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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: FASEB J. 2021 Jul;35(7):e21674. doi: 10.1096/fj.202100154R

Cooperativity between β agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation

Ajay P Nayak 1, John Lim 1, Eylon Arbel 2, Ruping Wang 2, Dominic R Villalba 1, Tahn L Nguyen 1, Niccole Schaible 3, Ramaswamy Krishnan 3, Dale Tang 2, Raymond B Penn 1
PMCID: PMC8802313  NIHMSID: NIHMS1773600  PMID: 34115899

Abstract

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Keywords: actin cytoskeleton, airway smooth muscle, asthma, β2 adrenoceptor, c-abl tyrosine kinase

INTRODUCTION

Effective asthma management requires regulating airway smooth muscle (ASM) contractile state to avoid or reverse bronchoconstriction. Whether this is attempted by use of direct bronchodilators (e.g., β-agonists), by anti-inflammatory agents (e.g., corticosteroids), or some combination of both, too often management is lacking, as an estimated 50% of all asthmatics have suboptimal control (1). Further, safety issues associated with β-agonist use in the form of either long acting (LABA) (2), or short acting (SABA) (3) β-agonists still persist. All current bronchodilator drugs have limited clinical effectiveness, due to multiple mechanisms mediating desensitization of their target receptor, or their inability to target the multiple receptors or intracellular signals that promote ASM contraction (4–7).

At the cellular level, ASM contractile state is controlled by two independent processes: 1) cross bridge cycling (promoted by pharmacomechanical coupling), and 2) cytoskeletal dynamics (dependent on actin polymerization state) (8). While β-agonists are effective in inhibiting the former by regulating phosphorylation of myosin light chain (MLC20), their ability to regulate the actin cytoskeleton in ASM is unclear, as is the ability to augment β-agonist-mediated ASM relaxation by agents that inhibit actin polymerization. For years, studies into the regulation of the actin cytoskeleton dynamics were primarily concerned with understanding cell motility/migration, most often in non-muscle cells. In recent years, we and others have demonstrated that ASM stiffness (owing to actin cytoskeleton reorganization) can be targeted to affect the response of ASM to contractile stimuli (8–16). Thus, targeting the actin cytoskeleton reorganization is an alternative means of inhibiting ASM contraction for which a strong conceptual and growing empirical basis exists.

The premise underlying the current study is that the current limitations of bronchodilator drugs can be overcome by an approach that targets both pharmacomechanical coupling and actin polymerization. Using cell, tissue and in vivo models of ASM contraction, we establish the cooperative nature of combined targeting of pharmacomechanical coupling and actin polymerization with emphasis on combined action of β-agonists and c-Abl tyrosine kinase inhibitors. We further elaborate the mechanisms by which β-agonists and inhibitors of c-Abl cooperatively effect bronchodilation.

METHODS AND MATERIALS

Antibodies and other reagents

Imatinib (IMAT) was purchased from LC Laboratories (Woburn, MA). GNF-5, isoproterenol (ISO) and histamine were purchased from Sigma-Aldrich (St. Louis, MO). Phospho-myosin light chain 2 (Thr18/Ser19; pMLC20), and phospho-myosin phosphatase target subunit 1 (Thr853; pMYPT1) antibodies were purchased from Cell Signaling Technology (Danvers, MA), anti-vasodilator-stimulated phosphoprotein (anti-VASP) antibody was purchased from BD Transduction Laboratories (BD Biosciences, San Jose, CA), monoclonal anti-β-actin and anti-vinculin antibodies were purchased from Sigma-Aldrich, phospho-heat shock protein 20 (Ser16; pHSP20) antibody was purchased from Abcam Inc. (Toronto, Canada), and all secondary antibodies conjugated to infrared dyes were purchased from LI-COR Biosciences (Lincoln, NE).

Cell culture, treatment conditions and lysate preparation

Primary ASM cultures were established from human tracheae obtained from de-identified non-asthmatic donors from NDRI as described previously (17). ASM cells were cultured in 12-well plates in Ham’s F12 medium supplemented with fetal bovine serum (FBS; 10%) until confluence and subsequently starved in plain F-12 medium devoid of FBS, as described previously (18).

