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Am J Physiol Lung Cell Mol Physiol 291: L932-L940, 2006. First published June 9, 2006; doi:10.1152/ajplung.00339.2004
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Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness

Fulvio R. Gil,1 Nedjma B. Zitouni,1 Eric Azoulay,1 Karim Maghni,2 and Anne-Marie Lauzon1

1Meakins-Christie Laboratories, Department of Medicine, McGill University, and 2Research Center, Sacré-Coeur Hospital, Université de Montréal, Montréal, Québec

Submitted 14 September 2004 ; accepted in final form 5 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(–)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (–)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC20) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC17) isoforms, h-caldesmon, and {alpha}-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC20 phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 ± 2.0%) (mean ± SE) than Lewis rats (53.0 ± 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC17 isoform, h-caldesmon, and {alpha}-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 ± 0.8 mN) than Lewis (1.9 ± 0.4 mN; P < 0.05) and greater LC20 phosphorylation level in Fischer (55.1 ± 6.4) than in Lewis (41.4 ± 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC20 lead to smooth muscle hypercontractility.

myosin heavy chain; 7-amino acid insert; phasic muscle; tonic muscle; myosin regulatory light chain; trachealis

AIRWAY HYPERRESPONSIVENESS is defined as an excessive narrowing of the airways in response to a bronchoconstrictive challenge. There are two major lines of evidence suggesting that abnormalities in smooth muscle directly contribute to airway hyperresponsiveness. First, hyperresponsive airway tissue exhibits a state of hypercontractility as evidenced by increased shortening velocity and extent of shortening in response to electrical or pharmacological challenges (5, 12, 25, 33, 46). Second, hyperresponsive airways have an impaired capacity to relax (35, 39). These two observations could be explained by abnormalities at many levels, including muscle cell organization (37, 38), resistance of the connective tissue (4, 28), mechanical properties of the contractile apparatus itself (27), and activation state of the contractile cells (44). The involvement of the molecular components of the contractile apparatus, particularly myosin, has yet to be directly assessed. A difference in the expression of myosin isoforms that vary in their contractile properties could plausibly underlie the enhanced contractile responses in hyperresponsive airway smooth muscle. Although the distribution of such isoforms is known to be influenced by developmental, pathological, and hormonal factors (6, 47, 49), little attention has been given to potential changes in myosin isoform expression in airway hyperresponsiveness.

Myosin is composed of two heavy chains and four light chains [two regulatory (LC20) and two essential (LC17) light chains]. Two isoforms of the smooth muscle myosin heavy chain (SMMHC) are generated by alternative mRNA splicing at the 5'-end (1, 48). These isoforms differ by the presence [(+)insert] or absence [(–)insert] of a 7-amino acid insert in the motor domain, near the ATPase site (48). The expression of these isoforms is tissue specific (1, 48). Slowly contracting tonic muscle is mostly composed of the (–)insert isoform, whereas rapidly contracting phasic muscle predominantly comprises the (+)insert isoform (16, 48). Similarly, two isoforms of LC17 have been reported to have a tissue specific expression, i.e., the acidic isoform LC17a is mostly expressed in phasic muscle, whereas the basic isoform LC17b is mostly expressed in tonic muscle (14, 31, 42). Regardless of the LC17 isoform, the presence of the 7-amino acid insert in the SMMHC motor domain has been shown to be necessary and sufficient to double the actin-activated ATPase activity and the rate of actin filament movement ({nu}max) in the in vitro motility assay (21, 22, 36). Thus a predominance of the fast (+)insert myosin isoform could potentially explain the increased rate and extent of shortening of hyperresponsive airway smooth muscle. Indeed, we recently purified myosin from multiple rat organs and showed, by the in vitro motility assay, that the rate of actin filament movement is proportional to the (+)insert isoform content and in accordance with the contractility of the corresponding organ (23).

Another potential contributor to airway smooth muscle hypercontractility is the extent of LC20 phosphorylation. LC20 phosphorylation is accomplished by a Ca2+-calmodulin complex that activates myosin light chain kinase (MLCK), which in turn phosphorylates LC20. A greater level of activation has been correlated with greater shortening velocity (2, 20). Increased content of MLCK and increased levels of LC20 phosphorylation in tracheal tissue from an allergic dog model of asthma have been reported (20), as well as elevated MLCK mRNA levels in asthmatic cells (25). The combination of elevated levels of LC20 phosphorylation and increased expression of the fast (+)insert SMMHC isoform could greatly potentiate the rate and extent of airway smooth muscle shortening.

