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Abstract
Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation–contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5–240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist–induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation–contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.
The salient clinical feature of asthma, bronchospasm, is due primarily to agonist-induced airway smooth muscle (ASM) shortening (1). In ASM cells, the intracellular signaling cascades, collectively known as excitation–contraction (EC) coupling, evoke force generation (2). EC coupling can be semiquantitatively assessed in HASM cells by measuring the markers: increase in cytosolic Ca2+ and phosphorylation of MLC (myosin light chain) and MYPT1 (myosin phosphatase regulatory subunit 1). A variety of extrinsic and intrinsic factors modulate EC coupling in ASM cells to amplify airway hyperresponsiveness (AHR) in asthma. Our previous studies showed that cytokines, toxicants, and allergens amplify EC coupling in ASM cells to mediate AHR (3, 4). Recently, we reported that human ASM (HASM) cells isolated from obese lung donors exhibited amplified contractile phenotype, characterized by elevated markers of EC coupling (5). However, the precise mechanisms driving amplified EC coupling in HASM cells remain poorly understood.
Glucose catabolism generates energy and intermediate metabolites critical for cellular functions. Glycolysis is regulated by three rate-limiting enzymes, namely hexokinase, phosphofructokinase, and pyruvate kinase (6). Among these, PFK1 (phosphofructokinase 1) catalyzes the first reaction that commits glucose to glycolysis, and its enzyme activity is regulated by the ATP/AMP ratio (6). F2,6BP (fructose 2,6 bisphosphate) is an allosteric activator of PFK1 and reverses the inhibitory effect of ATP to increase the affinity of PFK1 to fructose-6-phosphate (7). PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase) catalyzes formation of F2,6BP that could be targeted to regulate cellular glycolysis rates (8, 9). Prior studies have reported altered glucose metabolism in structural and inflammatory lung cells in airway diseases. Findings from those studies include increased oxidative phosphorylation in airway epithelial cells (10) and increased lactate in nasal epithelial cells (11) in asthma. In a house dust mite–induced murine model of asthma, dendritic cells critical for allergen sensitization showed elevated glycolysis upon activation and inhibition of glycolysis-attenuated dendritic cell migration and inflammatory mediator release (12). However, little is known about the role of glycolysis in the contractile function of ASM cell. To assess whether glycolysis regulates the contractile function of ASM, we hypothesized that glycolysis modulates EC coupling in HASM cells.
Using primary HASM cells and human precision-cut lung slices (hPCLS) obtained from human lung donors, we demonstrate that pharmacological inhibition of glycolysis depleted cellular ATP and attenuated agonist-induced EC coupling in HASM cells and carbachol-induced bronchoconstriction in hPCLS. Our studies suggest that glycolysis in HASM cells can serve as a novel target to transiently decrease cellular ATP and inhibit EC coupling in ASM cells and bronchoprotect human airways.
hPCLS were prepared from healthy (i.e., without lung pathology) human lung donors obtained from the National Disease Research Interchange (Philadelphia, PA) or the International Institute for the Advancement of Medicine (Edison, NJ). These were deidentified human tissue and therefore exempt from institutional review board (IRB) approval for human subject research. hPCLS were prepared and carbachol (CCh)-induced bronchoconstriction assays were conducted as described in our previous publications (15, 16).
HASM cells were isolated from the lungs of human donors without asthma procured from the National Disease Research Interchange or International Institute for the Advancement of Medicine. Donor characteristics are presented in Table E1 in the data supplement. Primary HASM cells were harvested, characterized, and grown in culture as described in our previous publications (13).
Based on preliminary experimental findings, HASM cells or hPCLS were treated with 10 μM of PFK15 for 10 minutes. Final DMSO concentration in vehicle control was 0.05%. In HASM cells, contractile agonists CCh (25 μM, 10 min) and histamine (2.5 μM, 10 min) were used to increase MLC, MYPT1, or AKT phosphorylation with or without PFK15.
HASM cells grown to confluence in 12-well format were transiently transfected with nontargeting or PFKFB3-targeting siRNA (40 nM) validated by previous publications (Ambion, siRNA ID: s10357) (14). Experiments were conducted in transfected cells at 48 hours after transfection.
In HASM cells and hPCLS, Seahorse XF Glycolysis Stress Test, Seahorse XF Cell Mito Stress Test, and Seahorse XF Real-Time ATP Assays were conducted according to manufacturer’s instructions (Agilent). Details are provided in the data supplement.