To examine the cooperative effects of β-agonists and c-Abl inhibitors on regulation of pharmacomechanical coupling, we pretreated cultured primary human ASM cells with ISO (1 μM) alone or in combination with c-Abl inhibitors (GNF-5 or IMAT; 10 μM) for 10 min at 37°C and subsequently simulated cells with histamine (1 μM) for 10 min at 37°C. Histamine was chosen as the contractile agonist for these cell-based studies given histamine provides more consistent responses with respect to contraction and contractile signaling than does methacholine, due to the rapid loss of m3 muscarinic acetylcholine receptors that occurs with passage of human ASM cultures (19). A set of wells with cultured ASM cells were treated with c-Abl inhibitors alone to assess regulatory effects on MLC20, while cells treated with vehicle (DMSO 0.001%) served as negative controls. Depending on downstream analysis targets for western blot analysis, protein lysates were prepared by two separate approaches. 1) For assessing regulation of pMLC20 and pMYPT1, following treatment conditions, cells were treated with 100 μl of stop buffer (1:1 v/v perchloric acid in dH2O) to arrest all cellular processes stored on ice for 10 mins as described previously (20). Cells were scraped off, collected in 1.5 ml Eppendorf tubes, and centrifuged for 5 min at 4°C at 15,000 RPM. Cells were rinsed with 1 ml ice-cold phosphate-buffered saline (PBS), centrifuged again and resuspended in 45 μl of RIPA lysis buffer containing 15 μl of NuPAGE sample buffer (Invitrogen, CA). Samples were stored in −20°C. 2) For assessing VASP and pHSP20 by western blotting, immediately following treatment/stimulation, cells were washed in ice-cold PBS, lysed with 100 μl of RIPA lysis buffer (containing 2x Laemmli buffer), harvested and stored at −20°C as described previously (18).

Western blotting

Cell lysates generated as described above were subjected to western blot analysis by two different approaches. pMLC20 regulation was assessed using Invitrogen western blotting system using methods described previously (20). Immunoblotting for pMYPT1, pHSP20 and for assessing VASP shift was performed using standard SDS-PAGE approach as described previously (18). Owing to the large size (140 kDa) of pMYPT1, SDS-PAGE utilized a 6 % acrylamide gel, necessitating analysis of vinculin (as opposed to β-actin) to assess loading. Briefly, following electrophoresis proteins were transferred onto nitrocellulose membranes and incubated for 1 h with Tris-buffered saline (TBS) containing Tween-20 (0.2%) and 3% bovine serum albumin (BSA; 3%) (blocking buffer). Membranes were incubated overnight at 4°C with primary antibodies to various proteins of interest. The next morning, blots were briefly washed with TBS-Tween buffer and incubated with secondary antibodies conjugated to infra-red (IR) dyes. Quantitative assessment of immunoreactive protein bands was performed by measuring integrated intensity for each protein band of interest using Odyssey software (18).

Animals and related procedures

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Albany Medical College. C57BL/6 mice 10–14 weeks old were housed in the animal facility at Albany Medical College and provided food and water ad libitum.

Murine precision cut lung slices (PCLS)

Mice were euthanized by intraperitoneal injection of pentobarbital. Tracheotomy and sternotomy were performed, and a cannula was carefully inserted through the trachea and fixed in place by suture. The lungs were inflated with 2% low-melting-point agarose (type VII) prepared in Hank’s Balanced Salt Solution (1x HBSS). Lungs were rinsed with ice-cold HBSS supplemented with HEPES, excised from the thoracic cavity and placed in a 4°C refrigerator for 30 min to allow for solidification of infused agarose. Lungs were cut transversely at 300 μm in HBSS supplemented with HEPES using a Vibratome (LeicaVT1200S). The PCLS were placed in warm Dulbecco’s Modified Eagle Medium (DMEM) supplemented with antibiotics and incubated at 37°C with 5% CO2. Prior to experiments, PCLS were placed in the modified physiological saline solution (PSS; 110 mM NaCl, 3.4 mM KCl, 0.8 mM MgSO4, 4.8 mM CaCl2, 25 mM HEPES, 1 g/L dextrose, pH 7.4) for 30 min in an incubator and subsequently placed in a 35-mm glass bottom culture dish with the modified physiological saline solution.

