help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on September 4, 2003, doi:10.1165/rcmb.2003-0254OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0254OCv1
30/3/326    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tuck, S. A.
Right arrow Articles by Lauzon, A.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tuck, S. A.
Right arrow Articles by Lauzon, A.-M.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 326-332, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0254OC

Time Course of Airway Mechanics of the (+)Insert Myosin Isoform Knockout Mouse

Stephanie A. Tuck, Karim Maghni, Annie Poirier, Gopal J. Babu, Muthu Periasamy, Jason H. T. Bates, Renaud Leguillette and Anne-Marie Lauzon

Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal; Sacre-Coeur Hospital, Research Center, Respiratory Division, Université de Montréal, Montréal, Quebec, Canada; Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio; and Departments of Medicine and Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont

Address correspondence to: Anne-Marie Lauzon, Ph.D., Meakins Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St., Montréal, PQ, H2X 2P2 Canada. E-mail: anne-marie.lauzon{at}mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two smooth muscle myosin heavy chain isoforms that differ by the presence ([+]insert) or the absence ([-]insert) of a 7–amino acid insert in the motor domain have a 2-fold difference in their in vitro actin filament velocity. We hypothesized that a preferential expression of the fast (+)insert isoform in airway smooth muscle would increase the rate of bronchoconstriction. To verify our hypothesis we measured the time course of bronchoconstriction following a bolus injection of methacholine (160 µg/kg) in (+)insert isoform knockout (KO) and corresponding wild-type (WT) mice. Neither baseline airway resistance (Raw) (0.424 ± 0.04 for WT and 0.374 ± 0.01 cm H2O·s·ml-1 for KO) nor peak Raw (4.1 ± 0.9 for WT and 4.0 ± 0.5 cm H2O·s·ml-1 for KO) differed between groups. However, the time to peak Raw was significantly longer in the KO (17.2 ± 0.6 s) compared with the WT (14.6 ± 0.8 s) mice (P < 0.05). Differentiating Raw with respect to time revealed a greater rate of bronchoconstriction for the WT during the initial 4 s, presumably reflecting the faster shortening velocities under these relatively unloaded conditions. Reverse transcriptase–polymerase chain reaction analysis revealed that the (+)insert myosin isoform mRNA content in the WT airways was 47.8 ± 5.6%. We conclude that the presence of the (+)insert myosin isoform in the airways increases the rate of bronchoconstriction.

Abbreviations: knockout, KO • methacholine, MCh • polymerase chain reaction, PCR • small animal ventilator, SAV • smooth muscle myosin heavy chain, SMMHC • wild-type mice, WT mice


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vertebrate smooth muscle exhibits a large range of contraction rates (1, 2). Indeed, based on electrophysiologic measurements, smooth muscle was originally divided into two categories: tonic, slowly contracting, multiunit, tone maintaining (as in most blood vessels), and phasic, rapidly contracting, single unit (as in the bladder and intestine) (1). At the molecular level, smooth muscle myosin heavy chain (SMMHC) isoforms are believed to contribute to these differences in rate of shortening. Two of the four SMMHC isoforms, generated by alternative mRNA splicing at the amino-terminus, (3, 4) differ by the presence ([+]insert isoform) or absence ([-]insert isoform) of a 7–amino acid insert in the motor domain, near the ATPase site. The presence of the insert doubles the actin-activated ATPase activity and the rate of actin filament movement in the in vitro motility assay (5–8). The (+)insert isoform is preferentially expressed in phasic muscle, whereas the (-)insert isoform is found predominantly in tonic muscle. Two other SMMHC isoforms are generated by alternative mRNA splicing at the carboxyl-terminus (3, 4), but no difference in their mechanical properties has been reported (9). Isoforms of the essential light chain coexpressed with the heavy chain isoforms (LC17a with the [+]insert and LC17b with the [-]insert isoform) were also once believed to contribute to the smooth muscle kinetic differences (see review from Ref. 10), but Rovner and coworkers (11) have since clearly demonstrated that the SMMHC insert is necessary and sufficient to double the rate of actin filament movement in the in vitro motility assay.