In the present study, we characterized SMMHC, LC17, h-caldesmon, and {alpha}-actin content in the trachealis muscle of Fischer and Lewis rats, a model of innate airway hyperresponsiveness. This model is based on differences in airway responsiveness elicited in the absence of airway inflammation. Furthermore, we quantified the level of LC20 phosphorylation of Fischer and Lewis rat tracheal muscle strips during isometric contraction induced by methacholine. Our results demonstrate that, in this model, a difference in myosin isoform expression and alterations in phosphorylation levels are associated with airway hyperresponsiveness. We report a greater expression of the fast (+)insert isoform in the Fischer hyperresponsive rat compared with the Lewis normoresponsive rat but no difference in LC17 isoforms, h-caldesmon, and {alpha}-actin. A greater level of LC20 phosphorylation is also observed in the hyperresponsive Fischer rats. Along with previously reported factors, such as increased Ca2+ mobilization (44) and increased smooth muscle mass (11) in the Fischer compared with the Lewis rat, these results suggest a genetically controlled mechanism by which multiple cellular and molecular functions are altered to produce an enhanced bronchoconstrictive response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Fischer (F344) and Lewis rats 8–12 wk of age were purchased from Harlan and housed in an animal care facility at McGill University before experimentation. All experimental protocols involving animals were approved by the McGill University Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care. For the pulmonary mechanics studies, the animals were given xylazine (7 mg/kg ip) as a preanesthetic agent and were then anesthetized with pentobarbital sodium (35 mg/kg ip). For all other studies, the animals were killed with an overdose of pentobarbital sodium (100 mg/kg ip).

Airway Responsiveness to Methacholine

Aerosolized methacholine (MCh) dose-response curves on airway resistance were performed to verify the previously reported hyperresponsiveness of Fischer vs. Lewis rats (11, 19, 46). Pulmonary mechanics measurements were made in the spontaneously breathing animals using the Quadra-t system (SCIREQ; Montreal, PQ, Canada). This system consists of a 200-ml Plexiglas box comprising a main chamber connected to the atmosphere by a pneumotachograph, and a reference chamber. The rat tracheae were intubated with a 6-cm polyethylene tube (PE240, Becton Dickinson) using a fiber-optic light source shone through the neck to view the vocal cords. The rats were then placed on a heating pad in the lateral decubitus position. Airflow was measured by inserting the tip of the endotracheal tube in the main chamber and measuring the pressure drop across the pneumotachograph (difference between the main and the reference chambers) with a differential pressure transducer (±0.5 kPa, HCX series; Sensortechnics). Esophageal pressure was measured using a saline-filled catheter (PE160, Becton Dickinson) inserted in the lower third portion of the esophagus. The tip of the catheter was connected to a pressure transducer (Transpac4, Abbott Laboratory). The position of the catheter was adjusted until clear cardiac artifacts could be observed. Transpulmonary pressure (PTP) was computed as the difference between esophageal and main chamber pressure. Pulmonary resistance (RL) was calculated by multiple linear regression from PTP and airflow using the Quadra-t software (SCIREQ).

A disposable Hudson nebulizer was used to aerosolize the animals with either saline or MCh for 30 s at an airflow of 8 l/min. Six Fischer and seven Lewis rats were first challenged with saline and then MCh. Starting with a dose of 0.0625 mg/ml, the MCh concentrations were then doubled until RL reached 200% of the saline value. The EC200 RL (the dose necessary to double baseline RL) values are reported.

RT-PCR

The relative proportion of the (+) and (–)insert SMMHC isoform mRNA present in both the Fischer and Lewis rat trachealis muscle was determined. Tracheae [note that the trachealis muscle has been shown to be mechanically representative of peripheral airways in the Fischer and Lewis rats (19)], bladders [positive control for the (+)insert], and aortas [positive control for the (–)insert] from each strain were harvested and kept at –80°C until assayed. Tissues were powdered in liquid N2, and total RNA was extracted from the homogenates with TRIzol (Invitrogen, ON, Canada), as previously described (30). Strand cDNA was made by reverse transcribing 2 µg of total RNA with SuperScript II (Invitrogen). The PCR experiment was conducted with 2.5 units of Platinum Taq polymerase according to the manufacturer’s instructions (Invitrogen). The following PCR primers for the rat SMMHC were designed (Genbank accession no. S61949) to amplify both myosin isoforms, i.e., with (+) or without (–) the 21-nucleotide insert sequence: 5'-sense primer 5'-CAGTATTTGGCTGTGGTGGC-3' (nucleotides 145–164) and 3'-antisense primer 5'-CATTGCCGAAAGCCTCCAGG-3' (nucleotides 280–261). The samples were amplified in a Programmable Thermal Controller (PTC-100, MJ Research) for 30 cycles (1-min denaturation at 92°C, 2-min annealing at 55°C, and 3 min of extension at 72°C) for the rat SMMHC and 25 cycles (1-min denaturation at 92°C, 2-min annealing at 60°C, and 3-min extension at 72°C) for the housekeeping gene cyclophilin: 5'-sense primer 5'-GGTCAACCCCACCGTGTTCTTCG-3' (nucleotides 45–67) and 3'-antisense primer 5'-GTGCTCTCCTGAGCTACAGAAGG-3' (nucleotides 598–576). PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis, and semiquantitative densitometry analysis of the agarose gels was performed using the Fluoro 800 Advanced Fluorescence Imager (Alpha Innotech). Cyclophilin bands were visualized and used to assess the quality of the mRNA. Results were expressed as the ratio of the densitometric values of the (+)insert over the sum of the densitometric values for both isoforms. Only ratios were compared between rat strains, as the determination of isoform expression as a ratio avoids the problem of loading variations. Given that the two primers were designed to amplify both the (+) and (–)insert and that the two amplified cDNA fragments differ by 21 bp, it is probable that the efficiencies of the PCR were very similar for the two isoforms. Therefore, the band intensities on agarose gel for the (+) and (–)insert isoforms should reflect the initial levels of expression of the mRNA transcripts coding for the SMMHC isoforms. Five rats of each strain were studied. The data are presented as means ± SE. To verify that the PCR reactions were performed in the linear range of amplification, the number of cycles was initially varied from 28 to 34. Visualization and semiquantitative analysis were performed as above.