For PCLS-based experiments, we used 2 models (detailed below) to study cooperative effects of c-Abl inhibitors and a β-agonist. For each experiment, PCLS were first primed with MCh (100 μM) for 20 min, briefly rinsed with PSS and subsequently used for experiments.

a) Bronchoprotective effects:

PCLS were stimulated with to MCh followed by washing. PCLS were then treated with either ISO (1 μM), IMAT (10 μM), or GNF-5 (10 μM) or a combination of ISO with either IMAT or GNF-5 for 20 min. Following incubation, murine PCLS were treated with MCh (100 μM) for 20 min and images of airways were captured to analyze changes in airway lumen area. Airway lumen areas were compared before and after each inhibitor treatment.

b) Bronchodilatory effects:

Murine PCLS were treated with MCh (100 μM) for 20 min, and subsequently treated with ISO (1 μM), IMAT (10 μM), or GNF-5 (10 μM) or a combination of ISO with either IMAT or GNF-5 for an additional 20 min and images of airways were captured to examine changes in airway lumen area.

Images were captured prior to treatment with any reagent and following treatment with each stimulation condition. Change in lumen area was examined relative to that induced by stimulation of PCLS with MCh. Images of airway lumen were captured using a time-lapse microscopy (Leica DMI 6000, 10x object, phase-contrast).

In vivo FlexiVent studies

For in vivo analysis, we examined the cooperative inhibition of the MCh-induced airway resistance by β-agonist (ISO and c-Abl inhibitors (IMAT, GNF-5) using Scireq flexiVent instrument (Scireq, Canada) as described previously (21). Mice were recorded for weight and anesthetized, tracheotomized, and interfaced with the flexiVent instrument. Mechanical ventilation was set at 150 breaths/min with a tidal volume of 0.16 ml/kg and a positive end-expiratory pressure (PEEP) of 3 cm H2O. Airway resistance ((Raw (cm2/H2O/ml/s)) was measured in each mouse after nebulization of PBS (i.e., the vehicle for MCh) for 10 seconds using an Aeroneb ultrasonic nebulizer (SCIREQ). Following establishment of baseline measurements, mice were challenged with nebulized MCh (50 mg/ml). After steady state contraction was achieved (i.e., resistance plateaus), mice were then exposed to nebulized ISO, IMAT or combination of both.

Cell-based analysis of ASM contraction

Contractile traction force changes were evaluated by Contractile Force Screening (CFS) as per methods described previously (22). Briefly, 96-well plates with miniaturized Polydimethylsiloxane (PDMS) substrates bearing surface-bound fluorescent microspheres were plated with primary human ASM cells. The ASM cells were treated with 10 μM histamine for 30 min followed by treatment with ISO (1 μM), the c-Abl inhibitor imatinib (10 μM), or a combination as indicated for an additional 30 min. In response to treatments, the ASM contracts/ relaxes and correspondingly, the fluorescence microspheres displace. From these displacements, and with knowledge of substrate stiffness (3kPa) and thickness (100 μm), we computed ASM contraction, represented by the strain energy (pico Joules), on a well-by-well basis using Fourier Transform Traction Microscopy (FTTM) (23), modified to the case of cell monolayers (24). Data are presented as a % of histamine-induced contraction.

Confocal imaging of actin cytoskeleton regulation

Regulation of actin cytoskeleton state by β-agonist and c-Abl inhibitors was examined by immunofluorescence approach as described previously (12). Cultured human ASM cells were treated with either MCh (1 μM), MCh + ISO (1 μM), MCh + IMAT (10 μM) or MCh + ISO + IMAT for 10 min. Following incubation, cells were fixed with 4% paraformaldehyde and subsequently stained with rhodamine-phalloidin (F-actin) and AlexaFluor488-DNase I (G-actin) (Life Technologies, Carlsbad, CA). Immunofluorescence imaging was performed using a Leica DMI 6000 microscope. Fluorescence signal intensities for rhodamine and AlexaFluor488 were compared to determine F-/G-actin ratio.

Regulation of β2AR-mediated cAMP generation

To assess the potential regulatory effect of IMAT on β2AR responsiveness per se, primary human ASM cultures were plated in 24 well plates and stimulated for 10 min with either vehicle, 10 μM forskolin (FSK) or ISO (10 nM-10 μM) in the presence/absence of 10 μM IMAT with all cells receiving isobutylmethylxanthine (100 μM). Media was aspirated and reactions terminated with 100% ethanol. Well contents were harvested and assayed for cAMP content using the cAMP-Screen ELISA (Thermo Fisher Scientific) as per (25).