The goal of the current study was to determine if the content in (+) and (-)insert SMMHC isoforms affects the rate of bronchoconstriction in vivo. Hyperresponsive airways exhibit an increased rate and extent of smooth muscle shortening when subjected to a bronchoconstrictive challenge (12–16). A greater content of the fast (+)insert SMMHC isoform in hyperresponsive airways could contribute to these enhanced mechanical properties. Furthermore, correlations have been established between development, hormone level, pathologies, and myosin isoform content (17–19). This suggests that the differential expression of the myosin isoforms must have a functional significance, although the implications for airway smooth muscle are unclear. Both the (+) and (-)insert isoforms have been shown to be expressed in airway smooth muscle (20–22), presumably causing alterations in its mechanical properties at the molecular level. However, whether myosin isoform expression can affect lung function at the whole organ level is unknown. We therefore chose to measure the rate of change of lung mechanics after a challenge of intravenous methacholine (MCh) in knockout mice (KO) deficient in the (+)insert myosin isoform and corresponding wild-type mice (WT) (23). The KO was engineered in a normo-responsive background to avoid any potentially confounding factors involved with airway hyperresponsiveness. We hypothesized that expression of the (+)insert isoform would be associated with a faster time course of bronchoconstriction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
KO mice genetically deficient in the (+)insert myosin isoform and the corresponding WT mice were created by targeted deletion of the 5b exon of the SMMHC gene (23). The (+)insert isoform was completely absent from these KO mice and the splicing switched to the (-)insert isoform (23). Both male and female mice were studied at 5–6 wk of age (24–26 g). Protocols were approved by an institutional ethics committee.

Respiratory Mechanics
Animals were sedated with xylazine hydrochloride (10 mg/kg intraperitoneally) and anesthetized with sodium pentobarbital (40 mg/kg intraperitoneally). The trachea was cannulated with a snug-fitting metal needle connected to a small animal ventilator (SAV, flexiVent, Scireq; Scientific Respiratory Equipment, Montreal, PQ, Canada). The mice were ventilated with sinusoidal inspiration and passive expiration with an amplitude of 0.18 ml and at a rate of 150 breaths/min. A positive end-expiratory pressure (PEEP) of 1.5 cmH2O was established by connecting the expiratory line of the ventilator to a water column. Before measurements, the mice were paralyzed with pancuronium bromide (0.8 mg/kg intravenously).

The SAV is a computer-controlled ventilator in which a piston in a cylinder of known diameter is driven by a linear motor (24). Specific volume (V) perturbations (see below) were applied by the SAV to the airway opening to evaluate respiratory mechanics in the mice. To estimate respiratory system mechanical parameters, measurements were made of piston volume displacement (V) and pressure inside the cylinder (P). Before each experiment, the SAV was calibrated as previously reported (25, 26).

Time Course of Bronchoconstriction
The time course of bronchoconstriction to intravenous MCh was measured in 10 WT and 17 KO mice. An inflation to a P of 30 cm H2O was given by closing the expiratory line followed 2 min later by a 4-s V oscillation signal (described below) applied to the tracheal opening to measure baseline mechanics. A second inflation to a P of 30 cm H2O was given followed 2 min later by a bolus injection of MCh (160 µg/kg). Immediately following MCh administration, a 24-s V oscillation signal was applied to the tracheal opening.

Both the 4- and 24-s V oscillation signals were composed of a repeating 2-s signal containing 13 sinusoids with frequencies ranging from 1–20.5 Hz. The sinusoid frequencies were mutually prime and their amplitudes decreased hyperbolically with frequency. The phases of the sinusoids were chosen to minimize the peak-to-peak volume excursion of the signal. The signal was scaled to have a maximum peak-to-peak volume excursion of 0.18 ml.

Respiratory system impedance (Zrs) was calculated by dividing the fast Fourier transform of P by the transform of V at the 13 frequencies present in V, and then dividing the result by -j2{pi}f. The following model described by Hantos and coworkers (27) was fit to (Zrs):

(1)
where Raw represents the frequency-independent airway resistance connected to a constant-phase tissue compartment, where I is inertance of the gas in the airways, Gti reflects the viscous dissipation of energy within the lung tissue during inflation and deflation, and Hti reflects energy storage in the respiratory tissues, and

(2)
For baseline measurements, lung impedance was calculated from the entire 4-s signal. For the 24-s signal used post-MCh, lung impedance was calculated using a 2-s window spaced every 32 points over the 24 s data record giving impedance measurements every 0.125 s. The resulting signals of Raw, Gti, and Hti versus time were then smoothed with a 2-s running mean. Differences in baseline and peak values and time to peak were tested by t test.

Airway Responsiveness to MCh
For sake of completeness, dose–response curves to intravenous MCh were also measured in 8 WT and 13 KO mice. An inflation to a P of 30 cm H2O was given, followed 2 min later by baseline measurements. A 2.5-Hz sinusoidal V oscillation of 0.18 ml amplitude and 1.2 s duration was applied to the airway opening. The 2.5-Hz oscillation was applied after the animal expired passively to PEEP. Respiratory system resistance (R) and elastance (E) were estimated by fitting the data to the single compartment model of the respiratory system where

(3)
where is flow. After baseline measurements, a bolus injection of MCh was given (volume of injection = 1 µl/g body weight) and estimates of R and E were obtained every 15 s until peak R was reached. Injection of the next highest dose of MCh was given 30 s later. Doubling doses of MCh from 10–320 µg/kg were used. Responsiveness was measured as the log dose of MCh required to double baseline R (logED200).