Gene Sequencing

To confirm that the products amplified from the tracheal tissues were the rat SMMHC (+) and (–)insert isoforms, the amplicon of size corresponding to the (–)insert was purified from one Lewis rat trachea, whereas the (+)insert was purified from one Fischer rat trachea, for gene sequencing. Briefly, an aliquot (4 µl) of each of four PCR reactions were run on agarose gel stained with ethidium bromide to check the result of the PCR amplifications before further purification. Three PCR reactions were then pooled and prepurified using the GFX purification kit (AP Biotech) as previously described (29). The eluted prepurified samples were then run on an agarose gel and visualized by crystal violet staining (Sigma). The purified cDNA was sequenced commercially. The sense and antisense gene sequence results for both isoforms were searched using the National Center for Biotechnology Information search engine GenBank (National Institutes of Health).

Trachealis Contractility

Tracheae were dissected from the carina to the bronchial bifurcation. They were immediately placed in ice-cold Krebs-Henseleit (KH) solution as in Ref. 13 with the modification to 10 mM glucose, cleaned, and cross-sectioned into four equivalent segments of ~4 mm width. A longitudinal cut was made through the ventral side of the trachea through the cartilage rings, and 4–0 silk suture thread passed through the cartilaginous portion of one of the tracheal segments. A loop was made of the thread and hooked onto a Grass FT03 C force transducer. The other cartilaginous end was affixed by an alligator clip and moved into a 25-ml Radnoti organ bath bubbled with 95% O2-5% CO2. The tissue was bathed in KH at 37°C, with fresh solution instilled approximately every 10 min, and incubated for 30 min. Data acquisition was performed using LABDAT software (RHT-InfoDat).

With the use of a dose response curve to MCh, a concentration of 10–6 M was chosen for producing only a submaximal response in both animals (to avoid saturation of the force and phosphorylation signals). The muscle was allowed to contract for 30 s in response to this dose. It was then freeze-clamped with forceps cooled in liquid N2 and was subsequently placed for 2 min in a mixture of 10 mM dithiothreitol (DTT) and 10% trichloroacetic acid in acetone and then placed for 2 min in a similar mixture without DTT. These tissues were then analyzed by Western blot for their total LC20 content and level of LC20 phosphorylation (Western Blot Analysis). Seven rats from each strain were studied in pairs. Two to three segments per rat were stimulated, and one was used as a control, mounted in the tissue bath, but not challenged with MCh. The average for all muscle strips is reported for each animal. Data are presented as means ± SE.

Western Blot Analysis

Myosin heavy chain isoforms. The (+)insert isoform and total SMMHC content present in the Fischer and Lewis rat trachealis muscle was determined at the protein level. Tracheae, bladders [positive control for total SMMHC and for the (+)insert], aortas [positive control for total SMMHC and negative control for the (+)insert], and skeletal limb muscles [negative control for total SMMHC and for the (+)insert SMMHC] from both strains were harvested, snap-frozen in liquid nitrogen, and kept at –80°C until assayed. For all dissections, great care was taken to remove all loosely associated connective tissue and blood vessels to reduce the influence of vascular smooth muscle myosin. However, it is likely that some residual vascular tissue may appear as a trace signal.