RhoA activation was measured using the RhoA Pull-Down Activation Assay Biochem Kit (Cytoskeleton Inc.). PFK15 (10 μM, 10 min) or the RhoA inhibitor Rhosin (3 μM, 10 min)-pretreated HASM cells were stimulated with recombinant human thrombin (0.1 U/ml) for 2 minutes. The lysates (600 μg total protein) were incubated (1 h) with 50 μg of Rhotekin beads, centrifuged, and the pellets were analyzed by Western blot for RhoA.
Agonist-induced [Ca2+]i in HASM cells was determined as previously described (3).
HASM cells were treated with vehicle or PFK15 (10 μM) for 10–60 minutes, then stimulated with the β-adrenergic receptor agonist isoproterenol (10 μM, 10 min) or adenylyl cyclase activator forskolin (10 μM, 5 min). The cells were lysed and analyzed by cAMP screen ELISA system from Applied Biosystems as previously described (15).
HASM cells or hPCLS from at least five independent lung donors (biological replicates) were used in experiments, with three technical replicates for each experimental condition (donor characteristics in Table E1). When applicable, experimental measurements were first normalized to vehicle control in each donor to obtain fold change and used to obtain group mean ± SEM. Interexperimental group comparisons were performed by unpaired, two-tailed Student’s t test, one-way ANOVA with multiple group comparison test, or two-way ANOVA with Sidak’s multiple comparisons test in GraphPad Prism 8.0, with significance set at P ⩽ 0.05.
PFK15, a small molecule inhibitor of glycolysis, selectively inhibits PFKFB3 and reduces F2,6BP formation (16). F-2,6-BP is an allosteric activator of PFK1, the key rate-limiting enzyme in glycolysis. Seahorse assay was used to measure the metabolic activity of HASM cells. In the control group, glucose injection increased extracellular acidification rate, which was then inhibited by rotenone and antimycin A, the inhibitors of mitochondrial electron transport chain complex I and III, respectively. The injection of 2-DG (2-deoxy-D-glucose), a glucose analog which competitively inhibits hexokinase, abrogates glycolysis. PFK15 inhibited glycolysis, maximal glycolytic capacity, and glycolytic reserve in HASM cells (Figures 1A–1D). PFK15 also attenuated maximal mitochondrial respiration and mitochondrial ATP production with little effect on basal respiration in HASM cells (Figures 1E–1H). LDH (Lactate Dehydrogenase) assays showed that PFK15 treatment had little effect on HASM cell viability (Figures E1A–E1C). These findings suggest that PFK15, at the concentrations used, inhibits glycolysis and ATP generation in HASM cells without cytotoxicity.
Figure 1. PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one) inhibits glycolysis in human airway smooth muscle (HASM) cells. (A) representative trace of ECAR in Seahorse XF assay. (A–D) PFK15 (10 μM) attenuates (B) glycolysis rates, (C) glycolytic capacity, and (D) glycolytic reserve in HASM cells (n = 10 donors). (E) representative trace of OCR in Seahorse XF assay. (E–H) PFK15 (10 μM) attenuates (G) maximal mitochondrial respiration and (H) mitochondrial ATP production in HASM cells, (F) with little effect on basal mitochondrial respiration (n = 10 donors). Data are presented as mean ± SEM; unpaired Student’s t test, compared with vehicle, **P < 0.01. 2-DG = 2-deoxy-D-glucose; ECAR = extracellular acidification rate; FCCP = trifluoromethoxy carbonylcyanide phenylhydrazone; OCR = oxygen consumption rate.
[More] [Minimize]In ASM cells, increased MLC phosphorylation promotes cell shortening; therefore, it can be measured as a biochemical surrogate of EC coupling (17). We tested whether inhibition of glycolysis by PFK15 altered agonist-induced MLC phosphorylation in HASM cells. PFK15 attenuated CCh-induced MLC phosphorylation in a concentration-dependent manner (Figures 2C and 2D), with the maximal inhibition at 5–30 minutes of exposure (Figures 2A and 2B). PFK15 pretreatment also attenuated histamine-induced MLC phosphorylation in HASM cells, suggesting that PFK15 effect on MLC phosphorylation is mediated in a contractile agonist–independent manner (Figures 2E and 2F). Pretreatment with β2AR (beta2-adrenergic receptor) agonist formoterol (10 nM) also significantly attenuated agonist-induced MLC phosphorylation in HASM cells.
Figure 2. Glycolysis inhibition attenuates agonist-induced MLC (myosin light chain) phosphorylation in HASM cells. (A and B) PFK15 (10 μM) attenuates carbachol (CCh)-induced MLC phosphorylation in HASM cells in a time-dependent manner (n = 5–7 donors). (C and D) PFK15 attenuates CCh-induced MLC phosphorylation in HASM cells in a concentration-dependent manner (n = 6–7 donors). (E and F) PFK15 (10 μM) attenuates histamine (Hist)-induced MLC phosphorylation in HASM cells (n = 5 donors). (G and H) PFK15 reverses CCh-induced MLC phosphorylation in HASM cells in a concentration-dependent manner (n = 6 donors). The positive control formoterol (Form, 10 nM, for 10 min) attenuated or reversed agonist-induced MLC phosphorylation in these experiments. Data are presented as representative images and mean ± SEM; one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with vehicle + CCh or vehicle + histamine, *P < 0.05, **P < 0.01, and ***P < 0.001. BL = baseline; pMLC = phosphorylated MLC; Veh = vehicle.
