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Airway Smooth Muscle Dysfunction Precedes Teratogenic Congenital Diaphragmatic Hernia and May Contribute to Hypoplastic Lung Morphogenesis

Division of Child Health, Royal Liverpool Children's Hospital (Alder Hey); School of Biological Sciences; and The Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom

Correspondence and requests for reprints should be addressed to Neil C. Featherstone, Medical Research Council Clinical Training Fellow, Division of Child Health, School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool L69 3BX, UK. E-mail: N.C.Featherstone{at}Liverpool.ac.uk

	Abstract

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Fetal intervention aims to improve lung growth and survival in congenital diaphragmatic hernia (CDH). Airway smooth muscle (ASM) is important in lung development: ASM progenitors produce a key growth factor for lung morphogenesis (fibroblast growth factor 10); ASM contractility is also coupled to growth. ASM hyperreactivity occurs in postnatal CDH and may exacerbate barotrauma via impaired lung compliance. We hypothesize that ASM hyperreactivity and its sequelae are based on an early developmental lesion of ASM activity in hypoplastic lung. Sprague-Dawley rats were fed 100 mg nitrofen on Day 9.5 of pregnancy to induce lung hypoplasia in offspring (controls had vehicle alone). Normal and hypoplastic lung primordia were cultured from Day 13.5 of gestation at 37°C in 5% CO₂ and loaded at 54 or 78 h with Ca²⁺-sensitive indicators: Fluo-4 for confocal imaging and Indo-1 or Fura-2 for photometric measurements of [Ca²⁺]_i. Hypoplastic lung features spontaneous propagating ASM Ca²⁺ transients with reduced frequency, increased amplitude, and significantly prolonged plateau duration, relative to control lung. Nonetheless, hypoplastic lung exhibits normal requirement for extracellular calcium entry and intracellular calcium release in initiation and regulation of ASM Ca²⁺ waves. Early ASM dysfunction in lung hypoplasia is apparent as specific anomalies of Ca²⁺ transients that indicate a problem with plasmalemmal ion channels/action potential generation. Elucidation of such an ASM lesion may allow pharmacologic amelioration not only of ASM hyperreactivity and its sequelae, but also of hypoplastic lung growth itself.

Key Words: airway smooth muscle • calcium • congenital diaphragmatic hernia • nitrofen

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Congenital diaphragmatic hernia (CDH) results in high mortality and long-term morbidity due to lung hypoplasia, pulmonary hypertension, and postnatal barotrauma (1). Ameliorating the latter via minimizing ventilatory pressures appears to improve outcome (2). Nevertheless, fetal interventions are under trial to improve hypoplastic lung function and growth (3, 4). Despite promising experimental and preliminary clinical data, conclusive evidence to back these interventions is awaited. To optimize such prenatal therapy, a better understanding of the determinants of lung growth is required (5).

Normal lung growth is governed by a combination of biochemical and mechanical factors (6, 7). For example, fibroblast growth factor-10 (FGF-10) is required for branching morphogenesis and proximodistal pulmonary differentiation, while fetal breathing movements, lung liquid–induced stretch, and airway smooth muscle (ASM) peristalsis appear to modulate pulmonary growth (7–10). Recent evidence that FGF-10 is produced by ASM progenitors provides a putative link between biochemical and mechanical stimuli to lung growth and underlines the importance of ASM in lung development (11). ASM undergoes spontaneous peristaltic contractions prenatally in a variety of species, including humans (12, 13). This activity is coupled to, and appears to regulate, normal lung growth (8); these peristaltic waves are underpinned by temperature sensitive, gap junction–dependent propagating ASM Ca²⁺ waves (14). Notably, transgenic pulmonary epithelial expression of an inhibitor of the Ca²⁺-binding protein, calmodulin, disrupts lung morphogenesis in vivo (15). If ASM function is important to normal lung growth, one might expect evidence of ASM abnormalities when lung growth is defective. Indeed, in both human and experimental lung hypoplasia, ASM-related dysfunction is apparent near term. Human hypoplastic lung exhibits abnormalities of serum response factor (SRF) isoforms (SRFs regulate ASM gene expression) (16). In the nitrofen model of CDH, ASM hypercontractility is apparent near term (17). Moreover, survivors of CDH suffer with long-term bronchial hyperreactivity (18).