Data presentation and statistical analysis

For experiments employing human ASM cell cultures in immunoblot analysis, data are presented as means ± SE (standard error) values for n observations; n observations reflect experiments employing ASM cultures derived from n different human donors. For FTTM experiments with human ASM cells, n observations reflect wells per treatment group. For experiments with murine PCLS and in vivo studies, data are represented as means ± SE values for n observations; n observations reflect different animals. Statistical analysis of the data was performed with GraphPad Prism (San Diego, CA) software. Statistically significant differences between experimental and control groups were assessed either by one-way ANOVA with Bonferroni’s post hoc analysis of multiple comparisons, with p < 0.05 being sufficient to establish significant differences between groups.

RESULTS

Functional cooperativity of β-agonist and c-Abl inhibitors in relaxing airway smooth muscle.

Regulation of ASM contraction in airways ex vivo.

To assess regulation of airway contractile state ex vivo, murine precision cut lung slices (PCLS) were employed. Murine PCLS were briefly stabilized in PSS solution, and subsequently primed for contraction by addition of MCh (100 μM), resulting in a significant reduction of airway lumen area, which was sustained for 20 min. Slices were washed and used to examine cooperative effects of c-Abl inhibitors (IMAT or GNF-5) and the β-agonist ISO on airway relaxation.

We first examined the bronchoprotective effects of ISO (1 μM), IMAT (10 μM), or GNF-5 (10 μM) or combination of ISO with IMAT or GNF-5 by pretreating PCLS for 20 min then subsequently challenging with MCh (100 μM). Individually, ISO, IMAT and GNF-5 significantly inhibited the MCh-induced reduction of airway lumen area (Figure 1A). Importantly, combining either IMAT or GNF-5 with ISO further enhanced the inhibitory actions on MCh-induced contraction, suggesting cooperative interaction.

Figure 1. Cooperativity of the c‐Abl inhibitors and ISO.

Figure 1.

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A. Bronchoprotection model. Mouse lung slices were treated with MCh (100 μM) for 20 min, washed with PBS for 20 min and treated with 1 μM ISO, 10 μM IMAT, or 10 μM GNF‐5 or combination of ISO with IMAT or GNF‐5 for 20 min. Mouse PCLS were then treated with MCh for 20 min. Airway contraction after inhibitor treatment was normalized to airway contraction before inhibitor treatment. Data are means ± SE (n = 5‐6). For all data ** p < 0.01; * p < 0.05 as determined by One-way ANOVA.

B. Bronchodilation model. Mouse lung slices were treated with MCh (100 μM) for 20 min, then treated with 1 μM ISO, 10 μM IMAT, or 10 μM GNF‐5 or combination of ISO with IMAT or GNF‐5 for 20 min. Airway contraction after inhibitor treatment was normalized to airway contraction before inhibitor treatment. Data are means ± SE (n = 5) and * denotes p < 0.05 as determined by One-way ANOVA. C. Kinetics of relaxation. Representative kinetics of IMAT or/and ISO-induced bronchodilation.

Next, we examined the bronchodilatory effects of the relaxing agents by first contracting murine PCLS with MCh (100 μM) for 20 min, then subsequently relaxing with either ISO (1 μM), IMAT (10 μM), or GNF-5 (10 μM) or the combination of ISO with IMAT or GNF-5 for 20 min. ISO, IMAT and GNF-5 each significantly reversed MCh-induced contraction of airway lumen area (Figure 1B). The combination of ISO with either IMAT or GNF-5 further enhanced the reversal of MCh-induced contraction of airway lumen area, underscoring cooperative effects of these drugs. A representative experiment showing the time-dependent relaxant effect of ISO, IMAT, and both are presented in Figure 1C.

Collectively, the results indicate that c-Abl inhibitors alone can sufficiently inhibit or reverse MCh-induced contraction of airways and their combination with β-agonist further enhances bronchoprotection and bronchorelaxation.

Regulation of airway resistance in vivo.

To further demonstrate the functional cooperativity between β–agonist and c-Abl inhibitor, we performed in vivo studies to examine the regulation of airway resistance induced by MCh challenge. In ventilated mice, nebulization with MCh (50 mg/kg) resulted in significant increases in airway resistance (Figure 2). Treatment with ISO or IMAT alone significantly reduced MCh-induced airway resistance. Combined treatment with ISO and IMAT resulted in significantly greater inhibition of airway resistance than that elicited by either drug alone.

Figure 2: In vivo analysis of c-Abl inhibitor (IMAT) and ISO in bronchodilation.