Tissues were powdered in liquid nitrogen and homogenized at 4°C in pyrophosphate extraction buffer. An equivalent amount of protein was loaded on the gel from assessment by Bradford assay. Electrophoresis was done on 7% polyacrylamide (0.06% bis-acrylamide) gels using a Laemmli buffer system. Proteins were electroblotted onto polyvinylidene difluoride membranes. After transfer, membranes were divided into two sections by cutting just below the 80-kDa marker. The upper portions of the membranes were first probed for the (+)insert SMMHC isoform using an antibody specific to the 7-amino acid insert sequence. For a high level of sensitivity, a biotinylated secondary antibody (DakoCytomation) was used. The secondary antibody was in turn detected by a streptavidin-biotinylated horseradish peroxidase complex (GE Healthcare Biosciences) and visualized with Enhanced Chemiluminescence (ECL) reagent (GE Healthcare Biosciences). These membranes were subsequently stripped in a solution containing 62.5 mM Tris·HCl, 10% SDS, and 0.7% 2-mercaptoethanol, and incubated in a heated water bath shaker for 30 min at 60°C. Next, the membranes were rinsed vigorously with Tris-buffered saline with Tween and then probed with an antibody recognizing all isoforms of the SMMHC. Detection was performed as above. To determine the amount of residual primary antibody left following stripping of the membranes, one of the three membranes was probed with the (+)insert antibody and visualized. The membrane was then stripped and directly reprobed with the secondary antibody as above. Only a trace level of luminescence was detected at exposures greater than that normally used. Last, the membranes were reprobed with an h-caldesmon-specific antibody and detected as above. The bottom portion of the membranes were sequentially probed for {alpha}-actin and GAPDH, although with a conventional horseradish peroxidase-linked secondary antibody detected with ECL. Quantification was performed with a Fluorchem 8500 imaging system using AlphaEase software (Alpha Innotech). The following antibodies were used: a polyclonal (+)insert antibody (provided by Dr. A. Rovner) made against a synthetic peptide based on the deduced amino acid sequence of the insert QGPSFAY (48); the polyclonal BT-562 (Biomedical Technologies) that recognizes all SMMHC isoforms; the monoclonal anti-smooth muscle caldesmon (Sigma); the monoclonal anti-smooth muscle {alpha}-actin (Sigma). GAPDH was used as a reference protein and probed with a monoclonal antibody (Ambion). Five rats of each strain were studied, and Western blots were performed in triplicate. The data are presented as means ± SE.

Myosin Essential Light Chain (LC17)

The LC17 isoforms were quantified by Western blot analysis in nondenaturing conditions following a previously described protocol (40) with modifications (A. Sobieszek, personal communication). Briefly, tracheae, bladders, and aortas from both strains were harvested as above and kept at –80°C until assayed. Proteins were extracted in 8.5 M urea and 0.5% 2-mercaptoethanol extraction buffer. The LC17 isoforms were separated by running on 8.5 M urea, 40% glycerol, 10% polyacrylamide, and 0.27% bis-acrylamide gels. The running buffer contained 50 mM Tris, 100 mM glycine, 2 mM EGTA, and 0.5% 2-mercaptoethanol. The isoforms were identified using a monoclonal antibody that recognizes both LC17 isoforms (kind gift from Dr. C. Kelley). Three sets of Fischer and Lewis organ extracts were run in pairs on separate Western blots, and detection was performed as above. The data are presented as the percentage of LC17a of the sum of the two isoforms.

Myosin Regulatory Light Chain (LC20) Phosphorylation

The total amount of LC20 and the level of phosphorylation were quantified by Western blot analysis as described above for myosin except for the following changes. Tissues were homogenized in 2-mercaptoethanol and SDS extraction buffer. Proteins were resolved on a 15% polyacrylamide gel. The following antibodies were used: polyclonal MLC2 FL-172 (Santa Cruz Biotechnology) that recognizes total LC20 and polyclonal 3671S (Cell Signaling Technologies) that specifically recognizes phosphorylation of the serine 19 residue of LC20. Baseline phosphorylation was determined in nonstimulated strips. Thiophosphorylated chicken gizzard was used as a positive control for LC20 phosphorylation. The results are expressed in relative units normalized to the darkest band on each blot.

Statistical Analysis

Airway responsiveness to MCh challenges: the trimmed means (i.e., the largest and smallest observations are omitted) and SE for each group were computed to present more robust statistics because the data set contained some extreme values (17). Group means were compared using Student’s t-test. For RT-PCR, mRNA expression of the SMMHC isoforms between the three organs and between Fischer and Lewis rats was compared by two-way ANOVA. Comparisons of the group means between Fischer and Lewis strains for each organ were performed by two-tailed Student’s t-tests. For all other experiments, comparisons of the group means between Fischer and Lewis strains were performed by two-tailed Student’s t-tests. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Airway Responsiveness to MCh

As previously reported in the literature (11, 19, 46), we found that the Fischer rats were hyperresponsive compared with the Lewis rats. The mean EC200 RL to MCh was 1.2 ± 0.2 mg/ml for the Fischer and 3.4 ± 0.8 mg/ml for the Lewis rats (P < 0.05).