We have hypothesized that rather than being the "appendix of the lung" (19), ASM helps regulate early lung morphogenesis (e.g., via its interrelationship with FGF-10 production) and also modulates later fetal lung growth, via effects on lung compliance. Certainly when normal neonatal lung is exposed experimentally to chronic positive pressure ventilation, ASM dysfunction (airway hyperreactivity) emerges in tandem with significant airway remodeling, again linking ASM activity and morphogenesis (20). If, therefore ASM plays a key role in regulation of fetal lung compliance and hence growth, it seems likely that abnormal ASM function in hypoplastic fetal lung may not only impair late prenatal growth, but also increase susceptibility to postnatal barotrauma and remodeling. In the present study we have tested the hypothesis that near-term ASM dysfunction seen in hypoplastic CDH lung has its origins not simply in compression of the fetal lung by a visceral hernia, but is apparent from the embryonic stages of hypoplastic lung development (21, 22).

We have used Ca²⁺ imaging of hypoplastic lung primordia to demonstrate early ASM dysfunction: Ca²⁺ transients in hypoplastic lung feature prolonged plateau phase, reduced frequency (at certain temperatures), and increased amplitude. Given that we have also shown that hypoplastic lung ASM Ca²⁺ transients have normal dependence on intra- and extracellular Ca²⁺ and gap junction–mediated propagation, the anomalous temporal characteristics of these Ca²⁺ transients suggest a key problem in the ASM ion channels/action potential generation. We propose that ASM dysfunction may underlie several clinically observed behaviors of the hypoplastic CDH lung.

	MATERIALS AND METHODS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Embryonic Lung Culture
Nitrofen (2,4-dichloro-4'-nitrodiphenylether) 100 mg (Zheijang Chemicals, Hangzhou, China) dissolved in olive oil was gavage fed to timed-pregnant Sprague-Dawley rats (Charles River Ltd, Margate, UK) on Day 9.5 of gestation (vaginal plug = Day 0, term = Day 22.5) to induce embryonic lung hypoplasia by Day 13.5 of gestation and left-sided CDH and pulmonary hypoplasia in newborn pups (23). In accordance with UK legislation, embryos were harvested on Day 13.5 of gestation (as previously described) (24). This is 24 h after the lung anlage forms as a diverticulum from the foregut. Upon retrieval, embryos were transferred to an isotonic saline bath cooled on ice. Microdissected from their extra-embryonic membranes, embryos were secured in a lateral position using cranial and caudal entomology pins. Using a Leica (Leica Microsystems [UK] Ltd., Milton Keynes, UK) MZ6 stereomicroscope and microsurgical instruments, a thoracic incision was performed to expose the embryonic heart and lungs. The heart was teased from the lung rudiments before the esophagus was freed carefully from the primitive carina. The trachea was then sectioned and the pulmonary complex transferred by pipette into serum-free culture medium (DMEM/F12 1:1; Gibco, Invitrogen Life Technologies, Paisley, UK). Lung rudiments were positioned on translucent polytetrafluorethylene membrane culture-dish inserts (Millicell; Millipore Corp., Bedford, MA) with serum-free culture media incorporating penicillin (100 IU/ml) and streptomycin (100 µg/ml; Gibco, Invitrogen Life Technologies). Lung primordia were incubated at 37°C in 5% CO₂ for periods up to 78 h. Using brightfield microscopy, specimens were inspected daily for histologic integrity, viability, and the periodic motion of luminal lung fluid. Culture media were changed every 48 h. Spontaneous airway contractions are seen in 100% of explants by 48 h in vitro using this established culture technique (8).

Loading Lung Explants with Ca²⁺-Sensitive Fluorophores
Control and hypoplastic lungs were cultured for 54 or 78 h before 4 h incubation at 37°C with the membrane-permeant form of Fluo-4 AM (15 µM; Molecular Probes, Invitrogen Life Technologies, Paisley, UK) with Pluronic F (for confocal imaging), or 2 h incubation at 37°C with the membrane-permeant forms of Indo-1 AM or Fura-2 AM (15 µM; Molecular Probes, Invitrogen Life Technologies) with Pluronic F (for ratiometric imaging of [Ca²⁺]_i). Explants were then allowed to equilibrate for 30 min in physiologic saline at 37°C.