Figure 2:

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Airway resistance measured in Balb/cJ mice treated with MCh (50 mg/kg) or MCh plus ISO (10 mg/kg) or IMAT (10 mg/kg) or ISO + IMAT. For all data n = 3 and * denotes p < 0.05 as determined by One-way ANOVA.

Regulation of cell traction forces in ASM cells.

To establish whether inhibitors of actin polymerization combined with β-agonist treatment could function in a cooperative manner to relax contracted ASM cells in culture, we assessed the regulation of ASM contraction in cultured human ASM cells by ISO and IMAT using the CFS approach. ISO (1 μM) and IMAT (10 μM) reduced histamine-induced contraction by ~ 23% and ~ 11% respectively (Figure 3). Combined treatment of ISO together with IMAT resulted in significantly greater ASM relaxation (32%) than that caused by either agent alone.

Figure 3: Cooperative regulation of ASM cell contraction in vitro by ISO and IMAT.

Figure 3:

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Human ASM cells were first contracted with histamine (10 μM) for 30 min and subsequently relaxed with ISO (1 μM), IMAT (10 μM) or ISO + IMAT and incubated for additional 30 min. Following each treatment, ASM contraction was measured using FTTM. Data are presented as a % histamine-induced contraction. n=4,* denotes p < 0.05 as determined by One-way ANOVA.

Inhibitors of actin polymerization do not affect pharmacomechanical coupling

Previous studies have shown that agonists of the β2AR relax contracted ASM via protein kinase A (PKA)-dependent regulation of regulatory myosin light chain (MLC20) (26), while c-Abl inhibitors have been shown to regulate ASM contraction by inhibiting the actin cytoskeleton polymerization state (11, 16). Treatment of cells with histamine resulted in significant increases in phosphorylation of MLC20 at threonine-18 and serine-19 residues (pMLC20) (Figures 4A and 4B). Pretreatment with ISO (1 μM) inhibited the increase in pMLC20 levels, to those observed at baseline. Consequently, further reductions in pMLC20 levels were not observed with concomitant pretreatment with IMAT (10 μM) or GNF-5 (10 μM). Interestingly, however, pretreatment of ASM cells with IMAT (10 μM) (but not GNF-5, 10 μM) alone significantly inhibited (~50%) histamine-induced pMLC20 levels (Figures 4A and 4B).

Figure 4: Cooperative effects of c-Abl inhibitors and ISO on mediators of pharmacomechanical coupling.

Figure 4:

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Human ASM cells were treated 10 min with either GNF-5 (10 μM) or IMAT (10 μM) ± ISO (1 μM), and subsequently stimulated with histamine (His; 1 μM). A. and B. Western blot analysis of pMLC regulation by c-Abl inhibitors and ISO. For all data n = 7 and * denotes p < 0.05 as determined by One-way ANOVA analysis. C. and D. Western blot analysis of pMYPT1T853 regulation by c-Abl inhibitors and ISO. For all data n = 4 and * denotes p < 0.05 as determined by One-way ANOVA.

We next examined regulation of phospho-MYPT1 (which regulates pMLC20 levels) (27) by β-agonist (ISO) and c-Abl inhibitors GNF-5 or IMAT. Pretreatment with ISO inhibited histamine-induced pMYPT1T853 expression (Figures 4C and 4D). There was no significant difference when cells were pretreated with ISO plus either of the c-Abl inhibitors. Finally, GNF-5 and IMAT alone failed to regulate pMYPT1.

Inhibitors of actin polymerization do not affect PKA-dependent signaling induced by β-agonists.

To assess whether c-Abl inhibitors affect signaling induced by β-agonists, we first examined the effect of GNF-5 or IMAT on ISO-induced changes in phosphorylation of vasodilator-stimulated phosphoprotein (VASP) determined by gel mobility shift of the phosphorylated VASP (pVASP) species (represented in the 50 kDa band) as described previously (18). Treatment of ASM cells with ISO (1 μM) increased pVASP species relative to basal and vehicle-treated conditions (Figures 5A and 5B). No increase in pVASP levels was observed following co-incubation with GNF-5 (10 μM) or IMAT (10 μM). Further, pVASP levels induced by ISO were unaffected by concomitant stimulation with histamine (1 μM). Finally, GNF-5 and IMAT alone did not alter pVASP levels.

Figure 5: Cooperative effects of c-Abl inhibitors and ISO on phosphorylation of PKA substrates VASP and HSP20.

