biochem Mol Biol 119: 451-458 Gunst SJ, Meiss RA, Wu MF, Rowe M (1995) Mechanisms for the mechanical plasticity of tracheal smooth muscle. Am J Physiol 268: C1267-C1276 Hargreave FE, Ryan G, Thomson NC, O'Byrne PM, Latimer K, Juniper EF, Dolovich J (1981) Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance. J Allergy Clin Immunol 68: 347-355 Heidenhain M (1900) Struktur der kontraktilen Materie. Ergeb Anat Entwick Herrera A M , Kuo K H , Seow C Y (2002) Influence of calcium on myosin thick filament formation in intact airway smooth muscle. Am J Physiol Cell Physiol 282: C310-C316 Hodgkinson JL, Newman T M , Marston SB, Severs NJ (1995) The structure of the contractile apparatus in ultrarapidly frozen smooth muscle: freeze-fracture, deep-etch, and freeze-substitution studies. J Struct Biol 114: 93-104 Hodgkinson JL (2000) Actin and the smooth muscle regulatory proteins: a structural perspective. J Muscle Res Cell Motil 21: 115-130 Huxley A F (1980) The muscular dystrophies. Future prospects. Br Med Bull 36: 199-200 99 Huxley HE, Hanson J (1954) Changes in the cross-striation of muscle during contraction and stretch and their structural interpretation. Nature 173: 973-976 Huxley HE (1957) The double array of filaments in cross-striated muscle. J Biophys Biochem Cyto 3: 631-647 Ishii N, Takahashi K (1982) Length-tension relation of single smooth muscle cells isolated from the pedal retractor muscle of Mytilus edulis. J Muscle Res Cell Motil 3: 25-38 Jakubiec-Puka A, Carraro U (1991) Remodelling of the contractile apparatus of striated muscle stimulated electrically in a shortened position. JAnat 178: 83-100 James A L , Pare PD, Hogg JC (1989) The mechanics of airway narrowing in asthma. Am Rev Respir Dis 139: 242-246 Jeffery PK, Gaillard D, Moret S (1992) Human airway secretory cells during development and in mature airway epithelium. Eur Respir J5: 93-104 Jiang H, Rao K, Halayko AJ, Kepron W, Stephens N L (1992) Bronchial smooth muscle mechanics of a canine model of allergic airway hyperresponsiveness. J Appl Physiol 72: 39-45 Jones AW, Somlyo AP, Somlyo A V (1973) Potassium accumulation in smooth muscle and associated ultrastructural changes. J Physiol 232: 247-273 Jones K A , Perkins WJ, Lorenz RR, Prakash YS, Sieck GC, Warner DO (1999) F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs. J Physiol 519 Pt 2: 527-538 100 Kapsali T, Permutt S, Laube B, Scichilone N, Togias A (2000) Potent bronchoprotective effect of deep inspiration and its absence in asthma. J Appl Physiol 89: 711-720 Kolliker A (1849) Beitrage zur Kentniss der glatten Muskeln. Z Wiss Zool 1: 48-87 Kuo K H , Wang L, Pare PD, Ford L E , Seow C Y (2001) Myosin thick filament lability induced by mechanical strain in airway smooth muscle. J Appl Physiol 90: 1811-1816 Kuo K H , Herrera A M , Wang L, Pare PD, Ford L E , Stephens NL, Seow C Y (2003) Structure-function correlation in airway smooth muscle adapted to different lengths. Am J Physiol Cell Physiol 285: C384-90 Kuo K H , Herrera A M , Seow C (2003) Ultrastructure of airway smooth muscle. Respir Physiolo Neurobiol Lambert R K (1991) Role of bronchial basement membrane in airway collapse. J Appl Physiol 71: 666-673 Lambert RK, Wiggs BR, Kuwano K, Hogg JC, Pare PD (1993) Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 74: 2771-2781 Lee CH, Poburko D, Kuo K H , Seow CY, van Breemen C (2002) Ca(2+) oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Circ Physiol 282:H1571-H1583 Lee RT, Yamamoto C, Feng Y, Potter-Perigo S, Briggs WH, Landschulz K T , Turi TG, Thompson JF, Libby P, Wight T N (2001) Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells. J Biol Chem276: 13847-13851 101 Martin C, Uhlig S, Ullrich V (1996) Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices. Eur Respir J 9: 2479-2487 McGill C (1909) The sturcture of smooth muscle in the resting and in the contracted condition. Am JAnat 9: 493-545 Mehta D, Gunst SJ (1999) Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J Physiol 519 Pt 3: 829-840 Moir RD, Sparm TP, Goldman RD (1995) The dynamic properties and possible functions of nuclear lamins. IntRev Cytol 162B: 141-182 Moore BJ, Verburgt L M , King GG, Pare PD (1997) The effect of deep inspiration on methacholine dose-response curves in normal subjects. Am J Respir Crit Care Med 156: 1278-1281 Moreno RH, Hogg JC, Pare PD (1986) Mechanics of airway narrowing. Am Rev Respir Dis 133: 1171-1180 Morris GE (2001) The role of the nuclear envelope in Emery-Dreifuss muscular dystrophy. Trends Mol Med 1: 572-577 Murphy T M , Mitchell RW, Phillips IJ, Leff AR (1991) Ontogenic expression of acetylcholinesterase activity in trachealis of young swine. Am J Physiol 261: L322-L326 Opazo Saez A M , Seow CY, Pare PD (2000) Peripheral airway smooth muscle mechanics in obstructive airways disease. Am J Respir Crit Care Med 161: 910-917 102 Orehek J, Charpin D, Velardocchio JM, Grimaud C (1980) Bronchomotor effect of bronchoconstriction-induced deep inspirations in asthmatics. Am Rev Respir Dis 121: 297-305 Paul RJ, Peterson JW (1975) Relation between length, isometric force, and 02 consumption rate in vascular smooth muscle. Am J Physiol 228: 915-922 Pease DC, Molinari S (1960) Electron microscopy of muscular arteries: pial vessels of the cat and monkey. J Ultrastruct Res 3: 447-468 Persson CG, Erjefalt I, Gustafsson B, Luts A (1990) Subepithelial hydrostatic pressure may regulate plasma exudation across the mucosa. Int Arch Allergy Appl Immunol 92: 148-153 Pratusevich VR, Seow CY, Ford L E (1995) Plasticity in canine airway smooth muscle. J Gen Physiol 105: 73-94 Qi D, Mitchell RW, Burdyga T, Ford L E , Kuo K H , Seow C Y (2002) Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation. Am J Physiol Cell Physiol 282: C1298-C1305 Rembold C M , Murphy R A (1990) Muscle length, shortening, myoplasmic [Ca2+], and activation of arterial smooth muscle. Circ Res 66: 1354-1361 Rice RV, Moses JA, McManus G M , Brady A C , Blasik L M (1970) The organization of contractile filaments in a mammalian smooth muscle. J Cell Biol Al: 183-196 Riley DE, Keller JM (1978) The ultrastructure of non-membranous nuclear ghosts. J Cell Sci 32: 249-268 103 Schollmeyer JE, Goll DE, Robson R M , Stromer M H (1973) Localization of alpha-actinin and tropomyosin in different muscles. J Cell Biol 59: 306a(Abstr.) Scichilone N, Kapsali T, Permutt S, Togias A (2000) Deep inspiration-induced bronchoprotection is stronger than bronchodilation. Am J Respir Crit Care Med 162: 910-916 Seidel JC (1969) The effects of monovalent and divalent cations on the ATPase activity of myosin. Biochim Biophys Acta 189: 162-170 Seow CY, Stephens N L (1987) Time dependence of series elasticity in tracheal smooth muscle. J Appl Physiol 62: 1556-1561 Seow CY, Ford L E (1993) High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state. J Gen Physiol 101: 487-511 Seow CY, Schellenberg RR, Pare PD (1998) Structural and functional changes in the airway smooth muscle of asthmatic subjects. Am J Respir Crit Care Med 158: S179-S186 Seow CY, Wang L, Pare PD (2000) Airway narrowing and internal structural constraints. J Appl Physiol 88: 527-533 Seow C Y (2000) Response of arterial smooth muscle to length perturbation. J Appl Physiol 89: 2065-2072 Seow CY, Pratusevich VR, Ford L E (2000) Series-to-parallel transition in the filament lattice of airway smooth muscle. J Appl Physiol 89: 869-876 Seow CY, Fredberg JJ (2001) Historical perspective on airway smooth muscle: the saga of a frustrated cell. J Appl Physiol 91: 938-952 104 Shen X, Wu MF, Tepper RS, Gunst SJ (1997) Mechanisms for the mechanical response of airway smooth muscle to length oscillation. J Appl Physiol 83: 731-738 Shoenberg CF (1969) A study of myosin filaments in extracts and homogenates of vertebrate smooth muscle. Angiologica 6: 233-246 Shoenberg CF, Goodford PJ, Wolowyk MW, Wootton GS (1973) Ionic changes during smooth muscle fixation for electron microscopy. JMechanochem Cell Motil 2: 69-82 Shoenberg CF, Needham D M (1976) A study of the mechanism of contraction in vertebrate smooth muscle. Biol Rev Camb Philos Soc 51: 53-104 Skloot G, Permutt S, Togias A (1995) Airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration. J Clin Invest 96: 2393-2403 Small JV (1977) Studies on isolated smooth muscle cells: The contractile apparatus. J Cell Sci 24: 327-349 Small JV (1985) Geometry of actin-membrane attachments in the smooth muscle cell: the localisations of vinculin and alpha-actinin. EMBOJ4: 45-49 Smith PG, Roy C, Fisher S, Huang QQ, Brozovich F (2000) Selected contribution: mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells. J Appl Physiol 89: 2092-2098 Somlyo A V , Somlyo AP (1993) Intracellular signaling in vascular smooth muscle. Adv Exp Med Biol 346: 31-38 Spector S, Fleisch JH, Maling H M , Brodie BB (1969) Vascular smooth muscle reactivity in normotensive and hypertensive rats. Science 166: 1300-1301 105 Stephens NL, Kong SK, Seow C Y (1988) Mechanisms of increased shortening of sensitized airway smooth muscle. Prog Clin Biol Res 263: 231-254 Thomson RJ, Bramley A M , Schellenberg RR (1996) Airway muscle stereology: implications for increased shortening in asthma. Am J Respir Crit Care Med 154: 749-757 Uvelius B (1976) Isometric and isotonic length-tension relations and variaitonsin cell length in longitudinal smooth muscel from rabbit urinary bladder. Acta Physiol Scand 97: 1-12 Vigouroux C, Auclair M , Dubosclard E, Pouchelet M , Capeau J, Courvalin JC, Buendia B (2001) Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin AJC gene. J Cell Sci 114: 4459-4468 Wang L, Pare PD, Seow C Y (2000) Effects of length oscillation on the subsequent force development in swine tracheal smooth muscle. J Appl Physiol 88: 2246-2250 Wang L, Pare PD, Seow C Y (2002) Changes in force-velocity properties of trachealis due to oscillatory strains. J Appl Physiol 92: 1865-1872 Warshaw D M , Fay FS (1983) Cross-bridge elasticity in single smooth muscle cells. J Gen Physiol 82: 157-199 Watanabe M , Takemori S, Yagi N (1993) X-ray diffraction study on mammalian visceral smooth muscles in resting and activated states. J Muscle Res Cell Motil 14: 469-475 Wiggs BR, Moreno R, Hogg JC , Hilliam C, Pare PD (1990) A model of the mechanics of airway narrowing. J Appl Physiol 69: 849-860 106 Wiggs BR, Bosken C, Pare PD, James A, Hogg JC (1992) A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 145: 1251-1258 Wilson K L (2000) The nuclear envelope, muscular dystrophy and gene expression. Trends Cell Biol 10: 125-129 Wingard CJ, Browne A K , Murphy RA (1995) Dependence of force on length at constant cross-bridge phosphorylation in the swine carotid media. J Physiol 488 ( Pt 3): 729-739 Woolcock AJ (1989) Epidemiology of chronic airways disease. Chest 96: 302S-306S Woolcock AJ, Peat JK (1989) Epidemiology of bronchial hyperresponsiveness. Clin Rev Allergy 7: 245-256 Xu JQ, Harder BA, Uman P, Craig R (1996) Myosin filament structure in vertebrate smooth muscle. J Cell Biol 134: 53-66 Xu JQ, Gillis JM, Craig R (1997) Polymerization of myosin on activation of rat anococcygeus smooth muscle. J Muscle Res Cell Motil 18: 381-393 107 

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