Confocal and Photometric Measurements
For confocal and photometric studies, lung primordia were secured in a 200-µl custom-made perfusion chamber by nylon mesh (Plastok, Birkenhead, UK) that holds the lung periphery against the coverslip without compressing principal airways (14).

Confocal imaging was used to determine spatiotemporal characteristics of intercellular Ca²⁺ waves and performed on an Ultraview LCI spinning Nipkow disc, widefield Olympus IX70 inverted microscope (Perkin-Elmer Confocal Microscope System, Cambridge, UK) with an ORCA ER cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). This permits image acquisition from a large area (from 2.1 x 1.6 mm with 4x objective to 160 x 110 µm with 60x objective) at 20–30 frames per second with relatively low intensity laser illumination and at good signal to noise ratio. Fluo-4–loaded explants were viewed at low-power (4x objective, numerical aperture [NA] 0.13; 10x objective, NA 0.17) or high-power magnification (60x water immersion objective, NA 0.9) and excited by argon/krypton laser at 488 nm. Resulting fluorescence was measured at 510 nm and is expressed as a function of maximum compared with basal values (F/F₀). To reduce photobleaching, Ca²⁺ transients from each lung were observed for not more than 90 s. Propagation speeds of the Ca²⁺ and contractile waves were measured using the Perkin-Elmer Microscope System: first, several regions of interest were selected randomly at discrete points in the airway. Separation of any two points was measured as pixel counts, and these figures were then converted to distance (µM) using calibration measurements for each objective and pixel binning option. Measurement of the interval (s) between initiation of the Ca²⁺ transient (or ASM contraction) at one point and the next, therefore, allowed propagation speed to be quantified.

To study intracellular Ca²⁺ transients, their temporal characteristics, and effects of their modulation by temperature and pharmacologic agents, calcium signaling was also measured photometrically using two ratiometric Ca²⁺-sensitive indicators, Indo-1 or Fura-2. The optical system has been described previously (25). Briefly, when Indo-1 was used, the photometric system consisted of an Olympus IX50 inverted microscope (Olympus UK Ltd., London, UK) and xenon lamp with an excitation wavelength of 340 nm. Light emitted by Indo-1–loaded explants at 400 nm and 500 nm was detected via photo-multiplier tubes and digitally recorded. The ratio of the fluorescence signal (F_400/500; 400 nm: 500 nm emission) was used as an indicator of [Ca²⁺]_i. This technique is less susceptible to movement artifact (than Fluo-4–based confocal imaging) as it integrates the signal (at two wavelengths) from a defined region. Photometric measurements of Fura-2–loaded explants employed a Cairn (Cairn Research Ltd., Faversham, UK) monochromator with dual excitation (340 and 380 nm) and the fluorescent signals were measured at 510 nm (F_340/380). With both indicators, Ca²⁺ signals were recorded using a 20x objective (N.A. 0.75) from the second bronchial division of the right lung (previously reported origin for most mechanical contractions) (8). For each lung, primordia Ca²⁺ transients were observed for at least 10 min. Temporal characteristics of the Ca²⁺ waves (duration of fast and slow phases, time to achieve 50% of peak amplitude, plateau duration, relaxation half-time [T₅₀], and duration at 50% relaxation) were determined from at least three representative transients per lung. Relative amplitudes of spontaneous Ca²⁺ transients and agonist-induced [Ca²⁺]_i responses were expressed as a percentage of the rise in [Ca²⁺]_i resulting from 1-min control applications of high-K⁺ solution (120 mM) or the muscarinic agonist, carbachol (100 µM); this permitted reproducible comparison between hypoplastic and control lung.