Myosin Isoform Determination by Multiplex Reverse Transcriptase–Polymerase Chain Reaction
The relative proportion of the (+) and (-)insert SMMHC isoforms present in the WT mice trachealis muscle, whole lung tissue, bladder (positive control for the [+]insert isoform), and aorta (positive control for the [-]insert isoform) was determined at the mRNA level. The mice were killed with an overdose of sodium pentobarbital administered intravenously, and the trachea, a caudal piece of lung, the bladder, and the aorta were rapidly harvested, immediately frozen, and kept at -80°C until assayed. Tissues were powdered in liquid nitrogen and total RNA was extracted from the homogenates with TRIZOL (Invitrogen, Burlington, ON, Canada) as previously described (28). Strand cDNA was made in 20-µl reactions using 2 µg of total RNA as template, oligo(dT)12–18 primer and Superscript II enzyme, and enzyme inhibitors (acetylated BSA and RNAguard RNase) (2 h incubation at 42°C). The polymerase chain reaction (PCR) mixture consisted of (final concentration) 1.5 mM MgCl2, 1x PCR buffer, 0.2 mM dNTP mixture, 2.5 U Platinum Taq polymerase (Invitrogen), 20 pmol of the upstream and downstream primers for the SMMHC gene, and 2 pmol of the upstream and downstream primers of cyclophilin, as well as the synthesized cDNA strand. PCR primers for the mouse SMMHC were designed (Genbank accession no. 1945079) to amplify both myosin isoforms, i.e., with (+) or without (-) the 21-nucleotide insert sequence: 5'sense primer 5'-GAA AGT CAT ACA GTA CTT GGC TGT G- 3' (671–695 nucleotides) and 3'antisense primer 5'-GAG TTG TCG TTC TTG ACG GTT TTC GCA TTG CC-3' (820–789 nucleotides). The SMMHC samples were coamplified with the housekeeping gene cyclophilin (primers 10-fold less concentrated to avoid saturation of cyclophilin amplification in our PCR conditions), used as an internal standard, in a Programmable Thermal Controller (PTC-200; MJ Research Inc., Watertown, MA) for 33 cycles (45 s denaturation at 92°C, 1.40 min annealing at 58°C, and 2.40 min of extension at 65°C). The product of the multiplex PCR was visualized by ethidium bromide staining after electrophoresis on agarose gel (3.5%) and the calculation of the size of the amplicons and the semiquantitative PCR analysis of the agarose gels was performed using the MultiGenius Bio Imaging System (Syngen, London, UK). The results are reported as mean ± SE.

Histology
Lung tissues from WT and KO mice were fixed in 10% neutral buffered formalin, dehydrated through a gradient of alcohols, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time Course of Bronchoconstriction
Animal weight did not differ between WT and KO mice (24.9 ± 1.4 g versus 25.8 ± 1.0 g for WT and KO mice, respectively). Baseline respiratory mechanics did not differ between WT and KO mice (Table 1). The time courses of Raw, Gti, and Hti after injection of 160 µg/kg MCh are shown in Figure 1. No differences in Raw, Gti, or Hti were found between groups at time = 0 s. After MCh injection, both Raw and Gti increased, reaching a maximum by 15–20 s after injection. Neither peak Raw (Figure 2) nor peak Gti differed between WT (Raw = 4.1 ± 0.9 cm H2O · s · ml-1; Gti = 11.5 ± 2.0 cm H2O · ml-1) and KO mice (Raw = 4.0 ± 0.5 cm H2O · s · ml-1; Gti = 11.2 ± 0.6 cm H2O · ml-1). The time to peak Raw was, however, significantly longer in the KO (17.2 ± 0.6 s) compared with the WT (14.6 ± 0.8 s) mice (Figure 2, P = 0.036). Similarly, the time to peak Gti tended to be longer in the KO (16.5 ± 0.6 s) compared with the WT (14.2 ± 0.9 s) but did not reach statistical significance (P = 0.054; Figure 1). Hti increased at a similar rate in both groups, but unlike Raw and Gti, did not reach a peak within the 24 s measurement period (Figure 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Baseline respiratory mechanics

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Time course of airway resistance (Raw), energy dissipation (Gti), and energy storage (Hti) after intravenous bolus injection of 160 µg/kg MCh. Each curve represents mean ± SE. Filled circles, WT mice; open circles, (+)insert KO mice. Data are plotted only every 5 points.

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. Peak Raw and time to peak Raw after intravenous bolus injection of 160 µg/kg MCh. Values are mean ± SE. *P < 0.05 compared with WT mice.

