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Asymmetrical length adaptation in airway smooth muscle : possible mechanisms Ali, Farah 2006

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Asymmetrical Length Adaptation in A i r w a y Smooth M u s c l e : Possible Mechanisms by FARAH ALI B . S c , M c G i l l University, 2002  A THESIS SUBMITTED I N P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE  in THE F A C U L T Y OF G R A D U A T E STUDIES (EXPERIMENTAL MEDICINE)  THE UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Farah A l i , 2006  Abstract  Airway smooth muscle ( A S M ) regulates flow resistance in the airways of the lung. Dysfunction of the smooth muscle is implicated in the exaggerated airway narrowing seen in asthma, possibly due to adaptation of A S M to excessively short lengths. In this study, we examined the behaviour of A S M in its adaptation to large changes in cell length and the underlying mechanisms for its ability to adapt and regain optimal contractility.  Isometric force measured immediately after a length change revealed  that the amount of decrease in force after the length change was very sensitive to the direction of the length change (more sensitive in release than in stretch), and relatively insensitive to the absolute lengths from/to which the muscle was stretched or released. Force decreased by 20.4+0.9 (%) when muscle length was doubled from an arbitrarily chosen reference length (L f, at which the muscle had been adapted); in the reversed re  direction with a halving of 2xL f, the decrease in force was 48.0±2.3 (%). re  Quantification of myosin filament density by electron microscopy revealed a similar asymmetry; a length increase from  L f r e  to 1.6xL f resulted in no significant decrease re  in filament density, but a length decrease from 1.6xL f to re  L f r e  left only 81.0±3.3 (%)  of the filaments intact (P<0.05). Velocity measurements after step changes in length revealed that velocity was proportional to muscle length, and the change in velocity was almost instantaneous after the length change (without full adaptation). We have developed a model to explain all the above results. It appears that length change leads to an immediate reconfiguration of the actin filament lattice so that the number of contractile units (appropriate to length) can be formed.  Formation of myosin  filaments within the actin filament lattice appears to be a separate process, which requires a longer time and tends to influence force and not velocity.  ii  Table of contents Page  Abstract  »  Table of Contents  i»  List of Figures  v  List of Abbreviations  vii  Acknowledgements  v  >'>  CHAPTER 1. Introduction 1.1  General introduction to airway smooth muscle  1  1.2  Cell structure of airway smooth muscle  2  1.3  Smooth muscle "sarcomeres"  4  1.4  Smooth muscle mechanics  7  1.5  Smooth muscle length adaptation  9  1.6  Mechanism of length adaptation  12  1.7  Length adaptation and disease  17  1.8  Hypotheses and specific aims  19  CHAPTER 2. Materials and Methods 2.1  Muscle strip preparation  23  2.2  Equilibration  24  2.3  Apparatus  26  2.4  Brief overview of experiment procedure  27  2.5  Change in force after quick-stretch or quick-release  30  iii  2.6  Change in myosin filament density after quick-stretch or quick-release..  32  2.7  Electron microscopy  33  2.8  Morphometric analysis  35  2.9  Change in velocity after quick-stretch or quick-release  36  2.10 Statistical Analysis  38  C H A P T E R 3. Results 3.1  Change in force after quick-stretch or quick-release  39  3.2  Change in thick filament density after quick-stretch or quick-release....  44  3.3  Change in velocity of shortening after quick-stretch or quick-release...  51  3.4  Summary of results for a 60% length change  56  C H A P T E R 4. Discussion 4.1  Result summary  57  4.2  Effects of quick-stretch and quick-release on force development  58  4.3  Effects of quick-stretch and quick-release on myosin filament density..  62  4.4  Effects of quick-stretch and quick-release on velocity of shortening  64  4.5  The intermediate state after quick-stretch or quick-release  67  4.6  Alternative mechanisms  72  4.7  Physiological relevance  73  References  75  iv  List of figures  Page Figure 1-1. Electron micrograph of a transverse section of a sheep tracheal smooth muscle cell  3  Figure 1-2. Muscle contractile units  6  Figure 1-3. Length-tension curves after length adaptation  11  Figure 1-4. Contractile unit rearrangement in response to length adaptation of a two-fold length change  16  Figure 1-5. Hypothesized model of immediate effects of length change on smooth muscle  20  Figure 2-1. Experimental protocol: change in force after quick-stretch or quick-release  29  Figure 2-2. Experimental protocol: change in myosin fdament density after quick-stretch or quick-release  29  Figure 2-3. Experimental protocol: change in velocity after quick-stretch or quick-release  29  Figure 3-1. Change in force (kPa) upon quick-stretch or quick-release Figure 3-2. Relative change in force (%F  max  41  ) upon quick-stretch or  quick-release Figure 3-3. Quick-stretch vs. quick-release  42 43  Figure 3-4. Electron micrograph of a sheep trachealis muscle cell in transverse section, and experimental data Figure 3-5. Magnified portion of previous image  46 47  Figure 3-6. Examples of electron micrographs from all experimental conditions  48  Figure 3-7. Thick filament density of four conditions sub-grouped by location along long axis of cell  49  Figure 3-8. Fractional change in force or thick filament density upon quick-stretch or quick-release  50  Figure 3-9. Velocity of shortening after quick-stretch and quick-release  54  Figure 3-10. Shortening velocities before length change, immediately after length change, and after adaptation to new length  55  Figure 3-11. Summary of results from quick-stretch or quick-release between L f and 1.6 L f r e  56  r e  Figure 4-1. Partial adaptation during activation post-QR  61  Figure 4-2. A proposed model to explain the rearrangement of contractile units in series in response to length change Figure 4-3. Modified model of structural changes after length change  66 71  Figure 4-4. Structural and functional outcomes at three states after a change in length from 1.5  71  L f r e  vi  List of abbreviations  Ach  Acetylcholine  ATP  Adenosine triphosphate  ASM  Airway smooth muscle  COPD  Chronic Obstructive Pulmonary Disease  ECM  Extracellular matrix  EFS  Electrical field stimulation  EM  Electronic microscopy  Fmax  Maximal force  F-V  Force-velocity  kPa  Kilopascal  L-T  Length-tension  Lref  Reference length .  Ljn situ  In situ length  MLCK  -  P PSS  Myosin light chain kinase P value  . -  Physiological saline solution  OR  Quick-release  QS  Quick-stretch  SE  Standard error of mean  SR  Sarcoplasmic reticulum  V  Volts  vii  Acknowledgements  In making my way through this degree, I owe a huge debt of gratitude to my supervisors. Dr. Chun Seow and Dr. Peter Pare were patient and effective teachers, who inspired me in science as well as in life. I would also like to thank my supervisory committee, Dr. Bob Schellenberg and Dr. Tom Podor, for lending me their time and knowledge, and for being helpful whenever I asked.  M y lab colleagues facilitated my experiments while spreading good humour and sharing their vast experiences. Gratitude is especially due Dennis Solomon and Leslie Chin for helping with the experiments in this dissertation. I also greatly appreciated the assistance of Dr. Apolinary Sobieszek who tried to help with my skinning experiments, alas, to no avail.  I have found all the members of the James Hogg iCapture Centre to be kind and generous with their knowledge and skills. I would especially like to thank Fanny Chu and Erin Tranfield for helping me get along with "Brutus," the transmission electron microscope, and Caroline Cheung and Hon Leong for teaching me the iCapture ways.  Finally, I would like to acknowledge my family and friends for supporting me, mind, body, and spirit, and for keeping me giggling.  viii  CHAPTER 1. Introduction  1.1 General introduction to airway smooth muscle  Vertebrates have two types o f muscle, striated and smooth, the function o f which is to generate force or motion. Striated muscle comprises skeletal muscle which is controlled by the somatic nervous system, and cardiac muscle which makes up the bulk o f the heart. Smooth muscle is controlled by the autonomic nervous system and is usually found lining the lumen o f hollow organs.  Examples o f these are the urinary bladder, stomach,  gastrointestinal tract, uterus, iris o f the eye, airways and blood vessels. Smooth muscle regulates the dynamic size and shape of the organ with which it is associated, thus dysfunction in smooth muscle contractility has been implicated in a variety o f conditions such as hypertension, pre-term labour and diseases of exaggerated airway narrowing, such as seen in asthma and C O P D .  The focus of this report is on airway smooth muscle ( A S M ) which controls the caliber o f the trachea and airways o f the lungs to regulate airflow. In mammalian trachea, smooth muscle bridges the dorsal side o f the C-shaped cartilaginous rings that run in transverse orientation to the airway length. Further down the airways, smooth muscle surrounds the entire lumen and in 4  th  to 7  th  generation bronchi, A S M bundles become smaller and  irregularly oriented (Daniel et al., 1986).  1  1.2 Cell structure of airway smooth muscle  A i r w a y smooth muscle cells are spindle-shaped, about 250pm long and 3-5pm wide at the centre o f the cell (Stephens, 2001). A cigar-shaped nucleus is located at the centre o f the cell and organelles such as mitochondria, the Golgi apparatus and sarcoplasmic reticulum (SR) tend to cluster near the poles o f the nucleus. The entire smooth muscle cell membrane (or sarcolemma) contains mutually exclusive areas o f caveolae and dense plaques (Fig. 1-1).  Caveolae are surface invaginations which greatly increase the cell  surface area, allowing molecules, ions and liquid to rapidly pass from extracellular to intracellular space (Stephens, 2001). Intimately associated with SR, they may provide a conduit for calcium transport within a cell (Kuo et al., 2003a).  Dense plaques are similar to dense bodies, but are located along the sarcolemma as opposed to throughout the cell cytoplasm. Both are electron dense areas that serve as anchoring sites for actin and intermediate filaments (Kuo et al., 2003a; Small and Gimona, 1998). Dense plaques paired between adjacent cells create a desmosome-like intermediate junction, or a "hemidesmosome" i f a single dense plaque is connected to the extracellular matrix ( E C M ) .  Hemidesmosomes are suggested to provide mechanical  coupling between myofilaments and the E C M (Stephens, 2001), and to have a role in mechanotransduction (Gunst et al., 2003; Zhang and Gunst, 2006).  2  Fig. 1-1.  Electron micrograph of a transverse section of a sheep tracheal smooth  muscle cell. Nucleus (N), dense plaque (DP), dense body (DB), caveoli (C) are shown, along with an intermediate junction that is squared off. Scale bar represents 0.5um.  3  1.3 Smooth muscle "sarcomeres"  A contractile unit (or sarcomere in striated muscle) is the basic functional unit o f muscle. It generates force and turns microscopic motion into macroscopic shortening. In striated muscle, sarcomeres consist o f thick filaments made of myosin, thin filaments made predominantly o f actin, and thin fdament anchoring structures called Z-disks (Fig. 1 -2a) (Gordon et al., 1966). Contractile filaments are arranged in parallel to the long axis o f a muscle bundle and in a repetitive fashion that gives striated muscle its "striated" appearance. Force and motion are generated by the ATP-dependent cyclical action o f cross-bridges that form between actin filaments and the head region o f myosin in nearby filaments, causing the thick and thin fdaments to slide past each other (Huxley, 1957).  