Solutions
Unless otherwise stated, lung explants were superfused at 37°C with buffered physiologic saline (pH 7.4) containing (in mM): 120 NaCl, 5.6 KCl, 0.12 MgSO₄, 2 CaCl₂, 11.7 glucose, 10.9 Hepes. Zero Ca²⁺ solution used in certain experiments consists of physiologic saline with CaCl₂ omitted and 2 mM EGTA added. Other additions to standard superfusate that were investigated include: the L-type Ca²⁺ channel antagonist, nifedipine (10 µM); the L-type Ca²⁺ channel agonist, Bay-K 8644 (1 µM); the gap junction uncoupler, 18- glycyrrhetinic acid (18-GA, 40 µM); the K⁺ channel antagonist, tetraethylammonium (TEA, 10 mM); the sarcoplasmic reticulum (SR) Ca²⁺ATPase inhibitor, cyclopiazonic acid (CPA, 20 µM); caffeine (1–10 mM), to facilitate calcium induced calcium release (CICR) via ryanodine receptors (RyR); ryanodine (20 µM), to inhibit ryanodine receptors; the muscarinic agonist, carbachol (1–100 µM); 2-aminoethoxy-diphenylborate (2-APB, 50 µM), a putative inhibitor of inositol triphosphate receptors (InsP₃R); and depolarizing high K⁺ solution (10–140 mM). Carbachol and ryanodine were dissolved in distilled water; CPA, 2-APB, and 18-GA were dissolved in dimethylsulfoxide (DMSO); nifedipine and Bay-K 8644 were dissolved in ethanol (ETOH). Calcium wave generation and airway contractility were unaffected by applications of vehicle alone (DMSO or ETOH). Ryanodine was obtained from Calbiochem (Merck Biosciences Ltd., Nottingham, UK). Sigma (Poole, Dorset, UK) supplied all other chemicals.

Histology
Cultured lungs were preserved after 78 h in 4% paraformaldehyde (0.1 M PBS, pH 7.4), rinsed in PBS, cryoprotected with 20% (wt/vol) sucrose, gelatin embedded (7.5% [wt/vol] gelatin, 15% [wt/vol] sucrose in PBS) before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap-frozen at –40°C. Lung sections were taken at 7 µm and mounted on chrome alum gel slides for storage at –40°C.

Slides were rinsed in PBS and incubated with -smooth actin antibody (monoclonal anti–-SMA Clone No.1A4, mouse; Sigma, Gillingham, UK) at 1:500 dilution with 0.1% BSA in PBS overnight at 4°C. They were then rinsed in PBS and the primary antibody labeled with FITC-labeled goat anti-mouse IgG (Sigma, Gillingham, UK) at 1:100 dilution. After washing, slides were mounted with Fluorescent mounting medium (S3023; Dako, Ely, UK).

Statistical Analysis
Data processing was performed using Origin 6.0 software (OriginLab Corporation, Northampton, MA). The SPSS v12.0 package (SPSS UK Ltd., Woking, UK) was used for statistical analysis. Parametric data are reported as mean ± SEM and compared using Student's t test. Nonparametric data are reported as medians (interquartile ranges) and compared with Mann Whitney U test. Statistical significance was taken at P < 0.05. Dose response curves were created using Origin Microcal sigmoidal fitting to the experimental points presented as means ± SEM.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Temporal Characteristics of ASM Ca²⁺ Waves Are Abnormal in Experimental Lung Hypoplasia
Hypoplastic lung primordia exhibited spontaneous regenerative intercellular Ca²⁺ waves similar to those previously demonstrated in control embryonic lungs (14). Confocal imaging of Fluo-4–loaded hypoplastic lungs with low-power magnification (4x or 10x) at 37°C, revealed intercellular Ca²⁺ waves propagating longitudinally through the principal airways. Similar to control lungs (14), hypoplastic ASM Ca²⁺ waves initiated from regions within the trachea, the left or right main airway, or occasionally from two foci simultaneously. High-power magnification (60x) revealed that the Ca²⁺ waves propagated via individual ASM cells, rather than undifferentiated mesenchyme or airway epithelium. Developing ASM cells were identified by their characteristic spindle shape, immediate subepithelial location, the orientation of the cell's long axis perpendicular to the adjacent airway long axis, and their smooth muscle actin immunopositivity (data not shown) as previously described (14). Hypoplastic lung intercellular Ca²⁺ waves propagated with an average speed of 241 ± 14 µm/s (n = 4, 24 individual Ca²⁺ waves), a speed similar to that of Ca²⁺ waves in control lung (219 ± 14 µm/s; n = 4, 10 individual Ca²⁺ waves) (P = NS).