[More] [Minimize]To assess whether PFK15 reverses CCh-induced phosphorylation of MLC, HASM cells were treated with CCh first for 10 minutes and then exposed to PFK15 for an additional 10 minutes. Addition of PFK15 reversed CCh-induced MLC phosphorylation in HASM cells (Figures 2G and 2H). The bronchodilator formoterol (10 nM) comparably reversed CCh-induced MLC phosphorylation.
To confirm the necessity of PFKFB3, the target of PFK15, in mediating agonist-induced MLC phosphorylation, PFKFB3 was silenced with transient siRNA transfection. siRNA transfection decreased PFKFB3 expression by ∼50% compared with that of nontargeting siRNA-transfected cells (Figures E2A and E2B). Partial silencing of PFKFB3, however, had little effect on agonist-induced MLC phosphorylation (Figures E2C and E2D). In these experiments, we included as a control overnight treatment with PFK15. Surprisingly, overnight exposure to PFK15 attenuated CCh-induced MLC phosphorylation in a concentration-dependent manner (Figures E2E and E2F).
PFK15 had little effect on agonist-induced cytosolic Ca2+ mobilization in HASM cells (Figures E3A–E3D), suggesting that attenuated Ca2+ mobilization cannot explain the effects of PFK15 on inhibition of MLC phosphorylation. In Ca2+ sensitization, MLC phosphorylation is maintained at elevated concentrations by RhoA-mediated inhibition of MLC phosphatase when MYPT1 is phosphorylated. We and other groups previously showed that M3 muscarinic cholinergic receptor mediates Ca2+ sensitization via activation of PI3K/Akt pathway (18–20). PFK15 attenuated carbachol-induced phosphorylation of Akt and MYPT1 (Figures 3A–3D). To confirm these findings, we measured the effects of PFK15 on small molecular weight G protein RhoA activation, which is upstream of MYPT1 phosphorylation. Thrombin-induced RhoA activation was also attenuated by PFK15 pretreatment (Figures 3E and 3F), whereas Rhosin, an inhibitor of RhoA activation, failed to significantly attenuate thrombin-induced RhoA activation in our experiments.
Figure 3. Glycolysis inhibition attenuates agonist-induced Akt, MYPT1 (myosin phosphatase regulatory subunit 1) phosphorylation, and RhoA activation in HASM cells. (A and B) PFK15 attenuates Cch-induced Akt phosphorylation in a concentration-dependent manner in HASM cells. Formoterol (10 nM) significantly attenuates Akt phosphorylation (n = 6–8 donors). (C and D) PFK15 (10 μM, 10 min) or formoterol (10 nM, 10 min) attenuates CCh-induced MYPT1 phosphorylation in HASM cells (n = 5 donors). (E and F) PFK15 (10 μM, 10 min) or Rhosin (3 μM, 10 min) attenuates agonist-induced RhoA activation in HASM cells (n = 5 donors). Data are presented as representative images of immune blots and mean ± SEM; one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with vehicle + CCh or vehicle + thrombin (Thr), *P < 0.05, **P < 0.01, and ***P < 0.001. NT = no treatment.
[More] [Minimize]Because PFK15 attenuates the key cellular markers of EC coupling in HASM cells, we tested whether PFK15 protects human small airways from carbachol-induced bronchoconstriction. PFK15 and the bronchodilator agent formoterol significantly attenuated the area under the curve of the carbachol concentration response curve (Figures 4A–4C). Carbachol-induced maximal bronchoconstriction was also significantly attenuated by PFK15 (Figure 4D), with little effect on sensitivity to carbachol (log half-maximal agonist concentration [log EC50]) (Figure 4E). In parallel experiments, we also demonstrated that PFK15 inhibited glycolysis (Figures 4F–4I) and oxidative phosphorylation (Figures 4J–4M) in a concentration-dependent manner in hPCLS.