While the sliding filament theory o f contraction is thought to be the mechanism for smooth muscle as well (Guilford and Warshaw, 1998), the exact structure of a contractile unit is as yet unknown. N o doubt, this understanding has been delayed by the "smoothness" or lack o f pattern in smooth muscle viewed under the microscope.  Therefore  information on smooth muscle contractile units has required a backward approach, using functional data and theoretical modeling to infer structure.  Herrera et al. (2005) have recently refined a model o f a smooth muscle "sarcomere" (Fig l-2b). In this model, thick filaments do not show the central bare zone characteristic of bipolar thick filaments in striated muscle. They are suggested instead to be row-polar or side-polar (Cooke et al., 1987; Hinssen et al., 1978; Small, 1977).  4  Z-disks are not  present in smooth muscle but are suggested to be replaced by dense bands or bodies which, like Z-disks, are composed mainly o f a-actinin (Small and Gimona, 1998; Stephens, 2001) and act as anchoring sites for thin filaments and intermediate filaments (Ashton et al., 1975; Small and Gimona, 1998). According to the model, thick filaments span the entire distance between dense bodies such that thick filaments completely overlap the thin filaments within the "sarcomere".  Though only two thin filaments are  shown in the smooth muscle "sarcomere", there are probably several others surrounding the thick filament.  5  &  thick filament  thick filament  thin filament  \  csi  thin filament  Z-line  J.  X  Dense body  Fig. 1-2. Muscle contractile units, (a) A striated muscle contractile unit. Cross-bridges are shown on a bi-polar thick filament,  (b) A smooth muscle contractile unit. Cross-  bridges are shown on a side-polar thick filament.  For simplicity, this thick filament is  shown to interact with two thin filaments o f opposite polarity, but i n fact, a thick filament could interact with a number o f thin filaments that surround it. Images are modified from Lambert et al., 2004.  6  1.4 Smooth muscle mechanics  Muscle contractions in situ are auxotonic, meaning that the muscle contracts against a varying load. Under experimentally controlled conditions, muscle contraction is often isometric or isotonic. In the former, the length o f the muscle remains constant by varying the external load to balance the tension generated by the muscle. In the latter, a constant external load is applied to the muscle such that the muscle w i l l shorten when the force generated by the muscle exceeds the external load.  Under these controlled loading  conditions, analysis o f muscle performance can be greatly simplified.  Upon contractile activation o f a smooth muscle cell, calcium enters the cytoplasm through extracellular calcium channels and from intracellular stores (ie. sarcoplasmic reticulum). Calcium binds to calmodulin, causing a conformational change that allows calmodulin to activate the myosin light chain kinase ( M L C K ) .  Activated M L C K can  phosphorylate the regulatory myosin light chain, turning on myosin's ATPase activity which allows cross-bridge  cycling (i.e., cyclic interaction o f myosin and actin).  Phosphorylation o f myosin also seems to play a role in stabilizing myosin thick filaments (Qi et al., 2002; Trybus and Lowey, 1984).  Total muscle force is generated by the summation o f active and passive forces.  Active  forces is generated by energy dependent contractile structures such as thin and thick filaments.  Passive force, or resting tension, is created by non-energy  dependent  structures within muscle cells and tissue such as the extracellular matrix and the  7  cytoskeleton.  Force can be normalized by the cross-sectional area o f the muscle; the  normalized force is called stress.  Based on the cross-bridge, sliding fdament theory o f contraction, the active tension development o f muscle is proportional to the amount o f overlap between thick and thin fdaments o f appropriate orientation or polarity (Gordon et al., 1966).  Thus force  generation o f a muscle is related to fdament length. It also infers that when the muscle is stretched or shortened (actively or passively), filament overlap decreases and tension generation falls.  This can be seen in a length-tension curve.  A length-tension curve  gives us information on the muscle's ability to stiffen, support load and also about the muscle's elasticity.  Another important measurement o f muscle function, along with force generation, is the velocity at which a muscle shortens.  A force-velocity curve can be constructed with  isotonic contractions at a series o f external loads. A s the external load decreases relative to the maximal tension the muscle can generate, the velocity o f muscle shortening increases because more cross-bridges can be devoted to shortening and less to counter the load. Force-velocity curves o f both striated and smooth muscle can be fitted with H i l l ' s hyperbola (Hill, 1938) o f the form (F+a)(V+b) = b(¥ +a) max  where F is the isotonic load,  Fmax is the maximal isometric force, V is the shortening velocity, and a and b are H i l l ' s constants. B y plotting H i l l ' s hyperbola, one can also find V o f the muscle which is the 0  extrapolated maximal velocity at zero load.  8  1.5 Smooth muscle length adaptation  During normal functioning, hollow organs such as the bladder, uterus and bowel undergo large changes in volume. Since smooth muscle lines the walls o f these organs, it suggests that smooth muscle cells themselves must be functional through a large length range. In 1976, Uvelius reported that rabbit urinary bladder muscle could undergo a greater than 7fold length change within which it retained its contractile ability (Uvelius, 1976). Since the change in muscle length could account for the overall change in volume o f the bladder, this suggested that it was indeed a change in muscle cell length, and not an additional tissue enclosed within the muscle tissue, that occurred upon change in organ circumference. Length adaptation has also been shown in other types o f smooth muscle such as swine carotid artery (Wingard et a l , 1995), rat anococcygeus muscle (Gillis et al., 1988) and airway smooth muscle (Gunst et al., 1995; Gunst et al., 1993; Pratusevich et al., 1995) with differing amounts o f length range.  A result o f length adaptation is that the active length-tension curve o f smooth muscle is not fixed (Fig. 1-3). A change in length w i l l initially cause a drop in force development, however, during the period o f adaptation, the entire length-tension curve w i l l shift such that maximal force production ( F  m a x  ) occurs at the new, adapted length.  The passive  length-tension curve also shifts during the period o f adaptation such that the passive force w i l l decrease after adaptation to a longer length, or increase after adaptation to a shorter length.  9  Smooth muscle length adaptation can be induced in a variety o f ways. Once set at a fixed length, it can occur with a single contraction (Gunst and W u , 2001; Seow et al., 2000), with a series of brief activations (Pratusevich et al., 1995) or with continuous submaximal activation for ten minutes (McParland et al., 2005). Length adaptation can also occur in relaxed muscle after a period o f hours or days (Martinez-Lemus et al., 2004; Naghshin et al., 2003; Wang et a l , 2001; Zeidan et al., 2000).  While skeletal muscle length adaptation has been shown to occur, it occurs only over long periods o f time. Such is the case in chronic obstructive lung disease where patients develop a shortened diaphragm (Rochester and Braun, 1985; Sharp et al., 1974), but in general, its functional length range is only 10-20% (Gordon et al., 1966).  10  Length (mm)  Length (mm)  Fig. 1-3. Length-tension curves after length adaptation. Both the active and passive  length tension curves of airway smooth muscle shift after a period of adaptation to a longer or shorter length, such that F  m a x  is produced over a large length range, (a) Closed  circles represent trachealis strips that have been passively shortened, open circles represent control strips from the same animal, (b) Closed circles represent trachealis strips that have been passively lengthened, open circles represent control strips from the same animal. Reproduced from Wang et al., 2001.  11  1.6 Mechanism of length adaptation  Large changes in smooth muscle length cannot be produced by a fixed array o f sliding filaments as found in striated muscle, thus the mechanism o f smooth muscle length adaptation  probably involves a structural reorganization  contractile apparatus.  o f the  cytoskeleton  and  A term to describe the ability to adapt and reform the filament  lattice such that optimal overlap o f thin and thick filaments (thus maximal force development) is achieved, was first coined by Ford et al. (1994) as plasticity o f smooth muscle.  To describe the cytoskeletal component o f length adaptation, upon contractile activation, the cytoskeleton becomes a highly dynamic structure with properties comparable to those of a soft glassy material (Bursac et al., 2005; Gunst and Fredberg, 2003).  In A S M ,  cytoskeletal proteins that regulate the structure and organization o f the actin cytoskeleton are sensitive to mechanical and contractile stimuli, and contraction o f the muscle at a short length results in a shorter, thicker array o f cytoskeletal filaments compared to contraction at a longer length (Gunst et al., 1995; Gunst et al., 2003; Gunst and W u , 2001). There is also evidence o f polymerization o f actin into thin filaments in response to contractile activation (Herrera et al., 2004; Mehta and Gunst, 1999) as well as to adaptation to a longer length (Herrera et al., 2004).  Thus, the dynamic state o f the  cytoskeleton seems to be key in allowing smooth muscle cells to adapt their shape to accommodate external forces.  12  The contractile element seems to allow smooth muscle length adaptation via thick filament regulation. This occurs during contractile activation and length adaptation. The amount o f myosin polymerization is substantially more than the amount o f actin polymerization under the same conditions (Herrera et al., 2004), and thick filaments, unlike thin filaments, show a linear correlation to force production (Kuo et al., 2001). Structural evidence o f thick filament evanescence,  with thick filaments dissolving  partially upon relaxation and reforming during activation was found in electron microscopic studies (Gillis et al., 1988; Herrera et al., 2002; Shoenberg, 1969), studies o f birefringence o f living tissue (Gillis et al., 1988; Godfraind-De Becker and Gillis, 1988a; Godfraind-De Becker and Gillis, 1988b; X u et al., 1997) and by X-ray diffraction (Watanabe et al., 1993).  Electron microscopic and birefringence studies also show  evidence o f an increase in thick filament density upon adaptation to a longer length (Herrera et al., 2004; K u o et al., 2003b; Smolensky et al., 2005).  In 1994, Ford et al. proposed a model by which A S M adapts to an increase in length by plastic alterations that place more myosin thick filaments in series.  