RT-PCR Analysis

Densitometric analysis of the (+) and (–)insert PCR products from both Fischer and Lewis rat trachealis, generated from 28 to 34 cycles of amplification, indicated that amplification at 30 cycles was within linear range (data not shown). The size of the (+) and (–)insert amplicons corresponded to the predicted sizes, i.e., 136 and 115 bp for the (+) and (–)insert, respectively (Fig. 1A). Analysis of the percent of (+)insert SMMHC isoform mRNA expression revealed a significant interaction of organ type with strain (P < 0.001), showing that the ratio of the (+)insert isoform between strains was different for different organs. In agreement with the literature (48), the aorta contained almost exclusively the (–)insert isoform, whereas the bladder predominantly contained (+)insert isoform mRNA. These results confirm the proper functioning of the primers designed. Comparisons of SMMHC isoform expression in the aorta and bladder revealed no statistically significant differences between the rat strains. The Fischer rat aortas contained 6.6 ± 2.7% of the (+)insert mRNA and the Lewis rats had 12.0 ± 2.2% (Fig. 1B). Conversely, the Fischer rat bladders contained 96.8 ± 1.5% of the (+)insert mRNA and the Lewis rats had 93.1 ± 1.1% (Fig. 1B). The trachealis muscle of both strains showed expression, at the mRNA level, of both isoforms but in different proportions (Fig. 1). The Fischer rat trachealis muscle had more of the (+)insert mRNA (69.4 ± 2.0%) than the Lewis rats (53.0 ± 2.4%; P < 0.001, Fig. 1B). This interstrain difference was specific to the airways and not observed in the aorta and bladder (Fig. 1B). Gene sequencing confirmed that the products amplified were indeed the rat (+) and (–)insert isoforms.


Figure 1
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  		Fig. 1. A: representative RT-PCR results of the (+) and (–)insert smooth muscle myosin heavy chain (SMMHC) isoforms in the Fischer and Lewis rat trachea, aorta, and bladder. The (+) and (–)insert isoform amplicons were 136 and 115 bp, respectively. B: RT-PCR semiquantitative analysis of the mRNA coding for the (+) and (–)insert isoforms in the Fischer and Lewis rat trachea, aorta, and bladder. (+)Insert, closed bar; (–)insert, open bar; n = 5 rats/strain. **P < 0.001.

 
Western Blot Analysis

Myosin heavy chain isoforms. The trachealis muscle of both strains showed protein expression for total myosin and for the (+)insert myosin isoform but in different proportions (Fig. 2A). Given that two different antibodies will invariably have different affinities for their respective target, the percentages of (+)insert isoform with respect to myosin cannot be calculated with this protocol. Thus the expression of (+)insert isoform and total SMMHC in the Fischer and Lewis rats is reported as relative ratios of (+)insert isoform to total SMMHC and total SMMHC to GAPDH, respectively. The amount of (+)insert myosin isoform over total SMMHC was greater in the Fischer than Lewis rat tracheae (1.84 ± 0.1 vs. 1.27 ± 0.06, respectively, P < 0.01, Fig. 2C). There was no significant difference in total SMMHC content with respect to GAPDH between the Fischer to Lewis rats (0.85 ± 0.07 vs. 0.75 ± 0.02, respectively, Fig. 2D). Furthermore, comparisons of Fischer and Lewis bladder smooth muscle showed no difference in (+)insert SMMHC isoform relative to total SMMHC (0.72 ± 0.01 vs. 0.74 ± 0.02, respectively). Total SMMHC content with respect to GAPDH in the bladder was also similar between the Fischer and Lewis rats (2.12 ± 0.22 vs. 1.77 ± 0.18, respectively). Controls for the (+)insert isoform and total SMMHC antibodies are shown in Fig. 2B. As expected, a positive signal for the antibody specific to the (+)insert SMMHC isoform was observed for bladder tissues, whereas no signal was detected in the aorta. Compared with the signal in bladder and aorta, only trace signals were detected for total SMMHC and the (+)insert isoform in skeletal muscle.


Figure 2
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  		Fig. 2. A: representative Western blots of the expression of (+)insert isoform and total SMMHC in the Fischer and Lewis rat tracheae. GAPDH was used as a loading control. B: Western blot controls for the (+)insert isoform and total SMMHC antibodies. Bladder (B) was a positive control for the (+)insert isoform and total SMMHC, skeletal muscle (S) was a negative control for the (+)insert isoform and total SMMHC, and aorta was a negative control for the (+)insert isoform. F, Fischer; L, Lewis rats. C: Western blot analysis of the Fischer and Lewis rat tracheal expression of the (+)insert isoform as a ratio of total SMMHC. Bars represent the mean of each group. N = 5 rats/strain. Western blots were performed in triplicate. **P < 0.01. D: Western blot analysis of the Fischer and Lewis rat tracheal expression of total myosin as a ratio of GAPDH. Bars: mean of each group. N = 5 rats/strain. Western blots performed in triplicate.

 
LC17 Isoform Expression

Western blot analysis of nondenatured protein extracts revealed no significant differences in the percentage of LC17a over total LC17 content between Fischer (39.5 ± 5.6%) and Lewis (40.5 ± 4.1%) rat tracheae (n = 3; Fig. 3A). The LC17 isoform content was not different either in the control organs for phasic and tonic muscle, bladder, and aorta, respectively (Fig. 3A).