Figure 5:

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Human ASM cells were treated 10 min with either GNF-5 (10 μM) or IMAT (10 μM) ± ISO (1 μM), and subsequently stimulated with histamine (His; 1 μM). A. and B. Western blot analysis of VASP phosphorylation by c-Abl inhibitors and ISO. For all data n = 5 and * denotes p < 0.05 as determined by One-way ANOVA. C. and D. Western blot analysis of pHSP20 regulation by c-Abl inhibitors and ISO. For all data n = 3 and * denotes p < 0.05 as determined by One-way ANOVA.

Although VASP phosphorylation is a faithful readout for Gs-coupled G protein-coupled receptor (GPCR) signaling through the cAMP/PKA axis, pVASP is not mechanistically involved in the regulation of ASM contraction. However, another PKA substrate, heat shock protein 20 (HSP20) when phosphorylated (pHSP20) contributes to β-agonist-mediated ASM relaxation (28). Treatment of ASM cells with ISO (1 μM) increased pHSP20 levels which were sustained upon subsequent stimulation with histamine (1 μM) (Figures 5C and 5D). Pretreatment of ASM cells with GNF-5 (10 μM) significantly enhanced ISO-induced pHSP20. IMAT (10 μM) modestly enhanced ISO-induced pHSP20 levels, although this increase was not significant. Treatment of cells with GNF-5 or IMAT alone was not sufficient to induce pHSP20.

Cooperative effects of β-agonists and c-Abl inhibitors on actin cytoskeleton regulation.

We next examined the regulation of changes in the actin cytoskeleton induced by contractile stimulus in the presence/absence of a β-agonist, IMAT, or both. Human ASM cells treated with different combination of drugs stained with phalloidin (staining F-actin) or DNase I (G-actin) were examined by immunofluorescence microscopy. Treatment of ASM cells with MCh significantly increased accumulation of F-actin filaments as demonstrated by increased phalloidin staining and an increased F-/G-actin ratio (Figure 6A and 6B). Treatment of MCh-stimulated cells with either ISO or IMAT alone reduced phalloidin staining and the calculated F-/G-actin ratio. Moreover, importantly, we observed cooperative regulation of F-actin with combined ISO + IMAT treatment, which resulted in a significant reduction in the F-/G-actin ratio.

Figure 6: Cooperative effects of β–agonist and c-Abl inhibitor on regulation of actin cytoskeleton.

Figure 6:

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Human ASM cells were treated 10 min with MCh (1 μM), MCh + ISO (1 μM), MCh + IMAT (10 μM) or MCh + both agents for 10 min. Cells were stained with phalloidin (F-actin) and DNase I (G-actin). A. and B. Examination of F-/G-actin pools by immunofluorescence imaging. Fluorescence intensity was analyzed using ImageJ software. For all data n = 3 and * denotes p < 0.05 as determined by One-way ANOVA.

β-agonist and c-Abl inhibitor cooperativity in bronchorelaxing, promoting actin depolymerization, occurs with submaximal β2AR activation.

We next examined whether imatinib could augment the bronchorelaxant effect of β-agonist, at submaximal levels of β-agonist stimulation. Whereas 10 nM ISO had almost no relaxant effect on contracted airways and the effect of 10 nM ISO +10 μM IMAT was not different from the effect of 10 μM IMAT alone, 100 nM ISO reduced the lumen area of MCh-contracted PCLS 24±4%, and combined 100 nM ISO +10 μM IMAT reduced lumen area by 59+3% (Figure 7A). Similarly, in MCh-treated human ASM cells, 100 nM ISO reduced the F/G actin ratio by 22±5%, and combined 100 nM ISO +10 μM IMAT reduced the F/G actin ratio by 51±5% (Figure 7B). Finally, to gain insight into whether increased β2AR responsiveness/decreased β2AR desensitization was a potential mechanism of the augmenting effects of IMAT, we analyzed β-agonist-stimulated cAMP accumulation in ASM stimulated with ISO + 10 μM IMAT. Results (Figure 7C) suggest IMAT does not regulate the acute responsiveness of the β2AR at the receptor locus, or of adenylyl cyclase as suggested by unaltered cAMP accumulation stimulated by forskolin.