Since actin thin  filaments are in excess o f thick filaments in smooth muscle [the ratio o f thin to thick filaments is 20-30 to 1 (Herrera et a l , 2004)], thick filaments are considered the limiting structure in a contractile unit and therefore an increase in thick filaments is assumed to be representative  o f an increase in contractile units.  Assuming that the properties o f  individual contractile units are unchanged, an increase in thick filaments in series explains the increased velocity o f shortening found at the longer adapted length. A 100%  13  increase in the length o f the adapted muscle resulted in a 67% increase in shortening velocity (Pratusevich et al., 1995).  The model of Ford et al. (1994) was corroborated and further examined by K u o et al. (2003b). After adapting a muscle strip to twice its original length K u o et al. found no change in isometric force production, a 69.4% increase in shortening velocity and a 76.0% increase in myosin fdament density. This was the first structural evidence o f a change in thick filament mass with length adaptation. They also found that muscle power output and energy consumption as measured by ATPase rate had a similar dependence on muscle cell length; 35.4% and 34.6% respective increase for a 50% increase in cell length. These data support the notion that myosin polymerization plays an important role in length adaptation and that a pool of monomeric myosin exists such that thick filaments can form in a matter o f seconds to minutes.  The data collected by K u o et al. could still be explained by a variety o f mechanisms, thus Herrera et al. (2005) narrowed down the possibilities by using additional ultrastructural and mechanical data. A schematic o f the model that explained all the available data on length adaptation is shown in F i g . 1-4.  According to this model, a doubling o f the  adapted cell length causes no net change in force production since the number o f contractile units in parallel decreases by 20% (which would predict a 20% decrease in force) and the length o f thick filaments increases by 20% (which would predict a 20% increase in force).  It also predicts the observed 67% increase in shortening velocity as  the number of contractile units in series increases by this amount, along with a similar  14  increase in power output, energy consumption, and thick fdament density as found by K u o et al. (2003b).  It agrees with the finding that a 100% increase in adapted length  caused a 34% increase in the number o f dense bodies per cell volume and that maximally shortened muscle length has a linear relationship with the isotonic load imposed on the muscle.  15  1 -5 Ljn situ  Fig. 1-4. Contractile unit rearrangement in response to length adaptation of a twofold length change.  Left-hand muscle cell is half the length o f the other.  Model  predictions are discussed i n text. Thick filaments are shown as thick lines, thin filaments as thin lines and dense bodies as open ovals.  16  Modified from Herrera et al., 2005.  1.7 Length adaptation and disease  Compared to normal airways, asthmatic airways have been shown to be more sensitive and constrict more in response to non-specific stimuli ( K i n g et al., 1999). mechanism of this hyperresponsiveness, however, remains unclear.  The  While chronic  asthma is characterized by an increased thickening o f all layers in the airway wall, computational modeling suggests that it is the increase in the A S M layer that contributes most to this non-specific hyperresponsiveness (Lambert et al., 2004).  This has been  corroborated by in vitro experiments using dynamically loaded smooth muscle strips to better mimic the in vivo effects o f breathing and deep inspiration (Oliver et al., 2006). Thus, a major motivation for A S M research lies in its involvement in diseases o f airway obstruction such as asthma.  The physiological function which requires A S M to undergo length adaptation is still unknown, however it may play a role in the pathology o f airway diseases. ASM  plasticity  may  bronchoprotective effects  explain  why  in response  asthmatics  show  less  For instance  bronchodilation  to a deep inspiration than control  (Scichilone et al., 2001; Wheatley et al., 1989).  or  subjects  Chronic airway inflammation may  decrease the diameter of the airway wall, thus resting length o f A S M , resulting in A S M adaptation to this shorter length. This may hinder the muscle's ability to undergo stretchinduced relaxation because o f higher passive force (Wang et al., 2001), and may also allow the muscle to shorten more as was shown by Herrera et al. (2005) and McParland et al. (2005). In the study o f Herrera et al. (2005), isolated porcine tracheal A S M strips  17  were shown to shorten more i f they were adapted at a shorter length.  The study by  McParland et al. (2005) showed a 1.57-fold increase in shortening amount when bronchial smooth muscle was adapted at approximately half its original length (compared to adaptation at the original length) along with at 1.93-fold increase in developed force and 1.75-fold increase in rate o f shortening. If this also occurs in vivo, it could play an important role in airway mechanics during or after an acute spontaneous asthma attack or any disease of chronic airway narrowing, such as C O P D .  18  1.8 Hypotheses and specific aims  While there has been a growing amount o f research into the functional and structural changes that occur when smooth muscle is adapted at different lengths, little is known about the changes occurring immediately after a large step-change in length. Thus, the purpose o f this dissertation is to fill in some of these gaps.  Force generation, velocity o f shortening, and thick filament density were evaluated shortly after quick-stretch (QS) or quick-release (QR) maneuvers in sheep tracheal smooth muscle strips. A Q S / Q R is a large lengthening or shortening step, respectively, which takes less than 2s and occurs 25s prior to electrical stimulation (allowing the passive viscoelastic tissue response to settle).  After taking the desired measurements for  the functional studies, the muscle is brought back to the adapted length as soon it returns to a relaxed state.  In regards to the immediate effects o f a length change on a smooth muscle strip, we hypothesize the following: 1) A quick-stretch will result in a small drop in force and thick filament density. 2) A quick-release w i l l result in a large and linear drop in force and thick filament density. 3) Both quick-stretch and quick-release w i l l result in no change in shortening velocity.  19  The rationale for the hypotheses are as follows: Assuming that a length change does not result in a change i n cross-bridge activity, there w i l l be an asymmetrical change in the muscle's mechanical properties and ultrastructure in relation to the direction o f length change, as illustrated in Fig. 1-5.  release to L Fig. 1-5.  r e f  adapted at 1.5 L«f  stretch to 2  Hypothesized model of immediate effects of length change on airway  smooth muscle. Thin lines represent thin filaments, thick lines represent thick filaments, cross-bars on thick lines represent cross-bridges, filled circles represent dense bands or bodies. For simplicity only one row o f contractile units is shown; in reality many exist in parallel in each cell.  The middle cell o f Fig. 1 -5 represents the contractile unit arrangement o f muscle adapted at 1.5 Lref-  According to the model o f contractile unit arrangement by Herrera et al.  (2005), thick filaments span the entire distance between dense bodies such that there are no regions o f non-overlap between myosin thick filaments and actin thin filaments. Though the model gives no information on the length o f thin filaments relative to thick filaments (as the lengths o f thick and thin filaments seem to be variable), we suggest that thin filaments are longer than thick filaments in the adapted state, thus there are segments o f actin filament that are not interacting with thick filaments (not shown i n Fig. 1-5). W e also suggest that a length stretch or release causes cell membrane attachments o f the thin  20  filament lattice to move further apart or closer together (respectively), pulling the dense bodies apart or allowing them to come closer together.  Hypothesis #1 is based on data from Herrera et al. (2005) where quick-stretches o f 10% and 30% caused minor decreases in force (-10%).  If the thin filaments are at least 33%  longer than thick filaments, a stretch from 1.5 Lref to 2 Lr f will cause no change in force e  as regions o f thin filaments that previously did not interact with thick filaments become overlapped with thick filaments, resulting in no net change in thick and thin filament overlap.  There may be some drop in force due to mechanical perturbation which has  been shown to cause disassembly o f myosin thick filaments (Kuo et al., 2001; Q i et al., 2002), however we assume this w i l l be minimal due to the gentle length change over a 2second period (see Materials and Methods).  Hypothesis #2 is also based on data from Herrera et al. (2005) where under non-adapted conditions the ability o f the muscle to generate force declines linearly as the muscle shortens. In our model, a 33% release (from 1.5 Lref to U-ef) causes a 33% loss o f overlap i  between thick and thin filaments as thick filaments either dissolve when they run into adjacent thick filaments or are unable to interact with adjacent thin filaments because o f the "wrong" polarity. A s such, force drops in proportion to the loss o f overlap.  If the changes in force are due to structural changes o f the contractile apparatus as opposed to another factor affecting force, we would expect force and thick filament mass to be influenced in a similar way.  Like force, a stretch should cause only a small  21  decrease in thick filament mass whereas a release should result in a large decrease, assuming thick filaments disassemble when they slide over dense bodies (this could prevent unwanted interaction o f thick filaments with thin filaments o f wrong polarity to generate negative force). Thick filament mass (the length o f each filament multiplied by number o f filaments) is quantified by the density o f thick filaments in a transverse section of an electron micrograph o f a smooth muscle cell.  While there have been no previous reports on velocity immediately following a length change, we speculate in hypothesis #3 that velocity will not change.  We expect no  immediate change in the number of contractile units in series upon stretch or release as this is the simplest case.  To examine these predictions, we will perform the following studies: 1) Measure the changes in force immediately after a quick-stretch or quick-release. 2) Measure the changes in thick filament density immediately after a quick-stretch or quick-release. 3) Measure the changes in shortening velocity immediately after a quick-stretch or quickrelease.  A l l experiments will be done between two lengths (L f, 1.6 Lref), and force measurements re  w i l l also be taken between three lengths (Lref, 1-5 Lref, 2 Lref).  22  CHAPTER 2. Materials and Methods  2.1 Muscle strip preparation  Sheep tracheas were obtained from a local abattoir (Pitt Meadows Halal Meats Ltd.). Upon sacrifice, tracheas were removed from the carcasses and immediately placed in 4°C physiological saline solution (PSS) (pH 7.4, contents in m M : 118 N a C l , 5 K C 1 , 1.2 N a H P 0 , 22.5 N a H C 0 , 2 M g S 0 , 2 C a C l , and 2g/l dextrose). Prior to experiment, a 2  4  3  4  2  tracheal segment was removed from the trachea.  