Despite these similarities, the temporal characteristics of hypoplastic lung ASM Ca²⁺ transients were altered compared with control ASM Ca²⁺ transients at both 37°C and 27°C (Figure 1). Hypoplastic lung ASM Ca²⁺ transients show characteristic 4-phase morphology: (1) fast phase, (2) slow phase to plateau, (3) plateau phase, and (4) relaxation phase. Hypoplastic lungs had an elongation of the plateau phase alone compared with normal control lung (2.45 ± 0.13 s versus 1.94 ± 0.15 s, P = 0.028); this prolongation was accentuated by cooling to 27°C (4.83 ± 0.29 s versus 3.32 ± 0.27 s, P = 0.005; Figure 1, Table 1) and accompanied by a significant reduction in the frequency of Ca²⁺ oscillations (0.49 ± 0.04 versus 0.7 ± 0.06 per minute, P = 0.003; Table 2). The frequency of Ca²⁺ oscillations at 37°C did not differ significantly between hypoplastic and normal lungs (0.67 ± 0.06 versus 0.81 ± 0.06 per minute, P = NS; Table 2).

View larger version (11K): [in this window] [in a new window]   Figure 1. Ca2+ transients within hypoplastic lung primordia have altered temporal characteristics. Superimposed normalized traces of typical Ca2+ transients recorded photometrically at 37°C and 27°C in nitrofen hypoplastic lungs and control lungs.

There were no gross differences between hypoplastic and normal lungs in the requirements of these three elements of Ca²⁺ signaling for the initiation or regulation of Ca²⁺ oscillations (Figures 2 and 3). Thus, in hypoplastic lung, propagating ASM Ca²⁺ transients (and consequent peristaltic contraction) require (1) external entry of Ca²⁺ via L-type Ca²⁺ channels, (2) SR Ca²⁺ uptake via the SR Ca²⁺ATPase (SERCA pump), (3) Ca²⁺ release via ryanodine (RyR) and InsP₃R receptors, and (4) functioning gap junctions.

View larger version (20K): [in this window] [in a new window]   Figure 2. Ca2+ transients within hypoplastic lung primordia are dependent on extracellular Ca2+ and gap junction integrity. (A) Reversible inhibition of Ca2+ oscillations by removal of external Ca2+. Ca2+-free solution (with 2 mM EGTA) decreased baseline intracellular calcium [Ca2+]i and rapidly abolished both intracellular Ca2+ transients and peristalsis in hypoplastic lung explants (n = 4). Readmission of Ca2+ quickly restored Ca2+ oscillations and mechanical activity. (B) Abolition of Ca2+ oscillations by L-type Ca2+ channel blocker (nifedipine, 10 µM). Nifedipine rapidly abolished spontaneous Ca2+ transients and peristalsis while reducing baseline [Ca2+]i (n = 4). (C) Increased frequency and amplitude of Ca2+ oscillations with L-type Ca2+ channel agonist Bay-K 8644 (1 µM). Bay-K 8644 (n = 4) raised [Ca2+]i, and increased Ca2+ wave amplitude (from 9 ± 2% to 18 ± 2% of peak obtained with depolarizing 120 mM K+ solution; P = 0.026) and frequency (from 0.48 ± 0.08 to 1.45 ± 0.25 per minute; P = 0.006). Data were obtained at 27°C. Amplitudes of the spontaneous Ca2+ transients (above) are at the lower end of the range quoted in Table 3. (D) Effects of K+ channel blocker (TEA, 10 mM) on Ca2+ oscillation frequency and relative amplitude. TEA (n = 5) did not significantly change Ca2+ wave amplitude (without TEA: 28 ± 10% versus with TEA: 44 ± 14% of peak with high K+ solution; P = NS) or frequency (without TEA: 0.34 ± 0.05 versus with TEA: 0.82 ± 0.21 per minute; P = NS). Data were obtained at 27°C. (E) Inhibition of Ca2+ oscillations by 18--glycyrrhetinic acid (40 µM). The gap junction uncoupler 18--glycyrrhetinic acid abolished both propagating Ca2+ waves and coordinated peristalsis after an initial brief excitatory phase (n = 4).