Figure 3
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  		Fig. 3. A: representative Western blot of smooth muscle LC17 isoforms from a nondenaturing urea-glycerol gel of bladder (B), aorta (A), and trachea (T), from Fischer (F) and Lewis (L) rats. Top: LC17b; bottom: LC17a. B: representative Western blot of smooth muscle caldesmon (h-CaD) and {alpha}-actin in the Fischer and Lewis rat tracheae. GAPDH was used as a loading control.

 
h-Caldesmon and {alpha}-Actin

The trachealis muscle of both strains showed similar protein expression of h-caldesmon (0.22 ± 0.01 for Fischer and 0.26 ± 0.02 for Lewis) and {alpha}-actin (1.89 ± 0.08 for Fischer and 2.13 ± 0.12 for Lewis), normalized to GAPDH (Fig. 3B).

Muscle Strip Contractility and LC20 Phosphorylation

The tracheal muscle strip tension generated at 30 s after a 10–6 M MCh challenge was greater for the Fischer (4.2 ± 0.8 mN) than for the Lewis rats (1.9 ± 0.4 mN; Fig. 4A; P < 0.05). The corresponding LC20 phosphorylation level was assessed by quantifying in the same muscle strips total LC20 expression, baseline LC20 phosphorylation level, and LC20 phosphorylation level 30 s after the MCh challenge. The total amount of LC20 did not differ between Fischer (71.8 ± 1.6) and Lewis (66.3 ± 4.3) rats (Fig. 4, B and C). Baseline phosphorylation level in the nonstimulated segments did not differ either between Fischer (17.3 ± 3.7) and Lewis (18.8 ± 5.9) rats (Fig. 4, B and C). However, the phosphorylation level at 30 s after MCh challenge was significantly greater for the Fischer (55.1 ± 6.4) than for the Lewis rats (41.4 ± 6.1; Fig. 4, B and C; P < 0.05).


Figure 4
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  		Fig. 4. A: Fischer and Lewis tracheal strip isometric force response to a submaximal (10–6 M methacholine) stimulus. N = 7 rats/strain. *P < 0.05. B: representative Western blot of smooth muscle myosin regulatory light chain (LC20) phosphorylation: total myosin regulatory light chain (Total LC20) and phosphorylated LC20 (pLC20) for the Fischer (F) and the Lewis (L) tracheal segments. Nonstimulated segments (ns) were used as negative control and thiophosphorylated chicken gizzard (pCG) was used as positive control. C: Western blot analysis of LC20 expression and phosphorylation. Left: total LC20 expression in Fischer and Lewis rat tracheal rings. Right: baseline phosphorylation measured in nonstimulated tracheal strips and phosphorylation 30 s after stimulation with methacholine. The average of all stimulated strips is reported for each animal. N = 7 rats/strain. *P < 0.05. Lewis, open bar; Fischer, closed bar.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study are that the airway hyperresponsiveness of the Fischer rats, compared with the normoresponsive Lewis rats, is associated with a greater expression of the fast (+)insert SMMHC isoform and a higher phosphorylation level of the LC20. The faster rate of cross-bridge cycling of the (+)insert isoform (22, 23) combined with enhanced myosin activation (20) provides two mechanisms to explain the greater muscle-shortening velocity observed in airway hyperresponsiveness.

The Fischer and Lewis Rat Model of Airway Hyperresponsiveness

Hyperresponsive airway smooth muscle contracts faster and to shorter lengths than normoresponsive smooth muscle (5, 12, 33, 46). The highly inbred Fischer 344 and Lewis rat strains have been commonly used to investigate potential genetic determinants of innate airway hyperresponsiveness (11, 19, 46). The Fischer rats are well known to have greater responsiveness to contractile agonists, like MCh, compared with the Lewis rat in the absence of inflammation and without prior sensitization (11, 19, 46). Fischer airway smooth muscle has been characterized as having a greater rate of shortening ex vivo and in vitro (3, 11). Our results also support these earlier findings: a MCh dose approximately threefold greater is required in the Lewis rats to achieve the same EC200 RL as in the Fischer animals. Our data suggest two mechanisms to explain the hypercontractility of the Fischer rat airway smooth muscle: altered myosin isoform expression and increased LC20 phosphorylation.

Although the hyperresponsiveness of the Fischer rat was shown to be associated with a greater quantity of airway smooth muscle (30% greater muscle mass in the Fischer rats), intraspecies differences in responsiveness has been shown to be uncorrelated with the airway smooth muscle mass (11). More recently, Wang et al. (46) studied MCh-induced bronchoconstriction in explants of subsegmental and smaller airways from agarose-inflated lungs from Fischer and Lewis rats. They showed a greater responsiveness to MCh in the Fischer rat airway explants and a higher rate of contraction of these ex vivo airways in the Fischer strain at all the inflation volumes studied. These results support the notion that the contractile properties of the smooth muscle itself contribute to the airway hyperresponsiveness of the Fischer rats. The mechanical properties of the Fischer and Lewis rat trachealis have also been studied in isolated muscle strip experiments by Blanc and coworkers (3). They reported a greater maximum shortening velocity and extent of muscle shortening for the Fischer compared with the Lewis rats, without any significant difference in maximal isometric tension. Similar mechanical properties have been reported in comparisons between asthmatic and normal airways (8, 25, 33). This demonstrates the relevance of the Fischer-Lewis rat model to the study of mechanisms underlying airway hyperresponsiveness and its role in asthma.