The in situ length o f tracheal smooth  muscle bundle connecting the C-shaped cartilage ring was measured. Caution was taken to ensure that the smooth muscle was relaxed when the measurement was made.  If the  epithelial layer was thrown into large folds, indicating that the underlying smooth muscle was contracted, the trachea was not used for the experiment. Relaxed tracheal rings were then cut open on ventral side. Connective tissue and epithelial layer covering smooth muscle were removed. Muscle bundles of approximately 8 m m long, 1 mm wide, and 0.3 mm thick were dissected out and clipped on both ends with aluminum foil clips for attachment to the force/length transducer.  23  2.2 E q u i l i b r a t i o n  A muscle strip was connected in a tissue bath to two hooks - one stationary and one connected to the lever arm o f a servo-controlled force/length transducer.  PSS in tissue  bath had previously been heated to 37°C, and p H stabilized by bubbling with carbogen gas (mixture o f 95% O2 and 5% CO2). During the equilibration and experimental procedures, the muscle strip was activated every 5 minutes with a 12-second electrical field stimulation (EFS) and the PSS in the muscle bath was changed.  Muscle  equilibration was done before beginning any experiment to allow the muscle to recover from mechanical and metabolic perturbations caused by dissection, lack o f perfusion and low temperature.  Equilibration was considered complete when stimulations produced a  stable maximal isometric force with low resting tension and took around 1.5 hours. During the equilibration period, an experimental reference length (Lref) for the muscle strip was determined using the in situ length as a guide, i.e., the amount o f stretch in muscle bath experienced by the muscle is approximately the same as that in situ.  It is important to note the difference between the adaptation and equilibration processes. In these experiments, equilibration and adaptation were both accomplished by 12-s isometric  contractions  at  5  minute  intervals, but  adaptation  occurs  only  after  equilibration. The main purpose of equilibration is to allow the muscle to recover from the trauma o f dissection while the purpose o f adaptation is to allow the muscle to recover from a length change. Thus during adaptation the muscle strip was set at a single length so that the structure  o f cytoskeleton and contractile apparatus could rearrange to  24  maximize contractility at that particular length (Kuo et al., 2003b; Pratusevich et al., 1995; Wang et al., 2001).  Adaptation was considered complete when isometric force  production stabilized at that length, and it usually took 0.5 hour.  25  2.3 A p p a r a t u s  The servo-controlled force/length lever system had a force resolution o f 10 u N and a length resolution o f 1 urn. The E F S was provided by a 60-Hz alternating  current  stimulator with platinum electrodes and a voltage o f 20-30 V that produced stable, maximal response from the muscle. The analog signals were converted to digital signals by a National Instrument analog-to-digital converter and then recorded by a computer which also controlled the onset and duration o f stimulation. This apparatus measured both isometric and isotonic contractions o f the muscle, i.e., muscle contractions at a constant length or muscle shortening at a constant load, respectively. More details about the apparatus can be found in publications from the Seow laboratory (Wang et al, 2000, 2002; K u o et al, 2003).  26  2.4 B r i e f overview of experiment procedure  The purpose o f this study was to examine ultrastructural and functional changes in a strip of airway smooth muscle immediately after a length change. A s discussed in Section 1.8, this was outlined in three specific aims:  1) Measure the changes in force immediately after a quick-stretch or quick-release. 2) Measure the changes in thick filament density immediately after a quick-stretch or quick-release. 3) Measure the changes in shortening velocity immediately after a quick-stretch or quickrelease.  For aim #1, quick-stretches (QS) and quick-releases (QR) were performed between three lengths (L f, 1.5 L-ef and 2 Lref). re  More specifically, the muscle was adapted at one o f  these preset lengths then quick-stretched or quick-released to another pre-determined length. The muscle was then adapted at another length and a Q S or Q R was performed from this new length.  A l l possible length permutations were studied.  The force  generated by the first contraction after Q S or Q R was recorded and compared to the force produced at the adapted lengths. (Fig. 2-1)  For aim #2, Q S and Q R maneuvers were performed between two lengths (Lref and 1.6 Lref). After adaptation at L-ef or 1.6 Lref, a Q S or Q R to the opposite length was applied, force was recorded and the muscle strip was fixed for transmission electron microscopy .  27  A control muscle strip that had undergone the same treatment, but without the Q S or Q R step, was fixed simultaneously. The control strip for Q S was fully adapted at Lref, and the control strip for Q R was fully adapted at 1.6 Lref- (Fig. 2-2)  For aim #3, the force-velocity curves o f a muscle strip adapted at Lr f and 1.6 Lref were e  determined, and at each adapted length, a Q S or Q R was performed to the opposite length. Force and velocity were recorded after the Q S or Q R , and the positions o f the F V points following the Q S or Q R were compared to the F - V curves at the adapted lengths. (Fig. 2-3)  28  Fig. 2-1.  Experimental protocol: change in force after quick-stretch or quick-  release. After equilibration, a single muscle strip was subjected to the following length manipulations.  The order o f length change was alternated.  Arrows pointing right  represent quick-stretches, those pointing left represent quick-releases.  Pair 2 Adapt at 1.6 L f . Adapt at 1.6 L^f then quick • released to Lref  Pair 1 Adapt at L i . Adapt at Lr f then quick-stretched  r e  r e  e  to 1.6 L f re  Fig. 2-2.  Experimental protocol: change in myosin fdament density after quick-  stretch or quick-release. After equilibration, two pairs of muscle strips from the same trachea were subjected to the following treatments (1 treatment per strip) before force was recorded and strips were fixed for electron microscopy.  Pairs 1 and 2 were  reversible in order.  Part 1 Adapt at L i Determine F - V curve at Lr f  Part 2 Adapt at 1.6 L f Determine F - V curve at 1.6 Lref  \  \  r e  r e  e  Q S tO 1.6 Lref  Plot F - V point o f Q R  Plot F - V point of Q S Fig. 2-3.  Q R tO Lref  Experimental protocol: change in velocity after quick-stretch or quick-  release. After equilibration, a single muscle strip was subjected to the following steps. Parts 1 and 2 were reversible in order .  29  2.5 Change i n force after quick-stretch or quick-release  After equilibration, the shortest and longest muscle lengths that produced nearly maximal isometric force ( F  m a x  ) were identified. Muscle length was recorded during the plateau o f  an isometric contraction. F  m a x  was identified by adapting the muscle at a length ~1 mm  less than in situ length. The muscle was then shortened by small increments until the maximal isometric force produced at that shortened length was around 90% o f F  m a x  after  adequate time for adaptation. This corresponded to the top o f the ascending limb o f the adapted length-tension curve and was taken as a reference length (Lref). B y lengthening the muscle in increments, the longest muscle length that produced >90% o f F  m a x  and <1.5  m N o f resting tension after adaptation was also identified. This low resting tension was to ensure that passive elastic components did not contribute excessively to the internal load during a quick length change.  A muscle strip was deemed suitable for our  experiment i f the long length was twice Lref or greater.  The muscle strip was adapted at 1 o f 3 lengths: Lr f, 1.5 L^f, or 2 L^f. A t each adapted e  length the muscle was manually quick-stretched (QS) or quick-released (QR) to the other experimental lengths.  Q S / Q R is a large lengthening or shortening step, respectively,  which takes less than 2 s and occurs 25 s prior to electrical stimulation (this was to allow the passive viscoelastic tissue response to settle).  The force produced by the muscle at  this quick-stretched or quick-released length was recorded and the muscle was brought back to adapted length within 25 seconds o f stimulation ending (when the muscle had  30  relaxed). Muscle recovery at the adapted length was achieved by stimulating the muscle. electrically every 5 minutes until force production stabilized, usually reaching the F x it ma  had attained prior to the length step, and for a minimum o f 3 stimuli.  The order o f  adapted lengths alternated for every experiment to control for the effects o f time or sequence. The experiment was repeated with 10 strips from 3 animals. (Fig. 2-1).  The force produced after Q S or Q R was expressed as a percentage o f the force when the \ muscle was adapted at the Q S or Q R length. For example, the force produced after a quick-release to L f would be normalized by the maximal force o f the muscle adapted at re  L f. This was to account for slight differences in maximal force at different lengths since re  the plateau of the length-tension curve o f smooth muscle is not perfectly horizontal (Pratusevich et al., 1995).  A n experiment example is as follows. A t each length (adapted, quick-stretched/released) the force was recorded.  Muscle was adapted at 1.5 Lref, quick-released to Lref, stimulated  by E F S then brought back to 1.5 L f within 25 s after stimulation. Force recovered at 1.5 re  Lref before a quick-stretch and stimulation at 2 Lr f, then it was adapted to 2 Lr f, quicke  e  released to 1.5 Lref and Lref, and finally adapted at Lref, then quick-stretched to 1.5 Lref and 2 Lref-  31  2.6 Change in myosin filament density after quick-stretch or quick-release  This experiment was performed with two pairs o f adjacent muscle strips from a single trachea.  A s previously described, the longest and shortest (Lr f) muscle lengths that e  produced near maximal force were found for each strip. The important lengths for this experiment were Lref and 1.6 L-ef thus the strips were deemed suitable i f each had a longest length (which maintained F  m a x  ) greater or equal to 1.6 Lref and i f F x and L f ma  re  were similar for strips in a pair. Force was recorded and strips were fixed for electron microscopy at four points; adapted at Lref, adapted at L-ef then Q S to 1.6 Lref, adapted at 1.6 Lref, and adapted at 1.6 L f then Q R to L f ( F i g . 2.2). Fixation was done 50 seconds re  re  after the final electrical stimulation, once the muscle had returned to its relaxed state. Three animals were used for this experiment.  32  2.7 Electron microscopy  Strips were fixed while they were still attached to the experiment apparatus for 15 minutes with a 37°C fixative solution of 2 % gluteraldehyde, 2% formaldehyde and 2% tannic acid, 0 . 1 M Na-cacodylate buffer, p H 7.3. Care was taken not to disturb the strips during this initial fixation. Strips were then removed from tissue bath, cut into 6-8 pieces so they measured less than 1 mm in length, and immersion fixed in fresh fixative for 2 hours. Samples were rinsed three times with 0.1 M Na-cacodylate buffer (pH 7.3), 10 minutes per rinse. Secondary fixation was done for 2 hours with 1 % osmium tetroxide in buffer (diluted from 2% osmium tetroxide on day o f use).  