View larger version (20K): [in this window] [in a new window]   Figure 3. Ca2+ transients within hypoplastic lung primordia are dependent on intracellular Ca2+ uptake via the SR Ca2+ATPase (SERCA pump) and release via RyR and InsP3R channels. (A) Agonist-induced SR Ca2+ release: hypoplastic ASM SR Ca2+ can be released via RyR and InsP3R channels. Caffeine was used to facilitate the Ca2+-induced Ca2+ release (CICR) properties of RyR. Likewise, carbachol was used to activate InsP3R-mediated SR Ca2+ release through inositol triphosphate (IP3) production. The experimental protocol used Ca2+-free solution to eliminate plasmalemmal Ca2+ entry. Caffeine (20 mM) transiently increased [Ca2+]i and produced brief tonic airway contraction. Carbachol (100 µM) generated high-amplitude Ca2+ transients and sustained tonic contraction. Data were obtained at 27°C. Amplitudes of the spontaneous Ca2+ transients shown are at the lower end of the range quoted in Table 3. (B) Abolition of Ca2+ oscillations by inhibition of the SR Ca2+ATPase. SR SERCA pump blockade with CPA (20 µM; n = 4) markedly increased baseline cytosolic [Ca2+]i and gradually abolished Ca2+ transients. Phasic mechanical contractions were progressively replaced by tonic airway contraction. Removal of CPA from the bathing solution reversed its effects. (C) Caffeine (1 mM) stimulates and subsequently abolishes Ca2+ oscillations. Facilitating CICR via caffeine resulted in a biphasic response, which may be due to activation of depolarizing chloride channels and/or hyperpolarizing K+ channels (see below) (n = 4). (D) Co-application of caffeine (1 mM) and TEA (10 mM) results in continued generation of Ca2+ transients. Blockade of K+ channels (voltage-gated [Kv] K+ channels and large-conductance [BK] Ca2+-activated K+ channels) by TEA prevented caffeine's inhibitory action, suggesting that caffeine-induced CICR does mediate K+ channel opening (n = 4). (E) Ryanodine (40 µM) abolishes Ca2+ transients. Inhibiting SR Ca2+ release via RyR using ryanodine abolished spontaneous Ca2+ waves and contractility (n = 4). (F) 2-APB (50 µM) abolished Ca2+ oscillations. Inhibition of InsP3R mediated SR Ca2+ release via 20-min application of 2-aminoethoxy-diphenylborate (2-APB), increased baseline [Ca2+]i, and gradually abolished both Ca2+ waves and contractility (n = 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Relative amplitudes of Ca2+ transients in hypoplastic lung primordia are elevated compared with controls. Experimental traces highlighting the amplitude of Ca2+ transients in nitrofen (upper trace) and control (lower trace) lungs relative to 1-min control applications of depolarizing high-K+ solution or 100 µM carbachol. Data were obtained at 27°C.

View larger version (10K): [in this window] [in a new window]   Figure 5. Hypoplastic ASM exhibits increased sensitivity to depolarizing KCl solution. Dose–response curve illustrating the activation of the Ca2+ transient by depolarizing high-K+ solution (10–140 µM) in hypoplastic lungs (circles) and control lungs (triangles). The dose–response curve was constructed from the phasic component only of the activated Ca2+ transient. The tonic component was inconsistent in hypoplastic (n = 10) and control (n = 12) lungs. Data are presented as mean ± SEM.

View larger version (11K): [in this window] [in a new window]   Figure 6. Effects of carbachol on [Ca2+]i are similar in hypoplastic and control lung ASM. Bar chart illustrating the activation of the Ca2+ transient by the muscarinic agonist, carbachol (1–100 µM), in hypoplastic lungs (open bars; n = 9) and control lungs (filled bars; n = 9). Data are presented as mean ± SEM.