 
Airway Responsiveness to MCh
To perform a complete characterization of the mechanics of bronchoconstriction in these animals, their responsiveness to MCh was also measured. Baseline R and E (before MCh injection) did not differ between WT (R = 1.29 ± 0.16 cm H2O · s · ml-1, E = 62.4 ± 4.7 cmH2O.ml-1) and KO (R = 1.04 ± 0.06 cm H2O · s · ml-1, E = 52.9 ± 2.4 cm H2O · ml-1) mice. MCh injection resulted in dose-dependent increases in both R and E in both groups (Figure 3). However, there was no difference in responsiveness to MCh between groups; the logED200 for R was 1.86 ± 0.07 for WT and 1.87 ± 0.03 for KO mice.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Respiratory system resistance and elastance in response to incremental intravenous bolus injections of MCh. Each curve represents mean ± SE. Filled circles, WT mice; open circles, (+)insert KO mice.

 
Myosin Isoform Determination by RT-PCR Analysis
To quantify the myosin isoform content in the airways of the WT mice, we measured by semiquantitative RT-PCR, the (+) and (-)insert isoform content in whole lung tissues, trachea, bladder, and aorta. The size of the (+) and (-)insert amplicons corresponded to the predicted sizes, i.e., 160 and 149bp for the (+) and (-)insert respectively. Also, the bladder, which is known to preferentially express the (+)insert myosin isoform, contained almost exclusively the (+)insert isoform mRNA (92.4 ± 1.4% [+]insert isoform [n = 2]; Figure 4A) whereas the aorta, which is known to preferentially express the (-)insert myosin isoform, contained almost exclusively the (-)insert isoform mRNA (23.4 ± 13.5% [+]insert isoform [n = 3]; Figure 4A). These two results confirmed that the primers functioned well. In the KO mouse bladder, as expected, there was a complete switch in expression to the (-)insert isoform (Figure 4A). This result confirms the previously reported smooth muscle phenotype change in this KO model (23). The WT mouse lung tissue and trachealis muscle expressed both isoforms but in different proportions. The WT mouse lung tissue expressed slightly more of the (-)insert than the (+)insert myosin isoforms (47.8 ± 5.6% [+]insert isoform [n = 4]; Figure 4B). The trachea, on the other hand, had more of the (+)insert than the (-)insert isoform (68.8 ± 4.4% [+]insert isoform [n = 4]; Figure 4C).



View larger version (102K):
[in this window]
[in a new window]
  		Figure 4. Multiplex RT-PCR results: (A) B: bladder, A: aorta. (B) L: lung. (C) T: trachea. Top band: cyclophilin; lower 2 bands: (+) and (-)insert SMMHC isoforms for WT (left panel) and KO (right panel) mice.

 
Histology
Lung histology showed no visually discernible differences between WT and KO mice sections (Figure 5).



View larger version (104K):
[in this window]
[in a new window]
  		Figure 5. Histologic staining of WT and KO lung sections. Sections were stained with hematoxylin and eosin, x40 magnification.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of this study is that the presence of the fast (+)insert SMMHC isoform in airway smooth muscle contributes to a faster time course of bronchoconstriction.

Significance of the Presence of the (+)Insert Myosin Isoform
Although the (+)insert SMMHC isoform is clearly associated with a greater rate of actin movement in the in vitro motility assay (5–8), there is controversy about its role in physiologic systems (18). Eddinger and Meer (29) showed that isolated rabbit stomach antral smooth muscle cells contained ~ 3 times more (+)insert isoform mRNA than fundic cells and contracted ~ 3 times faster. Sjuve and colleagues (30) reported that the maximal shortening velocity and the rate of force development were reduced by ~ 20% in hypertrophied rat bladders compared with control and that the relative amount of (+)insert isoform was decreased by 50%. Absence of correlations between mechanical performance and the (+)insert isoform content has also been reported. An increase in maximal shortening velocity was observed both in the pregnant hypertrophied rat myometrium (31) and in the mouse megacolon of Hirschsprung's disease (32) whereas the (+)insert isoform expression was decreased. Maximal shortening velocity of estradiol treated rat uterus was seen to increase, whereas the (+)insert isoform content decreased (19). Other reports have compared myosin isoform content and mechanical properties between different organs and showed no significant correlation (18). The discrepancies between these various reports addressing the role of the (+) and (-) insert SMMHC isoforms at the whole organ level most likely come from the multiple interactions between various factors such as inflammatory mediators, hormone levels, connective tissue attachments, etc.