Samples were again rinsed  with buffer 3 times, 10 minutes per rinse, and finally stored overnight at 4°C in fresh buffer.  Except for the initial fixation and overnight storage, all steps previously  described were performed on ice, on a shaker in fumehood. The following day, samples were rinsed 3 times for 10 minutes in distilled water, then en bloc stained with 1% uranyl acetate for 1 hour. Dehydrations were performed with 10 minutes each o f 50%, 75%, 80%, 90%, 95% ethanol, then 3 times in each 100% ethanol and propylene oxide. Infiltration with freshly mixed resin ( T A A B 812 mix, medium hardness) was done by diluting with propylene oxide in a ratio o f 1:3 then 3:1 for 30 minutes per dilution. Infiltration with pure resin was done for 1 hour, then overnight in fresh resin. A l l steps o f day 2 were done on a shaker in fumehood. Finally, tissue samples were placed in labeled moulds and baked in resin at 60°C overnight.  33  Blocks were sectioned on a Leica E M U C 6 ultramicrotome with a diamond knife at 5070 nm thickness and sections placed on 400-mesh copper grids. Grids were stained with 1% uranyl acetate for 4 minutes and Reynolds lead-citrate for 3 minutes before being viewed under a F E I Technai 12 transmission electron microscope. For each experimental group (example o f one group is "animal 3, quick-stretched from Lref"),  a  minimum o f two  blocks were sectioned and multiple grids per block were imaged.  In each group, images o f 15 cells in cross-section were collected; 5 cells smaller than 2.5pm in diameter, 5 cells larger than 2.5pm in diameter with no nucleus, and 5 cells with nucleus (usually larger than 2.5pm in diameter). This ensured that we gathered an equal amounts o f information from different areas o f the cell; near tapered cell end, near central cell segment, and at central segment with nucleus, respectively. Images were taken with a digital camera (Gatan BioScan, Model 792) at a magnification o f 37 000X, so to capture the cross-section of a single cell often required taking multiple images and reconstructing the whole cell cross-section in Photoshop®.  34  2.8 M o r p h o m e t r i c analysis  Image-Pro Plus software (version 4.0) was used to find thick filament density in cell cross-sections. Density is equal to mass divided by volume, and since cell volume was assumed to be independent o f cell length, thick filament density is an indirect measure o f the mass or content of thick filaments in a smooth muscle cell (Kuo et al., 2003b). Thick filaments were counted in a cell cross-section using the "manual tag" function. The cytoplasmic area o f a cell cross-section was found by subtracting the area taken up by the nucleus and organelles from the area o f the entire cell. Thick filament density was calculated by dividing the number o f thick filaments in a cross-section by the cytoplasmic area to give a value in #/pm . Thick filament density was compared within pairs. 2  Samples were "masked" on day 2 o f E M preparation protocol and remained masked until after morphometric data had been analyzed.  Thus, the group identity o f the samples  (adapted at Lref / Q S / adapted at 1.6 Lref / Q R ) were hidden from the experimenter until morphometric analysis was completed.  This ensured unbiased counting o f thick  filaments.  35  2.9 Change in velocity after quick-stretch or quick-release  After equilibration, the shortest (Lref) and longest muscle lengths that produced near maximal force were identified as in Section 2.5. This experiment was performed at two lengths, Lref and 1.6 Lref, thus the strip was deemed suitable for experiment i f the longest length was greater or equal to 1.6 Lref. Starting length of Lr f or 1.6 Lr f was alternated, e  e  with two experiments starting at L f and two at 1.6 Lref for a total o f four experiments or re  four animals.  The force-velocity ( F - V ) curve at Lr f and 1.6 Lref was determined for each tissue. This e  was accomplished by measuring the maximal shortening velocity during isotonic contractions at - 7 5 % , 50%, 30%, 20% and 15% o f maximal isometric force. [ F determined prior to every isotonic shortening.]  m a x  was  The F - V points at Lr f were fit to H i l l ' s  hyperbola (Hill, 1938) which is defined by the equation (F+a)(V+b) is the isotonic load, F  m a x  e  - b(F +a) max  where F  is the maximal isometric force, V is the shortening velocity, and  a and b are H i l l ' s constants.  To find the change in velocity from Lr f to 1.6 Lref, F - V e  points at 1.6 L-ef were fitted by scaling up the F - V curve at Lref. SigmaPlot® software (Version 8.02) was used to plot the curves.  After making the F - V curve at each length, the muscle was twice quick-stretched/released to the opposite length; once to measure the force produced after the Q S / Q R , and after readapting the muscle to the original length, the second time with the isotonic load set between 12%-24% o f the force found at the length step. Although changes in velocity  36  become more sensitive as % F  m a x  decreases (i.e., the slope o f the F - V curve increases in  absolute value), the velocity of Q S and Q R was measured between 12-24% F x to avoid raa  instability o f the force/length transducer at values below 10% F  m a x  . Velocity at the quick-  stretched/released lengths were examined and compared to the velocity o f the muscle at the adapted lengths. (Fig. 2-3)  37  2.10  Statistical analysis  In all experiments, data from each animal was averaged before averaging the means from different animals. Unless otherwise noted, data is shown as means ± standard error o f mean (SE).  Significant difference was determined using the Student's paired t-test,  significance being P O . 0 5 .  38  C H A P T E R 3 . Results  3.1 Change in force after quick-stretch or quick-release  The purpose o f this study was to examine the effects o f a large step-change in length on force generation in sheep tracheal muscle. This was done by subjecting a single muscle strip to quick-stretches and quick-releases from three adapted lengths (Lref, 1 5 Lref, and 2 L-ef) and measuring isometric force generation immediately after the length-step. average F  m a x  The  for the three animals in this group was 129.6±5.3 kPa. F i g . 3-1 shows the  changes in force immediately after a quick-stretch or quick-release in kPa.  F i g . 3-2  shows changes in force in relative terms such that the force produced immediately after a Q S or Q R is normalized by the maximal adapted force (Fmax) produced at that Q S / Q R length. In other words the Q S / Q R force was normalized by the force of the muscle when adapted to the Q S / Q R length. slight differences in F  m a x  This normalization simplifies results by accounting for  at different lengths.  Results o f these experiments suggest that a quick-release causes a larger decrease in force production than a quick-stretch, i.e., that shortening an A S M strip causes more disruption than stretching it.  F i g . 3-3 shows the P-values o f a Student's paired t-test when the  relative force change of a quick-stretch  was compared to that o f a quick-release  of a  similar length step. Significant difference ( P O . 0 5 ) between shortening and lengthening was seen especially when the change in length was large (i.e., 100%). The directional effect also seemed to be relatively insensitive to the absolute length to/from which it was  39  shortened. This was demonstrated at the intermediate length: the decrease in force from 1.5 Lref to 2 Lref was 8 . 7 5 ± 3 . 4 1 % and from 1.5 Lr f to L r was 2 0 . 4 4 ± 0 . 3 6 % (P=0.07). e  e f  From a structural point o f view, force generation is proportional to the number o f contractile units in parallel or a loss o f thick and thin filament overlap. Results o f this study suggest that a Q R causes a greater loss of these units than a Q S , assuming other factors influencing muscle force remain the same. This w i l l be further investigated in the next group o f experiments presented in this dissertation, using the density o f myosin (thick) filaments in a cell cross-section as a measure o f contractile unit mass (Section 3.2).  40  160  140  120 c3 U  100  o 1-  o HH  80  Adapted at L  —o-  60 4  ref  Adapted at 1.5 L Adapted at 2 L  ref  ref  40  L  r e f  l-5L  r e f  2 L ref  Length Fig. 3-1. Change in force (kPa) upon quick-stretch or quick-release. A single muscle strip was subjected to quick-stretches and quick-releases (as shown by grey arrows) from three adapted lengths. Isometric force generation was measured immediately after the length-step. Data shown are means ± S E .  41  s  80  Length Fig. 3-2. Relative change in force (%F  max  ) upon quick-stretch or quick-release.  Data from F i g . 3-1 is normalized to the maximal force (Fmax) produced at each Q S / Q R length. Data shown are means ± S E .  42  Quick-stretch L  ref  tO 1.5 L ef r  Lref tO 2 L 1.5L ,to2L  ref  r e  r e f  1.5L ,to2L , r e  r e  Quick-release 1.5 L tO L 2 L tO L ref  ref  ref  ref  2L e,to1.5L , 1.5 L tO Uef r  re  ref  P-value 0.01 <0.01 0.73 0.07  F i g . 3-3. Quick-stretch vs. quick-release. The change in relative force due to a similar length change but i n different directions (stretch vs. release) is examined v i a a Student's paired t-test.  43  3.2 Change in thick filament density after quick-stretch or quick-release  Four strips of trachealis from a single animal were fixed at four conditions; adapted at Lref, adapted at Lref then Q S to 1.6 L f, adapted at 1.6 Lr f, and adapted at 1.6 Lref then Q R re  to Lref.  e  This allowed the examination of the immediate effects o f length change on  myosin thick filament assembly and disassembly, and verification o f their effects on force generation.  To look for structural changes in the filament lattice, thick filament  density in cell cross-sections was measured by electron microscopy. Force was measured just prior to muscle fixation. Three animals were used for this study.  Fig. 3-4 is an example o f an electron micrograph o f a sheep trachealis smooth muscle cell in transverse section. Included is a detailed list of the data obtained from this cell, such as a breakdown o f cellular areas. Note that these data were obtained under "masked" conditions, thus the experimenter was without knowledge o f the experimental group the sample belonged to. F i g . 3-5 is a magnified portion of Fig. 3-4 with dense-bodies and thick, thin and intermediate filaments pointed out.  Thick filaments were identified by  their non-uniform cross-section of about 12-20 nm in "diameter", thin filaments by their 6-7 nm diameter and approximately circular shape, and intermediate filaments by their perfectly round shape and consistent 10 nm diameter.  F i g . 3-6 shows examples o f  electron micrographs from each experimental group and includes the thick filament density o f each cell.  44  A summary o f results from all three animals is shown in Figs. 3-7 and 3-8.  F i g . 3-7  shows the thick fdament density in every experimental group, sub-grouped by the area o f the cell at which the fdaments were counted.  F i g . 3-8 shows the average results o f  changes in thick fdament density and compares them to changes in force upon Q S or Q R . These data indicate that myosin fdament density does not decrease significantly from control upon Q S , but does decrease significantly from control upon Q R . The filament density drops by 19.0±3.2% for a 60% decrease in length.  