View larger version (12K): [in this window] [in a new window]   Figure 7. SR Ca2+ release is similar in normal and hypoplastic lung ASM. Bar chart illustrating the Ca2+ response to applications of caffeine 20 mM (in zero Ca2+ solution and 2 mM EGTA), carbachol 100 µM, and carbachol 100 µM (in zero Ca2+ solution and 2 mM EGTA). High K+ application (1 min) was applied to generate a 100% reference for [Ca2+]i. Hypoplastic lungs are represented by open bars (n = 8); control lungs are represented by filled bars (n = 7). Data are presented as mean ± SEM.

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
The hypoplastic lung and its susceptibility to barotrauma are key determinants of survival in congenital diaphragmatic hernia (1). We have postulated that ASM function is important not only in FGF-10 driven early lung morphogenesis but also in compliance-regulated late prenatal lung growth and potentially in regulating remodeling responses to ventilation. This overarching "smooth muscle" hypothesis is based upon several observations, including the following: (1) ASM progenitors produce FGF-10 (11); (2) ASM activity is coupled to embryonic lung growth in vitro (8); (3) human and experimental lung hypoplasia is associated with perinatal ASM dysfunction together with decreased lung compliance (16, 17); and (4) neonatal lung responds to chronic positive pressure ventilation with a combination of ASM dysfunction, impaired compliance, and airway remodeling (20). The present study examines the narrower hypothesis that ASM dysfunction observed near-term in human and experimental lung hypoplasia is not merely a late sequel of lung compression by a visceral hernia, but is instead an early indicator of (and perhaps contributor) to embryonic failure of lung growth. To test this, we have used the nitrofen model of CDH: embryonic lungs exposed in utero to nitrofen display hypoplastic growth before the visceral hernia supervenes (24). In contrast to the surgically created lamb model of CDH, nitrofen-induced CDH permits investigation of early pre-CDH events in the developing hypoplastic lung (26). To study early ASM dysfunction in embryonic lung explants, we have used confocal imaging and photometric measurements of [Ca²⁺]_i for two reasons. First, Ca²⁺ transients are known to underpin ASM peristalsis in normal lung explants (14). Second, spontaneously contracting cultured lung explants of < 0.5 mm length do not readily lend themselves to measurement of force.

Using these techniques, we have shown that the Ca²⁺ transients in hypoplastic lung exhibit significant abnormalities that together suggest a lesion of ASM plasmalemmal ion channels and/or action potential generation. Furthermore, we have shown that ASM peristalsis in hypoplastic lung is underpinned by spontaneous regenerative Ca²⁺ waves propagating via gap junctions that are dependent on not only extracellular Ca²⁺ entry but also intracellular Ca²⁺ release.

Normal and hypoplastic ASM Ca²⁺ transients appear to be action potential mediated, given their characteristic four-phase morphology, propagation speed, and transmission via gap junctions (27, 28). Hence, prolongation of the plateau phase of Ca²⁺ transients at 37°C and 27°C in hypoplastic lung ASM is likely to be due to alteration in the action potential (28). Further support for a prolongation of the action potential derives from the observed increase in relative amplitude of the Ca²⁺ transients in hypoplastic lung (relative to KCl-induced and carbachol-induced maxima) (28). Underlying abnormalities of membrane potential and ion channel functioning are also suggested by the significant reduction observed in the frequency of Ca²⁺ transients in hypoplastic lung at 27°C (28). Again consistent with an alteration in membrane potential/ion channel functioning, hypoplastic lung exhibited an elevated sensitivity to depolarizing KCl solutions. However, mechanisms controlling initial entry and/or release of Ca²⁺ and its subsequent SR uptake or extrusion from the cell appear unaffected in hypoplastic lung: the rates of rise and relaxation of the Ca²⁺ transients at 37°C and 27°C are comparable between normal and hypoplastic lung. Indeed, hypoplastic lung ASM appears to have normal SR activity: [Ca²⁺]_i dose–response to carbachol showed no significant difference between hypoplastic lungs and controls, even when external Ca²⁺ entry was eliminated. Similarly, there was no significant difference in the caffeine response in hypoplastic and control lung primordia. Unequivocal demonstration of the abnormalities of action and/or membrane potential necessitates micro-electrode recording (28). However, the embryonic lung presents considerable difficulties due to its small size and mechanical activity dislodging micro-electrodes. However, in the adult rat ureter preparation, prolongation of the plateau phase of the smooth muscle Ca²⁺ transient (by cooling) is similarly accompanied by increase in the amplitude of the Ca²⁺ transient and elongation of the action potential plateau phase (28).