Figure 7:

Figure 7:

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A. Dose-dependent inhibition of MCh-stimulated airway contraction by ISO in the absence or presence of 10 μM IMAT as described in Methods. Data are means ± SE (n = 5); * denotes p < 0.05 IMAT alone or ISO+IMAT vs ISO alone, and # denotes ISO vs ISO+IMAT as per One-way ANOVA plus Tukey post hoc test. B. Human ASM cells were treated 10 min with MCh (1 μM), MCh + ISO (0.01 – 10 μM) in the absence or presence of IMAT (10 μM) for 10 min. Examination of F-/G-actin pools by immunofluorescence imaging. Fluorescence intensity was analyzed using ImageJ software. n = 4; * denotes p < 0.05 IMAT alone or ISO+IMAT vs ISO alone, and # denotes ISO vs ISO+IMAT as per One-way ANOVA plus Tukey post hoc test. C. Regulation of β-agonist-induced cAMP accumulation in human ASM cultures by IMAT. Human ASM cultures in 24 well plates were stimulated with vehicle, the indicated concentrations of ISO, or 10 μM FSK, in the presence 10 μM IMAT or vehicle for 10 min. Reactions were quenched and cAMP isolated and quantified by ELISA as described in Methods. n=3

DISCUSSION

All current bronchodilator drugs target pharmacomechanical coupling, by either antagonism of pro-contractile GPCR (e.g., m3 muscarinic acetylcholine receptor (m3mAChR), cysteinyl leukotriene type 1 receptor) activation, or by inhibiting the intracellular signaling by these receptors that causes ASM contraction (as with β-agonists) (29). While antagonists of any one pro-contractile GPCR may fail to sufficiently block all relevant contractile signaling (29, 30), multiple factors can compromise effectiveness of β-agonists. Excessive cholinergic discharge in airways coupled with tissue inflammation may inhibit adenylyl cyclases (functional antagonism) (31), and heterologous desensitization (e.g., by intracellular PKA or PKC activity induced by airway inflammation) of the β2AR diminishes β2AR signaling and function (32–36). Further, β-agonists induce homologous desensitization of the β2AR, rendering it ineffective (7). Thus, multiple factors constrain drug effects, and monotherapies are often insufficient in targeting the multiple mechanisms influencing ASM contractile state; therefore, combination therapies are attractive given their multifactorial actions.

Our findings highlight an innovative approach to establish an asthma management strategy that helps overcome the limitations of current bronchoprotective/bronchodilatory drugs. Upon contractile activation, MLC20 undergoes phosphorylation which results in airway smooth muscle (ASM) contraction (pharmacomechanical coupling) and it has been well established that β-agonists regulate this phenomenon to relax ASM, in a PKA-dependent manner (26). The actin cytoskeleton also contributes to ASM contraction through addition of G-actin pools to existing filamentous F-actin (actin polymerization), thus promoting transmission of force between the contractile apparatus and the extracellular matrix (8, 11, 37). Using smooth muscle conditional knockout mice, we previously demonstrated that c-Abl knockout is sufficient to significantly inhibit contraction in murine ASM tissues, without affecting MLC20 phosphorylation (12). In this study, we examined whether combined targeting of pharmacomechanical coupling (by β-agonists) and actin cytoskeleton dynamics (by c-Abl inhibitors) could have a cooperative effect in relaxing ASM.

Our findings show that β-agonists and c-Abl inhibitors by themselves are capable of reversing or inhibiting airway contraction and combining these 2 treatments further enhances the relaxation of contracted ASM in a cooperative manner. The mechanistic details provide new insights into how to optimally disrupt the cooperation between cross bridge cycling and cytoskeleton stiffening that generates tension in ASM cells.

Pretreatment of ASM cells with ISO (1 μM) results in near complete inhibition of pMLC20 induction, thereby limiting any ability of c-Abl inhibitors to further affect this mechanism. Our studies demonstrate that IMAT alone can regulate histamine-induced pMLC20 levels by ~ 50%. However, given that the c-Abl inhibitor GNF-5 failed to inhibit MLC20 phosphorylation, this effect of IMAT is not likely via c-Abl inhibition, and is perhaps of function of IMAT being a competitive inhibitor of the ATP binding pocket (38). It has been previously demonstrated that IMAT can effectively inhibit phosphorylation of MLC20 of smooth muscle tissue in pulmonary arterial rings and inhibit contractile responses, possibly through activation of myosin light chain phosphatase (MLCP), a serine-, threonine-phosphatase (39). MYPT1 phosphorylation at threonine residues by kinases (including PKC) inactivates the enzymatic activity of PP1cδ, thus maintaining pMLC20 levels. We did not observe any effect of IMAT on regulation of MYPT1T853. These observations suggest that MYPT1T853 may not be regulated by IMAT. Finally, although we and others demonstrate that IMAT can regulate pMLC20, it has been suggested that at concentrations >1 μM IMAT (a tyrosine kinase inhibitor) may have off-target effect on other kinases including those involved in promoting contractile actions (40), again possibly related to imatinib’s mechanism of inhibition- competitive binding to the ATP-binding pocket- which limits specificity.