The decrease in force is  similar to what would be expected from the first study o f this dissertation, with Q R again causing a greater loss in force than a Q S (Section 3.1). Q S caused a decrease in force o f 5.0±2.5% and Q R caused a decrease in force o f 17.2±5.3%.  Since the change in force  shows a similar trend to the change in density, we can extrapolate to suggest that the changes in force seen between the three different lengths in Section 3.1 study may also be caused by a change in thick filament density.  However to be sure,  measuring thick filament density at these three lengths would be necessary.  45  experiments  Fig. 3-4. Electron micrograph of a sheep trachealis muscle cell in transverse section, and experimental data. Q S group, sectioned through central segment o f cell, animal 3. Total cell area, 7.283um ; area o f nucleus, 4.252um ; area o f mitochondria, caveolae and 2  2  other organelles, 1.505um ; number o f thick fdaments, 136. Therefore cytoplasmic area 2  o f cell is 1.546 u m and thick filament density is 89.12/um . Scale bar represents 0.5um. 2  2  46  F i g . 3 - 5 . M a g n i f i e d portion of previous image. Bottom right-hand corner o f Fig. 3-4. Arrowheads show dense bodies, large arrows point to thick filaments surrounded by thin filaments, small arrows point to intermediate filaments. Scale bar represents 0.5um.  47  Fig. 3-6. Examples of electron micrographs from all experimental conditions. (a) Adapted at L f, sectioned near tapered cell end as evidenced by the size, animal 3. re  Thick filament density is 90.36/um . Magnification 37000x. 2  (b) Q S , sectioned near  central segment as evidenced by the size, animal 2. Thick filament density is 51.52/um . 2  Magnification 23000x.  (c) Adapted at 1.6 L^f, sectioned near central segment as  evidenced by the centrally clustered mitochondria, animal 1. Thick filament density is 60.25/um . Magnification 23000x. 2  (d) Q R , sectioned near the central cell segment,  animal 1. Thick filament density is 42.82/um . Magnification 23000x. 2  represent 0.5um.  48  A l l scale bars  • central cell segment with nucleus • near central segment • tapered cell end • ALL areas  adapted at  Q S to  adapted at  Q R to  L f  1-6 Lref  L6 L f  L f  r e  re  re  Fig. 3-7. Thick filament density of four conditions sub-grouped by location along long axis of cell. Each solid-coloured bar shows averaged data o f 5 cells per animal, from 3 animals, thus 15 cells per bar.  Striped bars represent overall average o f other 3  bars o f the group, thus 45 cells each.  Thick filament density is in number/urn . Data  shown are means ± S E .  49  1.1  I  CO  I Control  VTA Quick Stretch  E3  Quick Release  c  CD  Q 1.0 CD O  .?  0.9  CD  g  °-  8  O "CD  g 0.7 O CO .  0.6  Filament Density  Force  Fig. 3 - 8 . Fractional change in force or thick filament density upon quick-stretch or quick-release. Control bars represent pre-QS or pre-QR values (i.e., adapted at L f or re  1.6 Lref respectively). Fractional change i n force is calculated from single muscle strips. Fractional change i n filament density is calculated from paired muscle strips from the same trachea.  P-values show results o f Student's paired t-test against their respective  controls. Data shown are means ± S E .  50  3.3 Change in velocity of shortening after quick-stretch or quick-release  In this group of experiments, we examined the acute effects of a large length-step on the shortening velocity o f trachealis muscle. A single strip of muscle was adapted at two lengths, Lref and 1.6 Lref. A t each adapted length, force-velocity curves were plotted by subjecting the strip to isotonic contractions against loads o f 10-75% F xma  A t each  adapted length, the strip was also subjected to two quick-stretch or quick-release maneuvers to the alternate length, and immediately stimulated. The first maneuver was to measure the force (Fmax) generated after Q S / Q R , and the second was to find the velocity of shortening by contracting against an isotonic load o f 12-24% o f the found Fmax. Thus, each Q S or Q R had a F - V point associated with it and the position o f these points relative to the F - V curves at adapted lengths was examined.  These experiments were carried out using four tracheas yielding an average F x o f ma  162.2±14.3 kPa.  Averaged F - V points are shown in F i g . 3-9, along with curves fitted to  these points. Filled circles represent F - V points from strips that were adapted at Lr f and e  open circles represent F - V points adapted at 1.6 Lref. Solid line is H i l l ' s hyperbola that best fits all points adapted at Lref (thus all filled symbols) and the dashed line represents the curve fitted to Lref scaled vertically by a factor o f 1.42. This was the scaling factor that most closely fit the points adapted at 1.6 Lref, and is also the average scaling factor (±0.05) for all four experiments. It suggests that a 60% increase in adapted length causes a 42%o increase in shortening velocity.  This is close to what would be expected.  Pratusevich et al. (1995) showed a 67% increase in velocity after a 100% increase in  51  adapted length in canine trachea, and K u o et al. (2003b) and Herrera et al. (2005) showed a 69% and 64% velocity increase after a similar length change in porcine trachea.  The average change in velocity immediately after Q S and Q R are shown as red squares in Fig.  3-9; the filled square represents the change after stretch and the open square  represents the change after release. The results of these experiments suggest that a quick length-step causes an immediate change in shortening velocity in airway smooth muscle, such that the velocity becomes identical to the velocity at the adapted Q S / Q R length. For example, when a strip is adapted at Lr f and quick-stretched to 1.6 Lref, the velocity is e  closer to that o f a strip adapted at 1.6 Lref than it is to a strip that has been adapted at Lref, and vice-versa.  The  average velocity after Q S and Q R are presented as bar graphs in F i g . 3-10.  Shortening velocities immediately after Q S at loads between 18-24% F x (average ma  21.2±1.2%) was found to be 0.265±0.011 UJs (middle bar o f Fig. 3-10a). The expected velocity o f strips adapted at L f and 1.6 Lr f were calculated at the same load by re  substituting the % F  m a x  e  o f the Q S into the equations o f the fitted curves of each  experiment. The average expected velocity o f a strip adapted at Lr f was 0.157±0.009 e  Lref/s and that o f a strip adapted at 1.6 Lref was 0.228±0.015 Lr f/s (respectively, left and e  right bars o f Fig. 3-10a). Therefore even though the strip had been adapted at the lower length, upon Q S the velocity immediately increased to 7 2 . 2 ± 9 . 4 % above the value measured at this lower length and even 2 0 . 4 ± 5 . 6 % above the velocity measured at the longer adapted length. [We suggest that the velocity after Q S went beyond the velocity at  52  1.6 Lref due to an artifact o f elastic recoil.  This would have caused an increase in the  passive tension upon quick-stretch. If it had been corrected for, it would have shifted the Q S point further left on the F - V curve and closer to the expected velocity at 1.6 Lref]  The results of Q R experiments were similar. The average velocity following a Q R was found at an average % F  m a x  o f 15.3±1.2. This velocity value was 0.197±0.005 Lref/s. The  velocities when adapted at L f and 1.6 Lref (found at % F re  method  described  above)  was  found  to be  m a x  0.196±0.013  between 12-18% by the and  0.284±0.022  Lref/s  respectively (Fig. 3-10b). Thus, there was a 25.1% ±4.5 drop in velocity after Q R from 1.6 Lref and no significant difference between the velocity after Q R and after adaptation to Lref- In other words, the velocity upon Q R immediately dropped to the velocity o f the strip when adapted that shorter length. The expected velocities are higher in the case o f Q R than in Q S because they were calculated at a slightly lower, but not significantly different, % F x and therefore further left along the adapted F - V curves. ma  Since velocity was shown to be related to the number o f contractile units in series (Lambert et al., 2004), the results shown here suggest that a length change has an immediate impact on contractile unit placement.  In the case o f muscle shortening,  contractile units are immediately taken out of series and require no additional change (in terms o f number) during the adaptation process.  In the case o f muscle lengthening,  contractile units are immediately added in series. A proposed model of how this occurs is presented in the discussion (Section 4.5).  53  Average F-V and QS/QR points 0.6 -i  %F ax m  Fig. 3-9. Velocity of shortening after quick-stretch and quick-release. Filled circles represent velocity measured from trachealis strips that were adapted at L f and re  open circles represent velocity when same strips were adapted at 1.6 L f. Solid line is re  H i l l ' s hyperbola that best fits all points adapted at L f and dashed line represents the re  curve fitted to L f scaled vertically by a factor o f 1.42. Q S and Q R points are shown re  as squares; the filled square represents velocity immediately after a QS and the open square represents velocity immediately after a Q R .  54  0.35  expected V at L .  Fig. 3-10.  V after QS  expected  expected V after QR expected  V a t 1.6 L  V a t 1.6 L„  VatL  Shortening velocities before length change, immediately after length  change, and after adaptation to new length.  Middle bars show actual measured  velocity (Lret/s) after QS or Q R . Velocity values from remaining bars were calculated from curves fitted to F - V measurements o f length adapted muscle at the same relative load (%Fmax) as Q S or Q R measurement was taken.  In both cases, Q S or Q R velocity  was closer to that observed when adapted at the stretched or released length. A l l values are significantly different (example is marked by asterisk) except V after Q R and expected V at Lr f (P-value is shown), (a) Velocity comparison after Q S , i.e., velocity at e  21.2 ±1.2 % F  m a x  (b) Velocity comparison after QR, i.e., velocity at 15.3±1.2 %Fmax.  55  3.4 Summary of results for a 60% length change  The results of a length change between L ^ f and 1 . 6 L r f are summarized in Fig. 3 - 1 1 . This e  highlights airway smooth muscle's asymmetrical response to length change, shortening being more disruptive to force and filament density than lengthening, and also shows the correlation between force and myosin filament density.  Finally, it shows that velocity  changes immediately and significantly upon a 6 0 % length change.  Quick-stretch (L f to 1.6 L ) re  rei  Quick-release (1.6 L  r e i  force  0.95 ± 0.02  0.83 ±0.05  myosin filament density  0.98 ±0.01  0.81 ± 0 . 0 3 *  velocity  1.72 ±0.09 *  0.75 ± 0.04 *  Fig.  3-11.  control  to L ,) re  1  Summary of relative change of quick-stretch or quick-release from  (Lref and 1.6 L f respectively). re  value (Student's t-test, P O . 0 5 ) .  T  * Statistically significant difference from control  Statistically significant difference from control value  (Student's t-test, P O . 1 0 )  56  C H A P T E R 4. Discussion  4.1 Result summary  In examining the mechanical behaviour and ultrastructure o f airway smooth muscle immediately following a change in length, we have found that these are  affected  asymmetrically. A passive shortening o f the muscle results in a larger drop in force and thick filament density than an equivalent stretching o f the muscle. For example, a 60% length increase causes no significant drop in force, unlike the 17.0±5.0% drop in force with a similar length decrease.  