Embryonic nitrofen-exposed lung, therefore, features abnormal Ca²⁺ transients that accompany early hypoplasia and precede supervention of the visceral hernia. Since propagating Ca²⁺ waves underpin ASM peristalsis (14), this indicates that ASM function is already abnormal at the embryonic stages of hypoplastic lung development. The latter conclusion is significant given that (1) ASM progenitors furnish the FGF-10 required for early branching morphogenesis; and (2) nitrofen-exposed lung primordia exhibit branching abnormalities in tandem with FGF-10 deficiency (11, 29). Together, this supports the thesis that ASM contributes to early FGF-10–driven lung morphogenesis and that this relationship is disrupted before CDH supervening. ASM dysfunction also persists after CDH develops: nitrofen-exposed fetal lungs display increased ASM contractility and human CDH survivors manifest reactive airways disease (17, 18). Therefore, the embryonic ASM dysfunction we have shown (increased duration and amplitude of embryonic ASM Ca²⁺ transients) may underlie, in part, later post-CDH ASM abnormalities (airway hyperactivity).

Having shown that early ASM dysfunction accompanies embryonic growth failure and is consistent with late ASM lesions, it is worth considering the mechanisms whereby ASM dysfunction might contribute to impaired lung growth. The "smooth muscle hypothesis" postulates that ASM abnormalities may impair not only embryonic lung growth (related to FGF-10–producing ASM progenitors) but also fetal pulmonary growth (via dysregulated lung compliance) (21). This later role of ASM dysfunction in fetal and postnatal lung development merits review. ASM contractility generates tension in the airway wall (13). It is reasonable to assume that such changes are accompanied by alterations in intraluminal pressure. The latter is known to influence several aspects of lung cell and organ growth (7, 30). Hence ASM activity can plausibly affect lung growth by virtue of its wider mechanical effects on lung compliance. Experimental support for this concept emerges from observation that in vitro inhibition/stimulation of Rho-associated kinase results in decreased/increased lung growth, respectively (presumably via modulation of cytoskeletal tension) (31). Certainly if unrecognized ASM dysfunction impairs compliance of hypoplastic lung, one might expect more unpredictable results from prenatal tracheal occlusion (32). Indeed, the results of tracheal occlusion in previously normal fetal lung may be less easily extrapolated to effects in hypoplastic lung. Moreover, the reported benefits of dynamic rather than static tracheal occlusion may arise in part due to simulation of the pressure waves generated by ASM peristalsis (33). Finally, if ASM participates in the regulation of neonatal lung compliance, observed ASM dysfunction may help explain the increased susceptibility of the hypoplastic lung to barotrauma (2).

In summary, we have shown that despite normal dependence on extracellular Ca²⁺ entry, intracellular Ca²⁺ release, and propagation via gap junctions, ASM Ca²⁺ transients in hypoplastic lung primordia feature abnormal ASM Ca²⁺ transients that are consistent with a lesion of membrane ion channels and/or action potential. These abnormalities precede CDH and yet are congruent with ASM dysfunction observed perinatally. We have proposed that such early ASM lesions may be important to defective lung development not only in embryogenesis but also in late fetal life and after postnatal ventilation. Testing this "smooth muscle hypothesis" may therefore help us to explain the survival benefits of limiting postnatal ventilatory pressures and emphasize the need for similar consideration during tracheal occlusion in human fetuses with CDH (21).

	Footnotes

 
This study was supported by the Medical Research Council, The Royal College of Surgeons of England, The Academy of Medical Sciences, The Health Foundation, and Birth Defects Foundation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0079OC on May 25, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 22, 2006

Accepted in final form May 9, 2006

	References

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 

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