We expected to find multiple factors contributing to the hyperresponsiveness of the Fischer compared with the Lewis rats since genetic studies have demonstrated that hyperresponsiveness is a polygenic trait (9). Indeed, two potential causes of the hyperresponsiveness in the Fischer-Lewis rat model have already been found. First, the airway NO-cGMP-relaxant pathway has been shown to be impaired in Fischer rats (19). Second, differences in intracellular Ca2+ mobilization have also been implicated in determining airway smooth muscle contractility in the Fischer and Lewis rats (44), likely due to differences in protein kinase C regulation (43). Our current results showing an increased LC20 phosphorylation reflect, at the myosin level, this report of increased Ca2+ mobilization. Together with greater expression of a faster myosin isoform, all these factors contribute to the notion of multifactorial alterations in the contractile machinery leading to airway hyperresponsiveness in the Fischer rats.

Based on their force-velocity data, Blanc and coworkers (3) used Huxley’s mathematical model of muscle contraction to predict myosin kinetics in the Fischer and Lewis rat trachealis muscle. They reported a shorter duration of cross-bridge attachment and detachment in the Fischer trachealis, thereby explaining its greater rate of shortening. Using the laser trap, we previously showed that the difference in kinetics between the myosin isoforms is due to a shorter attachment time for the (+)insert SMMHC isoform (22). Our findings of a greater expression of the (+)insert isoform in the Fischer rat trachealis provide a suitable explanation for these differences in myosin cross-bridge kinetics between the Fischer and Lewis rat trachealis predicted by Blanc et al. (3).

Physiological Significance of the (+)Insert Myosin Isoform

The presence of the (+)insert SMMHC isoform is clearly associated with a greater rate of actin filament movement ({nu}max) in the in vitro motility assay (21, 22, 36), although this correlation has been disputed at the physiological level in a few studies (see Ref. 47 for review). Nonetheless, Eddinger and Meer (10) showed a good correlation between the (+)insert myosin isoform content and velocity of shortening in isolated rabbit stomach smooth muscle cells. They showed that total myosin mRNA content was 95% (+)insert isoform in the antral region, compared with 38% in the fundic region, which was correlated to approximately three times greater rate of shortening of the antral cells. In canine airways, Ma et al. (27) also showed that the presence of the fast (+)insert myosin isoform was correlated with greater mechanical performance. They reported that tracheal smooth muscle expressed 2x more (+)insert mRNA than the bronchi. They also showed an approximate twofold greater maximum shortening capacity and more than twofold greater maximum shortening velocity for the trachealis muscle than for bronchial muscle from the sixth generation (26). The absolute (+)insert isoform mRNA content reported was very small, however, 13% in the trachea and 6% in the bronchi. More recently, we used the in vitro motility assay to measure {nu}max for myosin molecules purified from multiple rat organs expressing different proportions of the (+) and (–)insert SMMHC isoforms (23). We found that the (+)insert SMMHC isoform expression and {nu}max increased approximately linearly from the most tonic, and therefore slowest, to the most phasic, and therefore fastest muscle tested. Furthermore, to investigate the role of the (+)insert SMMHC isoform at the whole animal level, we previously measured the pulmonary mechanics of (+)insert isoform knockout mice (45). We found that the absence of the (+)insert isoform resulted in a significantly slower time to peak airway resistance in the knockout mice compared with the parent strain.

In this study, we report a strain-related difference in SMMHC isoform expression in the tracheae that is not observed in other organs. Similarly, Wetzel and coworkers (47) observed a difference in SMMHC isoform expression in precapillary cardiac arterioles between innately normotensive and hypertensive rats, whereas the isoform content in other organs was not different. These observations suggest that the expression of SMMHC isoforms for a given organ is not fixed; rather, variations arise from genetic origins. As such, differences in the expression pattern of the SMMHC isoforms for a given organ could result in the abnormal functioning of that organ.

Previous studies have also shown that the (+)insert SMMHC isoform is coexpressed with the acidic LC17a isoform, whereas the (–)insert SMMHC isoform is mostly expressed in conjunction with the basic LC17b isoform (18, 31, 42). Szymanski and coworkers (42) have also shown that the expression of various other contractile proteins differ between phasic and tonic portions of the opossum esophagus, i.e., the phasic circular muscle of the esophageal body in opossums contains more (+)insert SMMHC, LC17a, h-caldesmon, and a greater {gamma}- to {alpha}-actin isoform ratio than the tonic-lower esophageal sphincter. We investigated this further in the Fischer and Lewis rat trachealis, by quantifying by Western blot analysis, in addition to the SMMHC isoforms (Figs. 1 and 2), the h-caldesmon, {alpha}-actin, and the LC17 isoform expression (Fig. 3). The expression of h-caldesmon and {alpha}-actin was not different between Fischer and Lewis rat trachealis. The expression of LC17 isoforms was not different either between Fischer and Lewis rat trachealis, nor were they differently expressed in control organs for rat phasic (bladder) and tonic (aorta) muscles (Fig. 3). Others have also reported a lack of correlation between LC17 isoforms and contractility (18, 31, 40). Our data, therefore, do not support a shift in contractile proteins toward a more phasic phenotype for the Fischer trachealis but an isolated increase in the faster (+)insert SMMHC isoform along with an increased LC20 phosphorylation level.