Changes in force are reflected by changes in myosin  filament density, suggesting that the mechanical responses observed are due to structural changes within the muscle as opposed to other factors that affect force. The response of velocity to length change was opposite to what was hypothesized. While we expected to see no change in velocity o f shortening within the first contraction after a length step, in fact the velocity rapidly changed to its value i f adapted at the Q S or Q R length.  A s w i l l be argued in the following sections we suggest that the results from this study occur after thin filament lattice adaptation is complete, but while thick filament polymerization is as yet incomplete. Therefore the changes in velocity and force, and thus adaptation of the thin filament lattice and polymerization o f thick filaments within the lattice, are regulated by separate mechanisms and occur at different speeds.  57  4.2 Effects of quick-stretch and quick-release on force development  A s hypothesized, quick-release resulted in a larger drop in force compared to a quickstretch o f a similar length change.  However the decrease in force due to Q R was not  linear, nor as much as was expected.  These observations came from two studies that  measured force directly after changes between 2 or 3 lengths.  Possible mechanisms  underlying the observations are stipulated below.  Stretch can cause a loss o f force by two mechanisms; 1) mechanical disruption which causes thick fdament disassembly in relaxed muscle (Qi et al., 2002) and 2) stretch pulls fdaments apart, reducing thin and thick fdament overlap. The contribution o f the second mechanism depends on the thin filament length relative to the thick filament length, and the amount o f stretch.  If thin fdaments are 50% longer than thick fdaments, this  mechanism would not be important in a stretch o f 50% or less.  However there is no  quantified relationship between the amount o f length change and the amount of filament disassembly, nor do we know the ratio o f thin to thick filament length. Thus we cannot differentiate whether stretch causes a reduction in force due to one or both mechanisms.  Release also affects force due to mechanical disruption, but there appears to be another mechanism which makes release much more disruptive than stretch. W e suggest that this additional mechanism stems from the structure o f the contractile unit o f smooth muscle as described by Herrera et al. (2005).  A lack o f non-overlap areas o f thick and thin  58  filaments in the contractile units o f adapted muscle necessitates that shortening o f muscle leads to a reduction o f the overlap zone, and hence force production.  The drop i n force associated with Q R is not as much as was expected from data by Herrera et al. (2005), nor is it linear with respect to the magnitude o f length release. B y superimposing data from Section 3.1 on the L - T curve from Herrera et al., the difference in predicted and actual results can be quantified (Fig 4-1). This difference is attributed to a small amount o f adaptation that occurs in our specimen during the contractile activation following Q R . Thus, the specimen is neither fully adapted to its pre-release length, in which case its L - T points would be along the predicted (dashed) line, nor is it fully adapted to its Q R length, in which case it would contract at 100% Fmax (dotted line). The specimen recovered by 2 0 . 3 ± 3 . 2 % o f isometric force after a 25% Q R (2 Lr f to 1.5 Lref), e  2 1 . 7 ± 0 . 4 % aftera 33% Q R ( 1 . 5 Lrefto L ^ ) and 1 5 . 3 ± 2 . 3 % after a 50% Q R (2 L-efto Lref).  The force recovery therefore generally seems to be less with greater length step o f quickrelease.  Because o f the rapidity o f the length adaptation process, designing an experiment that disallows reorganization o f the sub-cellular structures (i.e., that is capable o f observing the immediate effects o f length change) but takes functional measurements is not always possible.  Herrera et al. (2005) circumvented this issue in their Q R experiments by  measuring the maximally shortened muscle length at isotonic loads between 10-90% Fmax, taking advantage o f the fact that there is little time for adaptation during continuous active muscle shortening.  The protocol for the present experiments  59  involves b i -  directional changes in length. Since imposing a large stretch on an activated muscle can cause damage to the muscle, we have adopted a protocol o f changing muscle length during the relaxed state.  Because one o f the primary objectives is to determine the  symmetry o f adaptation in stretched and shortened muscle, length changes in both directions were carried out in the relaxed state to ensure that any asymmetry observed was due to direction of length change and not other factors such as state of activation. One shortcoming o f this approach is that partial adaptation occurs between the time o f length change and the time isometric force is measured, as shown in Fig. 4-1.  The current Q S results are comparable to the data presented in Herrera et al. (2005) probably because both groups performed these experiments in a similar way. Herrera et al. found that a 10% Q S caused a 6% decrease in the force associated with that stretch, and a 30% QS caused - 1 0 % decrease in force. In the current data, a Q S o f 33% (from 1.5 Lref to2 Lref) caused a drop in force o f 8.7±3.4%. These data may also have come from muscle that had undergone partial adaptation to the new length, however for the purpose o f the model presented later, we assume for simplicity that Q S causes only this minor drop in force with or without adaptation having occurred.  60  120 Fully adapted  100 O |  80  Adapted at L  E o *  only  60  03  2  m s r m  % F recovery  40  o 20  h  0 0.0  0.2  0.4  0.6  0.8  1.0  Muscle Length (L  in  Fig. 4-1. Partial adaptation during activation post-QR.  1.2 8  1.4  1.6  J Dashed line shows length-  force relationship o f A S M strip fully adapted only at L i situ (equivalent to Lr f) and not n  adapted at the shortened lengths (solid symbols with error bars.  e  From Herrera et al.,  2005). Open circles show length-force results from Section 3.1 normalized to Lin situ and assumed to be only partially adapted to a particular length (see text).  The  difference in % isometric force o f open circles and reference line represents the % isometric force recovery in the current experiments.  For example, the % force recovery  after a 50% Q R (shown by bracket) corresponds to 15.3±2.3%. If A S M strip was fully adapted at 0.5 L j situ, the % isometric force would be 100%, as indicated by the dotted n  horizontal line.  61  4.3 Effects of quick-stretch and quick-release on myosin fdament density  In this group o f experiments we looked at the effects o f a 60% length change on force production and myosin fdament density, and found that the changes in force were reflected in the changes in density (Figs. 3-8 and 3-11). This provides strong evidence that the change in force we observed were indeed caused by a change in the structure o f the contractile apparatus as opposed to other variables that influences force.  [More  discussion on factors influencing force w i l l be presented later.] The correlation between force and myosin fdament density also implies that myosin fdaments have undergone partial adaptation (i.e., re-polymerization) along with force.  Ultrastructural data from this study shows that myosin polymerization in response to a length change is not complete by the first contraction after the length change. Based on data by K u o et al. (2003b), a 60% increase in length should result in a 33.6% increase in myosin fdament density after adaptation to the stretched length, but the present results showed that no additional myosin polymerization occurred immediately after the 60% stretch.  Length  adaptation  in  the  opposite  direction  should  cause  myosin  (iepolymerization o f thick filaments, and while some depolymerization had occurred after the initial contraction at the short length, the extent o f depolymerization was less than expected.  Adaptation from 1.6Lr f to I ^ f should cause myosin fdament density to e  decrease by 25% (Kuo et al, 2003), however it only decreased by 19.0%. This could be due to a less-than-expected degree of depolymerization with the length change, or due to some recovery after the length change.  62  To have a better correlation between force and myosin density, it would have been preferable to measure myosin filament density in the activated state.  This could also  have reduced the effects o f length adaptation as the time after stimulation at the new length would have been reduced. However to fix a strip in the activated state requires the plateau o f contraction to be maintained. This cannot be accomplished by electrical field stimulation (EFS), which is how all the previous measurements were made, but by stimulation with acetylcholine (Ach).  Since acetylcholine also causes a higher level o f  muscle activation than E F S , fixing the tissue in the activated state would have made it difficult to compare our previous force measurements by nullifying the baseline force as measured by E F S . We could not compare force measurements within a strip, or with force measurements made in our other studies. Therefore fixing in a relaxed condition was a useful compromise.  Although it would have been expected by previous reports, we saw no increase in thick filament density o f muscle strips adapted and fixed at Lref compared to strips at 1.6 Lref (Herrera et a l , 2004; K u o et a l , 2003b). This could be because strips adapted at Lref and 1.6Lref were not paired in this study.  They were controls for Q S and Q R groups,  respectively, therefore they were paired with their experimental equivalent.  Pairs may  have differed i f their absolute lengths were not identical. They may also have differed i f structural changes occurred due to time or treatment, as one pair o f dissected strips was placed at 4°C while waiting for the other to be  fixed.  The order o f Q S and Q R  experiments was alternated to control for treatment differences between pairs.  63  4.4 Effects of quick-stretch and quick-release on velocity of shortening  While the results o f the force and density experiments did not stray too far from what was expected, the immediate changes in velocity upon length change was contrary to what was hypothesized.  Shortening velocity immediately changed to the value it would be  after adaptation to its new length. However, i f our force results have been affected by some length adaptation (Section 4.1), it would also seem likely that the velocity results have been similarly affected.  We propose that the rearrangement o f contractile units in  series is actually complete by the first contraction after a length step and that no further rearrangement occurs during the adaptation process.  The idea that reorganization o f contractile units in series is much faster than a change in thick filament number or length does not seem unreasonable; thin filaments are much less structurally labile and in vast abundance over thick filaments in a smooth muscle cell (Herrera et al., 2004).  It is possible that thin filaments are pre-arranged into a lattice  which is easily rearranged, maybe via regulation by intermediate filaments (Fig. 4-2), whereas thick filaments, which require re-polymerization after mechanical perturbation or contractile unit shortening, require  more time to adapt to a length  change.  