Significance of the Presence of the (+)Insert Myosin Isoform for Airway Responsiveness
Evidence is accumulating to suggest that airway smooth muscle relaxes with each tidal breath (33). It is also well known that if prevented from taking deep inspirations, normal humans have an exaggerated response, similar to that of subjects with asthma, when challenged with bronchoactive agents (34, 35). Gunst (36) and Solway and Fredberg (33) have therefore suggested that the kinetics of muscle contraction must be crucial in bronchial hyperresponsiveness. That is, tidal breathing and deep inspirations presumably detach cross-bridges, which would hinder the contraction of slowly contracting muscles more than that of fast ones, i.e., rapidly contracting muscle is able to constrict more fully between each breath or deep inspiration (33, 36). A greater proportion of the (+)insert fast isoform would likely result in a greater rate of contraction of hyperresponsive muscle. As correlations have already been established between pathologies and myosin isoform content (17–19), it is reasonable to expect that myosin isoform expression could also be altered in airway hyperresponsiveness.

The increased rate of smooth muscle shortening of hyperresponsive airways has also been addressed at the muscle strip level by Lecarpentier and coworkers (37). They studied the mechanical properties of isolated trachealis muscle from the Fisher and Lewis rats, and reported a greater maximum shortening velocity and extent of shortening for the hyperresponsive Fisher compared with the less responsive Lewis rat (37). Furthermore, we recently measured the (+) and (-)insert isoform content in the trachealis muscle of Fisher and Lewis rats both at the mRNA and protein levels (38) and found a greater content of the fast (+)insert isoform in the Fisher rat. Taken together, these data suggest a role for the (+)insert isoform in the rate and extent of shortening of airway smooth muscle and consequently for airway responsiveness.

Time Course of Airway Mechanics and Myosin Isoform Content
To directly assess the influence of myosin isoform content on the time course of bronchoconstriction and airway responsiveness, we studied the pulmonary mechanics of mice deficient in the (+)insert isoform. We found that Raw increased more slowly in the KO mice as evidenced by a delayed peak value (Figures 1–2). Although these differences are subtle, they demonstrate a measurable effect of myosin isoform content on airway smooth muscle physiology. Of course, this conclusion can only be drawn if any potential differences in the kinetics of drug access to airway smooth muscle have been ruled out. There are two ways by which such differences between the WT and the KO animals could be introduced. First, there could be differences in circulatory parameters altering the delivery time of the drug to the airway smooth muscle. However, circulatory parameters and blood pressure (data not shown) and heart rate and cardiac function (23) have been measured before and shown to be the same between these WT and KO mice. Second, there could be differences in smooth muscle thickness again altering the delivery time of the drug. However, our tissue histology showed no visually discernible differences between WT and KO mouse lungs (Figure 5). We therefore conclude that the differences seen in the time course of bronchoconstriction between KO and WT reflect differences in airway smooth muscle physiology.

Further insight into the functional differences between KO and WT mice is obtained if we differentiate Raw with respect to time (Figure 6). The initial rate of contraction ({delta}Raw/{delta}t) is slower in the KO compared with the WT (Figure 6), which we speculate reflects the faster shortening velocities in the WT animals under the relatively unloaded conditions immediately following MCh injection. As shortening progresses, however, the distortion of the airway wall and surrounding parenchyma present an increasing load opposing the smooth muscle. The rate at which smooth muscle shortening proceeds then becomes a function of its force–velocity relationship. We know that these two myosin isoforms generate the same average force (6), so it is quite possible that the load becomes an equalizing influence after the first few seconds of contraction. In other words, because the WT muscle starts off shortening more rapidly than the KO, it faces an opposing parenchymal load sooner. In fact, it can be seen from Figure 6 that the rates of contraction cross over at ~ 4 s, after which the KO contracts faster. The rate of recovery is initially faster for the WT animals at ~ 15–20 s, agreeing again with a greater opposing load to contraction, and then becomes similar to that of WT at ~ 21–24 s. Thus, it seems that, in trying to understand the role of smooth muscle isoforms on bronchial responsiveness, we cannot consider the effects of the muscle independently of the mechanical load against which it must act.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 6. Rate of change of Raw with respect to time ({delta}Raw/{delta}t) after intravenous bolus injection of 160 µg/kg MCh. Each curve represents mean ± SE. Filled circles, WT mice; open circles, (+)insert KO mice. Data are plotted only every 5 points.