Physiological Significance of Increased LC20 Phosphorylation

The role of enhanced LC20 phosphorylation in airway hyperresponsiveness has been addressed in the allergic dog model (20) as well as in human asthmatics (25). Both MLCK and LC20 phosphorylation were shown to be elevated in the allergic dog (20). Increased MLCK mRNA expression was found in asthmatic compared with control subjects (25). Our model of innate airway hyperresponsiveness behaved similarly in that increased contractility was also associated with a greater level of phosphorylation. However, the specific role of this increased phosphorylation to enhanced Fischer airway smooth muscle contractility remains unclear given that recent reports have disputed the association between LC20 phosphorylation and rate of shortening (32, 34).

Another observation that we made and that underlies the sensitivity of the contractile machinery to the level of phosphorylation is the fact that the phosphorylation level was not constant among muscle strips down the trachea but was greater in the more caudal sections of the trachea (data not shown), with constant total LC20 content. These results predict enhanced contractility for the most caudal segments of the trachea. This is indeed what has been reported in dose-response curves in dogs (7), guinea pigs, and Fischer and Lewis rats (13). Our data for force of contraction of muscle strips when considered per tracheal segment followed a similar trend but did not reach statistical significance (data not shown). These findings could provide further insight into airway hyperresponsiveness but will require future investigation.

Smooth Muscle and Airway Hyperresponsiveness

There is ample evidence suggesting that airway lumen area gets bigger with each tidal breath (41). It has also been shown that if prevented from taking deep inspirations, normal humans respond similarly to asthmatic subjects when challenged with bronchoconstrictive agents (35, 39). From those observations, Gunst (15) and Solway and Fredberg (41) suggested that the kinetics of muscle contraction may play a crucial role in bronchial hyperresponsiveness. That is, tidal breathing and especially deep inspirations putatively allow for the detachment of cross-bridges. This must have a more pronounced relaxing impact on slowly contracting muscles than on fast ones, i.e., rapidly contracting muscle probably constrict to a greater extent between each breath or deep inspiration (15, 41). A greater activation state via increased levels of LC20 phosphorylation combined with a shift in myosin isoform content toward a greater proportion of the fast (+)insert isoform would certainly contribute to a greater rate of contraction of hyperresponsive muscle. Because correlations have already been established between stages of development, hormone levels, pathologies, and the (+)insert isoform content (6, 47, 49), it is reasonable to expect that its expression could also be altered in airway hyperresponsiveness and asthma. In a recent study, Ma and coworkers (25) measured a greater rate of shortening for single smooth muscle cells from asthmatic bronchial biopsies compared with those from normal individuals. They also observed a greater MLCK mRNA level in the asthmatics but they did not detect any (+)insert isoform mRNA in either of the asthmatic or control bronchial biopsies. We have since sequenced the human (+)insert isoform and, using primers specific to the human smooth muscle myosin, demonstrated unequivocally its expression at the mRNA and protein levels in multiple human organs including human trachea (23). Furthermore, in a preliminary study, we recently showed that the (+)insert isoform expression is increased in asthmatic bronchial biopsies compared with normals (24). These findings suggest a role for the myosin isoforms in mediating the altered airway response in human asthma.

In conclusion, our study proposes that in innate airway hyperresponsiveness, two mechanisms produce enhanced muscle contraction. First, a greater expression of the faster (+)insert isoform results in a faster rate of shortening. Second, an increased extent of LC20 phosphorylation for a given stimulus recruits more activated myosins to participate in contraction, thus leading to its hypercontractility. To date, there is no evidence linking (+) and (–)insert isoform expression with the extent of LC20 phosphorylation, suggesting that these phenomena occur independently. Further investigations might reveal a cascade of events, likely to be under genetic control, which together generate the smooth muscle hypercontractile state observed in airway hyperresponsiveness.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funds from the Banting Foundation, Natural Sciences and Engineering Research Council of Canada Grant 217457-00, and the Canadian Institute of Health Research Grant MGC-42667. A.-M. Lauzon and K. Maghni were supported by Scholarships from the Fonds de Recherche en Santé du Québec and F. R. Gil was supported by the Québec Respiratory Health Training Program.


   ACKNOWLEDGMENTS
 
We thank Dr. S. White for help in initial protocol design, A. Poirier for technical expertise, and Marvid Poultry for providing chicken gizzards.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A.-M. Lauzon, Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St., Montréal, Québec, Canada H2X 2P2 (e-mail: anne-marie.lauzon{at}mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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