As noted earlier, contractile activation of ASM is driven by pharmacomechanical coupling and actin cytoskeleton reorganization. β-agonists have been shown to mediate ASM relaxation in PKA-dependent manner (26). While PKA-mediated inhibition of MLC20 phosphorylation is believed critical to the relaxant effect of β-agonists, it has been suggested that β-agonists regulate actin polymerization through (PKA-phosphorylated) HSP20 (28). pHSP20 promotes dephosphorylation of cofilin, which is essential in the disassembly of actin filaments, resulting in relaxation. In the current study we observed potentiation of ISO-induced pHSP20 by both inhibitors of c-Abl. Yet, we did not observe similar increases in VASP phosphorylation (another substrate of PKA), suggesting compartmentalized regulation of PKA signaling by GNF-5 and IMAT.

Previous studies have shown β-agonists are capable of de-polymerizing actin as a means of promoting ASM relaxation (9, 10). Herein, we demonstrate that c-Abl inhibitor IMAT and β-agonist ISO promote disassembly of filamentous actin (lower F-/G-actin ratio) to a comparable extent. Combining these two drugs further enhanced actin depolymerization, beyond the magnitude of what is achieved individually.

Limitations of the current study include limited insight into the regulation of human ASM ex vivo and in vivo. Although the currently employed murine models enable a convenient, and paired, analysis of in vivo and ex vivo ASM contractile regulation, differences between murine and human ASM biology and physiology are well-established. Future studies employing either isolated human airway or precision cut lung slices, as well as the testing of other agents (e.g., inhibitors of other kinases (e.g., zipper-interacting kinase (41, 42) or LIM kinase (43)) capable of disrupting actin cytoskeleton integrity will help provide a robustness analysis of such drugs and their cooperativity with more conventional bronchodilator drugs. Although (oral) imatinib is currently being tested in human trials of severe asthmatics by the PrecISE network, the focus of these studies is not on acute bronchodilation but instead relates to imatinib’s anti-inflammatory effects. With a sound preclinical foundation establishing the cooperative effects of imatinib, and the formulation of imatinib enabling effective delivery by inhalation in humans, future clinical research/trial studies will be justified to clarify the therapeutic utility of targeting the actin cytoskeleton in ASM.

In summary, our data demonstrate c-Abl inhibitors and β-agonists each regulate the actin cytoskeleton of ASM via distinct mechanisms. This effect, in addition to the inhibition of pharmacomechanical coupling by β-agonist, translate into cooperative inhibition of ASM contraction when both agents are used in combination. Thus, a therapeutic reserve can be tapped by the addition of a c-Abl inhibitor to β-agonist therapy.

Acknowledgements

The authors thank Alyssa Rezey, Thomas Brown, and Sarah MacMullan for technical assistance. This study was funded by National Heart, Lung, and Blood Institute (NIH/NHLBI) Grant R01-HL145392 (to RBP, DT, RK), and Grants HL110951 and HL130304 (to DT).

Nonstandard Abbreviations

ASM

airway smooth muscle

c-abl

Abelson tyrosine kinase

CFS

contractile force screening

FTTM

Fourier Transform Traction Microscopy

GPCR

G protein-coupled receptor

HSP20

heat shock protein 20

IMAT

imatinib

ISO

isoproterenol

LABA

long acting β2 adrenoceptor agonists

MCh

methacholine

MLC20

myosin light chain 20

MLCP

myosin light chain phosphatase

MYPT1

myosin phosphatase target subunit 1

NDRI

National Disease Research Interchange

PBS

phosphate-buffered saliine

PCLS

precision cut lung slices

PDMS

polydimethylsiloxane

PKA

Protein kinase A

PKA

Protein kinase C

SABA

short acting β2 adrenoceptor agonists

VASP

vasodilator-stimulated phosphoprotein

Footnotes

Conflicts of interest

No conflicts of interest, financial or otherwise, are declared by the authors.

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