 
Airway Responsiveness to MCh
On the basis of our above speculations, we would predict that the KO and WT mice would not necessarily have different maximal responses to MCh because of the limiting influence of parenchymal load. In fact, this turned out to be the case; myosin content did not affect the magnitude of response in Raw (Figure 2) or its dose–response behavior (Figure 3). We must point out, however, that a large change in responsiveness was actually not expected because this KO was made on a C57BL/6 background that has previously been reported to be hyporesponsive when compared with A/J, Balb/C, and C3H/HeJ mice (39). Therefore, any difference in pulmonary mechanics due to a further reduction in responsiveness would be small. Decreases in rate of shortening and maximal force generation have been reported in this KO model in bladder and mesenteric vessels (23) but these muscles are phasic and predominantly express the (+)insert isoform (18, 20). According to our RT-PCR data, this is not the case for the pulmonary airways of these hyporesponsive WT mice with only 47.8 ± 5.6% (+)insert isoform. The content in the trachealis muscle was greater (68.8 ± 4.4%), but the tracheal contribution to the magnitude of bronchoconstriction remains to be determined. Therefore, a relatively low amount of (+)insert isoform in the WT pulmonary airways may have contributed to our observation of no difference in bronchial responsiveness between KO and WT mice. Testing this possibility will have to await the development of a KO mouse constructed on a hyperresponsive mouse background.

In conclusion, by knocking out the fast (+)insert SMMHC isoform we were able to observe a significant decrease in the early rate of bronchoconstriction following MCh challenge, likely due to the reduced shortening velocity of the unloaded muscle compared with WT. We hypothesize that, with progression of shortening, the increasing opposing load presented by the distorting parenchyma equalized the rates of shortening. Our study thus shows that differences in molecular structure of smooth muscle can have a measurable effect on physiologic function.


   Acknowledgments
 
The authors thank Dr. Heberto Ghezzo for his many suggestions. This research was supported by an operating grant from the Association Pulmonaire du Québec. A.-M.L. and K.M. are recipients of salary awards from the Fonds de la recherche en santé Québec; G.J.B. is supported by the American Heart Association grant 0365173B; M.P. is supported by NIH grant HL 38355-15; J.H.T.B. is supported by NIH grants R01 HL-62746, R01 HL-67273, and NCRR-COBRE P20 RR-15557; S.A.T. received a postdoctoral fellowship from the Research Institute of McGill University Health Center; and R.L. received a postdoctoral fellowship from the Montreal Chest Institute.