Intermediate filaments are a good candidate for rearranging the actin filament lattice because they are abundant in smooth muscle, associate with dense bodies and dense bands, and have previously been shown to affect mechanical properties of smooth muscle (Small and Gimona, 1998).  There is also an increasingly popular notion that thin  filaments (or thin filament associated proteins such as caldesmon) provide a framework  64  upon which myosin monomers can polymerize into thick filaments (Gunst and Tang, 2000; Katayama et al., 1995; Kudryashov et al., 2002; Seow, 2005; Small et al., 1992). This suggests that the thin filament lattice must be in place before myosin polymerization can occur, also backing up the idea that alterations in the thin filament lattice are faster than those o f the thick filament lattice.  65  adapted at L f re  adapted at 1.5 L  adapted at 2 L  Fig. 4-2.  ref  ref  A proposed model to explain the rearrangement of contractile units in  series in response to length change. Intermediate fdaments (shown as thin grey nonstraight lines) are attached to dense bodies in sequence such that stretching the cell brings additional contractile units into the series with little disruption o f existing units.  66  4.5 The intermediate state after quick-stretch or quick-release  We believe that the current results may not reflect the most immediate effects o f a length change, i.e., when the muscle has not yet undergone any adaptation to its new length. Our findings may instead reflect a state o f partial adaptation of the muscle to its new length.  A t this intermediate  state, the rearrangement o f contractile units in series is  complete, but the repolymerization o f thick filaments is not.  We have modified our  hypothesized model o f structural rearrangements occurring upon length change to include this intermediate state (Fig. 4-3).  Mechanical behaviour (force and velocity) and  structural outcomes (myosin filament density) o f each o f the three steps after length change shown in our model are described in Fig. 4-4.  There are most likely several steps that occur during length adaptation, but we highlight three in our modified model.  The first step after length change is identical to that  hypothesized in Section 1.8 (Fig. 1-5). We believe this is the outcome o f length change from 1.5 Lref i f no adaptation to the new length has occurred. The force, velocity and filament density associated with this state are shown with a dotted line in Fig. 4-4.  The  second step after the length change would be the intermediate state that we observed in our protocol by the first contraction after length change. line in F i g . 4-4.  This is represented by a pink  Finally, full adaptation to the new length is shown as the last step o f the  length change and is represented as a solid black line in Fig. 4-4.  67  The first step after length change is expected to look as such for the reasons discussed in the Section 1.8. Briefly, the expected force after Q S or Q R comes from data by Herrera et al. (2005) where a 33% Q R caused a 40% decrease in force (shown in F i g . 4-1) and a 30% Q S caused a ~10% drop in force. Myosin filament density shows the same relative response as force since the current and previous data show that they are correlated. To simplify the modified model by showing only one row o f contractile units, the decrease in force is shown as a decrease in thick filament length (but in fact, the decrease in force and density could be accomplished by either a decrease in thick filament length, a decrease in contractile units in parallel, or a combination o f both).  The velocity o f  shortening while the muscle has not undergone any adaptation to the new length has yet to be shown, but we speculate it is as described in the graph (discussed in Section 1.8 and earlier in this section), and is represented in the model by no change in the number o f contractile units in series.  The intermediate state is expected to look as such because o f the data presented in this dissertation. The force data is shown as in Fig. 3-2, and the filament density data is again assumed to match the force by changing thick filament length in the model. A s velocity at this intermediate state was shown to be identical to the value at its newly adapted length (Section 3.3), the velocity graph shows the pink line following the black line, and the model shows that the number o f contractile units in series is the same as after full adaptation to the new length.  68  The structural manifestations o f a Q S at the intermediate state (compared to the prestretched state) would be the addition of contractile units in series to accommodate the increase in velocity. There would also be a concomitant decrease in fdament overlap o f the original contractile units to account for the small drop in force and myosin fdament density. A t the intermediate state, a 33% stretch (ie. from 1.5 Lref to 2 Lref) would result in a 22% increase in velocity and contractile units in series, since a length doubling causes a 67% velocity increase (Herrera et al., 2005; K u o et al., 2003b; Pratusevich et al., 1995). This is shown in our model as a ~22% increase in the number of contractile units in series. According to data from Section 3.1, this same 33% stretch would cause a force decrease o f 8.8% ± 5 . 9 (Fig. 3-2) which is shown in our modified model as a thick filament shortening o f 10% due to mechanical perturbation (as opposed to a loss o f available thin filament length).  Upon Q R , this intermediate state is caused by both a decrease in contractile units in series as well as a reduction in the overlap o f thick and thin filaments as compared to the prerelease state. The thick filaments in the intermediate state are slightly longer than those immediately after the quick-release to account for the partial force recovery seen in the graph of F i g . 4-1.  However, they still require additional polymerization before  adaptation is complete. In the intermediate state, a Q R o f 33% (i.e. from 1.5 Lref to Lref) would result in a 22% decrease in the number of contractile units in series according to the previous model (Herrera et al., 2005; K u o et al., 2003b; Pratusevich et al., 1995), and a 20.4% ± 0.6 decrease in force according to data from the present study (Fig. 3-2) shown by a 20% reduction in thick filament length.  69  The final state o f length change, i.e., full adaptation, is shown as described by Pratusevich et al. (1995), K u o et al. (2003b) and Herrera et al. (2005).  Again, they  suggested that a 100% increase in adapted length caused no change in force generation, a 67% increase in velocity and a 67% increase in myosin filament density. Therefore, a length change o f 33% (ie. from 1.5 Lref to 2 Lref or 1.5Lref to Lref) would result in no change in contractile unit length, a 22% change in velocity and contractile units in series and a 22% change in myosin filament mass. F i g . 4-3 shows the structural arrangement after adaptation to a new length as presented by K u o et al. (2003b).  70  adapted at 1.5 U f  adapted at 1.5 L  release, no adaptation to new length  intermediate L  stretch, no adaptation to new length  re(  adapted at L  re(  intermediate 2 L  re(  adapted at 2 L  re(  ref  Fig. 4-3. Modified model of structural changes after length change. Top panel shows effects o f a length decrease. Bottom panel shows effects o f a length increase.  Force (%F )  Myosinfilamentdensity (% change)  max  L f  1.5L f  re  re  2L f re  Lref  1.5L f re  2L f re  Velocity (% change)  L f re  1.5Lref  2L r re  Fig. 4-4. Structural and functional outcomes at three states after a change in length from 1.5 L f . re  Values are reflective o f model i n F i g . 4-3. Dotted line with diamonds  represent outcomes after length change when muscle is still fully adapted to original length (1.5 L f ) , grey line with squares represent the findings i n this study, i.e., the re  intermediate state, and solid black line with triangles represent full adaptation to the new length.  71  4.6 Alternative mechanisms  Other possible interpretations o f the immediate change in velocity upon length change could be related to changes in calcium levels or M L C K activity. A n increase in calcium levels or M L C K activity would cause an increase in myosin phosphorylation and therefore more rapid cross-bridge cycling. This would cause an increase in velocity o f muscle shortening and could therefore account for the changes observed after stretch. Similarly, a decrease in M L C K activity or calcium levels could cause the decreased velocity after release. More experiments would be needed to determine whether these interpretations are valid, but our model assumes that they are not. The current theories regarding calcium and M L C K regulation cannot explain the structural changes observed in the present study. The changes in myosin fdament density observed suggest the force and  velocity alterations seen after a length change is likely due to restructuring o f  contractile apparatus at the contractile fdament level.  72  4.7 Physiological relevance  The purpose o f this dissertation is to examine the structural and functional state o f airway smooth muscle after a length change but before full adaptation to the new length. This intermediate state is perhaps more physiologically relevant than the fully adapted state which can only be observed under static conditions.  Under in vivo conditions, the  airways are continuously perturbed by the action o f breathing because o f the tethering o f lung parenchyma to the airway wall.  The perpetual oscillatory strain on the airway  smooth muscle disallows the muscle to adapt fully to any particular length. The present findings have provided us a clearer picture o f the dynamic state o f mechanical performance o f airway smooth muscle during adaptation. If maintaining airway patency is the goal, then reduction o f active muscle force is a positive outcome o f length oscillation.  The asymmetry o f force decrease observed in this study also points out  another positive aspect o f airway smooth muscle behavior, that is, greater force loss is associated with shortening compared with that associated with lengthening. Occlusion o f the airways is more likely to occur i f force is not diminished at short lengths. Yet another positive aspect o f muscle behavior is the immediate reduction in shortening velocity upon reduction in muscle length, even before the muscle force has a chance to recover.  A  reduction in muscle length is presumably associated with a narrowed airway. A greater shortening velocity in a narrowed airway would increase the likelihood o f airway closure, whereas in a distended airway the velocity o f shortening may not be as crucial. Considering the fact that airway smooth muscle is capable o f shortening by an amount equivalent to >80% o f its original length (Herrera et al., 2005), it is perhaps not difficult  73  to understand that the dynamic in vivo environment where airway smooth muscle reside and how the muscle react to the externally imposed forces all worked together to prevent excessive shortening o f the muscle. It is not difficult either to imagine that this delicate balance could be disturbed under pathological conditions leading to airway obstruction.  74  References  Ashton, F. T., Somlyo, A . V. and Sondyo, A . P. (1975). The contractile apparatus o f vascular smooth muscle: intermediate high voltage stereo electron microscopy. 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