Received in original form July 2, 2003

Received in final form August 20, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Somlyo, A. V., and A. P. Somlyo. 1968. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmacol. Exp. Ther. 159:129–145.[Abstract/Free Full Text]
  2. Malmqvist, U., and A. Arner. 1991. Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle. Pflugers Arch. 418:523–530.[CrossRef][Medline]
  3. Eddinger, T. J., and R. A. Murphy. 1988. Two smooth muscle myosin heavy chains differ in their light meromyosin fragment. Biochemistry 27:3807–3811.[CrossRef][Medline]
  4. Nagai, R., M. Kuro-o, P. Babij, and M. Periasamy. 1989. Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J. Biol. Chem. 264:9734–9737.[Abstract/Free Full Text]
  5. Kelley, C. A., and R. S. Adelstein. 1994. Characterization of isoform diversity in smooth muscle myosin heavy chains. Can. J. Physiol. Pharmacol. 72:1351–1360.[Medline]
  6. Lauzon, A. M., M. J. Tyska, A. S. Rovner, Y. Freyzon, D. M. Warshaw, and K. M. Trybus. 1998. A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap. J. Muscle Res. Cell Motil. 19:825–837.[CrossRef][Medline]
  7. Lauzon, A. M., K. M. Trybus, and D. M. Warshaw. 1998. Molecular mechanics of two smooth muscle heavy meromyosin constructs that differ by an insert in the motor domain. Acta Physiol. Scand. 164:357–361.[CrossRef][Medline]
  8. Rovner, A. S., Y. Freyzon, and K. M. Trybus. 1995. Chimeric substitutions of the actin-binding loop activate dephosphorylated but not phosphorylated smooth muscle heavy meromyosin. J. Biol. Chem. 270:30260–30263.[Abstract/Free Full Text]
  9. Kelley, C. A., J. R. Sellers, P. K. Goldsmith, and R. S. Adelstein. 1992. Smooth muscle myosin is composed of homodimeric heavy chains. J. Biol. Chem. 267:2127–2130.[Abstract/Free Full Text]
  10. Somlyo, A. P. 1993. Myosin isoforms in smooth muscle: how may they affect function and structure? J. Muscle Res. Cell Motil. 14:557–563.[CrossRef][Medline]
  11. Rovner, A. S., Y. Freyzon, and K. M. Trybus. 1997. An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms. J. Muscle Res. Cell Motil. 18:103–110.[CrossRef][Medline]
  12. Antonissen, L. A., R. W. Mitchell, E. A. Kroeger, W. Kepron, K. S. Tse, and N. L. Stephens. 1979. Mechanical alterations of airway smooth muscle in a canine asthmatic model. J. Appl. Physiol. 46:681–687.[Free Full Text]
  13. Fan, T., M. Yang, A. Halayko, S. S. Mohapatra, and N. L. Stephens. 1997. Airway responsiveness in two inbred strains of mouse disparate in IgE and IL-4 production. Am. J. Respir. Cell Mol. Biol. 17:156–163.[Abstract/Free Full Text]
  14. Jiang, H., K. Rao, A. J. Halayko, X. Liu, and N. L. Stephens. 1992. Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle. Am. J. Respir. Cell Mol. Biol. 7:567–573.
  15. Mitchell, R. W., E. Ruhlmann, H. Magnussen, A. R. Leff, and K. F. Rabe. 1994. Passive sensitization of human bronchi augments smooth muscle shortening velocity and capacity. Am. J. Physiol. 267:L218–L222.
  16. Wang, C. G., J. J. Almirall, C. S. Dolman, R. J. Dandurand, and D. H. Eidelman. 1997. In vitro bronchial responsiveness in two highly inbred rat strains. J. Appl. Physiol. 82:1445–1452.[Abstract/Free Full Text]
  17. Samuel, J. L., L. Rappaport, J. J. Mercadier, A. M. Lompre, S. Sartore, C. Triban, S. Schiaffino, and K. Schwartz. 1983. Distribution of myosin isozymes within single cardiac cells: an immunohistochemical study. Circ. Res. 52:200–209.[Abstract/Free Full Text]
  18. Wetzel, U., G. Lutsch, H. Haase, U. Ganten, and I. Morano. 1998. Expression of smooth muscle myosin heavy chain B in cardiac vessels of normotensive and hypertensive rats. Circ. Res. 83:204–209.[Abstract/Free Full Text]
  19. Calovini, T., H. Haase, and I. Morano. 1995. Steroid-hormone regulation of myosin subunit expression in smooth and cardiac muscle. J. Cell. Biochem. 59:69–78.[CrossRef][Medline]
  20. White, S., A. F. Martin, and M. Periasamy. 1993. Identification of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the S1 head region. Am. J. Physiol. 264:C1252–C1258.
  21. Ma, X., and N. L. Stephens. 1997. Difference in expression of myosin heavy chain head (with 7-amino acid insert) isoform between canine tracheal and bronchial smooth muscle. Am. J. Respir. Crit. Care Med. 155:A370. (Abstr.).
  22. White, S. L., M. Y. Zhou, R. B. Low, and M. Periasamy. 1998. Myosin heavy chain isoform expression in rat smooth muscle development. Am. J. Physiol. 275:C581–C589.
  23. Babu, G. J., E. Loukianov, T. Loukianova, G. J. Pyne, S. Huke, G. Osol, R. B. Low, R. J. Paul, and M. Periasamy. 2001. Loss of SM-B myosin affects muscle shortening velocity and maximal force development. Nat. Cell Biol. 3:1025–1029.[CrossRef][Medline]
  24. Schuessler, T. F., and J. H. Bates. 1995. A computer-controlled research ventilator for small animals: design and evaluation. IEEE Trans. Biomed. Eng. 42:860–866.[CrossRef][Medline]
  25. Bates, J. H., T. F. Schuessler, C. Dolman, and D. H. Eidelman. 1997. Temporal dynamics of acute isovolume bronchoconstriction in the rat. J. Appl. Physiol. 82:55–62.[Abstract/Free Full Text]
  26. Gomes, R. F., X. Shen, R. Ramchandani, R. S. Tepper, and J. H. Bates. 2000. Comparative respiratory system mechanics in rodents. J. Appl. Physiol. 89:908–916.[Abstract/Free Full Text]
  27. Hantos, Z., B. Daroczy, B. Suki, S. Nagy, and J. J. Fredberg. 1992. Input impedance and peripheral inhomogeneity of dog lungs. J. Appl. Physiol. 72:168–178.[Abstract/Free Full Text]
  28. Maghni, K., R. Taha, W. Afif, Q. Hamid, and J. G. Martin. 2000. Dichotomy between neurokinin receptor actions in modulating allergic airway responses in an animal model of helper T cell type 2 cytokine- associated inflammation. Am. J. Respir. Crit. Care Med. 162:1068–1074.[Abstract/Free Full Text]
  29. Eddinger, T. J., and D. P. Meer. 2001. Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity. Am. J. Physiol. Cell Physiol. 280:C309–C316.[Abstract/Free Full Text]
  30. Sjuve, R., H. Haase, I. Morano, B. Uvelius, and A. Arner. 1996. Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder. J. Cell. Biochem. 63:86–93.[Medline]
  31. Haase, H., and I. Morano. 1996. Alternative splicing of smooth muscle myosin heavy chains and its functional consequences. J. Cell. Biochem. 60:521–528.[CrossRef][Medline]
  32. Siegman, M. J., T. M. Butler, S. U. Mooers, L. Trinkle-Mulcahy, S. Narayan, L. Adam, S. Chacko, H. Haase, and I. Morano. 1997. Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation. Am. J. Physiol. 272:G1571–G1580.
  33. Solway, J., and J. J. Fredberg. 1997. Perhaps airway smooth muscle dysfunction contributes to asthmatic bronchial hyperresponsiveness after all. Am. J. Respir. Cell Mol. Biol. 17:144–146.[Free Full Text]
  34. Skloot, G., S. Permutt, and A. Togias. 1995. Airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration. J. Clin. Invest. 96:2393–2403.
  35. Moore, B. J., L. M. Verburgt, G. G. King, and P. D. Pare. 1997. The effect of deep inspiration on methacholine dose–response curves in normal subjects. Am. J. Respir. Crit. Care Med. 156:1278–1281.[Abstract/Free Full Text]
  36. Gunst, S. J. 1983. Contractile force of canine airway smooth muscle during cyclical length changes. J. Appl. Physiol. 55:759–769.[Abstract/Free Full Text]
  37. Blanc, F. X., S. Salmeron, C Coirault, and Y. Lecarpentier. 1999. Strain related differences in airway responsiveness are associated with differences in the shortening capacity of isolated tracheal smooth muscle. Am. J. Respir. Crit. Care Med. 159:A471. (Abstr.).
  38. Lauzon, A.-M., E. Azoulay, and K. Maghni. 2001. Smooth muscle myosin heavy chain isoform expression in airway hyperresponsiveness. Am. J. Respir. Crit. Care Med. 163:A539. (Abstr.).
  39. Duguet, A., K. Biyah, E. Minshall, R. Gomes, C. G. Wang, M. Taoudi-Benchekroun, J. H. Bates, and D. H. Eidelman. 2000. Bronchial responsiveness among inbred mouse strains: role of airway smooth-muscle shortening velocity. Am. J. Respir. Crit. Care Med. 161:839–848.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
A. Cojocaru, C. G. Irvin, H. C. Haverkamp, and J. H. T. Bates
Computational assessment of airway wall stiffness in vivo in allergically inflamed mouse models of asthma
J Appl Physiol, June 1, 2008; 104(6): 1601 - 1610.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. H. T. Bates, A. Cojocaru, H. C. Haverkamp, L. M. Rinaldi, and C. G. Irvin
The Synergistic Interactions of Allergic Lung Inflammation and Intratracheal Cationic Protein
Am. J. Respir. Crit. Care Med., February 1, 2008; 177(3): 261 - 268.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
R. Leguillette and A.-M. Lauzon
Molecular Mechanics of Smooth Muscle Contractile Proteins in Airway Hyperresponsiveness and Asthma
Proceedings of the ATS, January 1, 2008; 5(1): 40 - 46.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
J. G. Martin and T. Jo
Genetic Differences in Airway Smooth Muscle Function
Proceedings of the ATS, January 1, 2008; 5(1): 73 - 79.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. H. T. Bates, A. Cojocaru, and L. K. A. Lundblad
Bronchodilatory effect of deep inspiration on the dynamics of bronchoconstriction in mice
J Appl Physiol, November 1, 2007; 103(5): 1696 - 1705.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. J. Eddinger and D. P. Meer
Myosin II isoforms in smooth muscle: heterogeneity and function
Am J Physiol Cell Physiol, August 1, 2007; 293(2): C493 - C508.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. H. T. Bates and A.-M. Lauzon
Parenchymal tethering, airway wall stiffness, and the dynamics of bronchoconstriction
J Appl Physiol, May 1, 2007; 102(5): 1912 - 1920.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
F. R. Gil, N. B. Zitouni, E. Azoulay, K. Maghni, and A.-M. Lauzon
Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness
Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L932 - L940.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. H. T. Bates and A.-M. Lauzon
Modeling the oscillation dynamics of activated airway smooth muscle strips
Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L849 - L855.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
R. Leguillette, F. R. Gil, N. Zitouni, S. Lajoie-Kadoch, A. Sobieszek, and A.-M. Lauzon
(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues
Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1277 - C1285.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
C. Duracher, F.-X. Blanc, P.-Y. Gueugniaud, J. S. David, B. Riou, Y. Lecarpentier, and C. Coirault
The Effects of Isoflurane on Airway Smooth Muscle Crossbridge Kinetics in Fisher and Lewis Rats
Anesth. Analg., July 1, 2005; 101(1): 136 - 142.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Wahl, T. J. Eddinger, and C.-M. Hai
Sinusoidal length oscillation- and receptor-mediated mRNA expression of myosin isoforms and {alpha}-SM actin in airway smooth muscle
Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1697 - C1708.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0254OCv1
30/3/326    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tuck, S. A.
Right arrow Articles by Lauzon, A.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tuck, S. A.
Right arrow Articles by Lauzon, A.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.