Figure 4. Glycolysis inhibition attenuates agonist-induced bronchoconstriction in human precision-cut lung slices (hPCLS). (A) Representative image of CCh-induced bronchoconstriction. (B) Concentration-response curve of CCh-induced bronchoconstriction (Mean ± SEM of 5 donors). (A–E) CCh-induced bronchoconstriction was attenuated by 10-minute pretreatment with PFK15 (10 μM) or formoterol (10 nM). PFK15 pretreatment decreased the (C) area under the curve and (D) maximal agonist-induced contraction (Emax) with little effect on (E) log half-maximal agonist concentration (log EC50) (n = 7 donors). (F) Representative trace of ECAR in hPCLS. (F–I) PFK15 attenuated (G) glycolysis rate, (H) glycolytic capacity, and (I) glycolytic reserve in a concentration-dependent manner in hPCLS (n = 5 donors). (J) Representative trace of OCR in hPCLS. (J–M) PFK15 attenuated (K) maximal, (L) basal mitochondrial respiration, and (M) mitochondrial ATP production in hPCLS (n = 5 donors). Data are presented as mean ± SEM, one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with vehicle, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
[More] [Minimize]Although not an efficient ATP-generating step, glycolysis may be a source of ATP under certain circumstances. To test whether PFK15 attenuates phosphorylation of proteins involved in EC coupling by reducing glycolysis-generated ATP, we measured ATP in HASM cells in the absence or presence of PFK15 using Seahorse assay. In the control group, oligomycin injection inhibited ATP synthase (complex V) to decrease oxygen consumption rate after basal measurements. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone), a potent uncoupler of oxidative phosphorylation in mitochondria, collapses the proton gradient and disrupts the mitochondrial membrane potential to maximize oxygen consumption rate. The third injection is a mixture of rotenone and antimycin A that shuts down mitochondrial respiration. PFK15 treatment attenuated both mitochondrial and total cellular ATP production rates in HASM cells, with a modest inhibitory effect on glycolysis-generated ATP (Figures 5A–5C). Furthermore, PFK15 attenuated basal mitochondrial ATP production (Figure 5D) and isoproterenol or forskolin-induced cAMP production (Figures 5E and 5F) in a time-dependent manner. Cellular ATP concentration regulates AMPK (AMP-activated protein kinase) activity. To determine whether AMPK is activated in response to reduced ATP concentration, we measured phosphorylation of AMPK in HASM cells with and without PFK15 pretreatment. PFK15 trended toward increased AMPK α phosphorylation at 4 hours of treatment (Figures 5G and 5H), with little effect on ACC (Acetyl CoA Carboxylase) or PFKFB3 phosphorylation (Figure E4).
Figure 5. Glycolysis inhibition attenuates ATP production and agonist-induced cAMP production in HASM cells. (A–D) PFK15 (10 μM) attenuated (A) total and (C) mitochondrial ATP (Mito ATP) production, with little effect on (B) glycolytic ATP (Glyco ATP) production in HASM cells (n = 10 donors). A represents the combined glycolytic and mitochondrial ATP production data as shown in B and C; data are presented as mean ± SEM, unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (D) PFK15 (10 μM) attenuated mitochondrial ATP production in a time-dependent manner in HASM cells (n = 3 donors). Data are presented as mean ± SEM, one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with vehicle, **P < 0.01, and ***P < 0.001. (E and F) PFK15 (10 μM) pretreatment attenuated cAMP induced by (E) isoprenaline (Iso, 10 μM, 10 min), or (F) forskolin (FSK, 10 μM, 5 min) in a time-dependent manner in HASM cells (n = 6 donors). Data are presented as mean ± SEM, one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with NT + Iso, or NT + FSK, *P < 0.05, **P < 0.01, and ***P < 0.001. (G and H) PFK15 treatment trended toward increased AMPK α (AMP-activated protein kinase α) phosphorylation in HASM cells at 4-hour treatment (n = 3 donors). Data are presented as representative immunoblot image and mean ± SEM, one-way ANOVA analysis with Dunnett’s multiple comparison test, all groups compared with Veh + CCh, *P < 0.05, **P < 0.01, and ***P < 0.001.
[More] [Minimize]Amplified EC coupling in ASM cells leads to elevated tension development and AHR. We previously reported that HASM cells from obese human lung donors exhibit amplified agonist-induced EC coupling and shortening force, although the underlying mechanisms are unclear. In the current study, we demonstrate that inhibition of glycolysis impedes cellular ATP production, attenuates agonist-induced EC coupling in HASM cells, and elicits bronchoprotection in small airways. The primary mechanism appears to be decreased cellular ATP and phosphorylation of various proteins in the EC coupling pathway (summarized in Figure 6).
Figure 6. A conceptual model for the role of glycolysis in excitation–contraction (EC) coupling in HASM cells. PFK15, a glycolysis inhibitor, attenuated glycolysis and total ATP production in HASM cells. We posit that ATP deficiency results in decreased agonist-induced MLC, MYPT1, and Akt phosphorylation and RhoA activation—the key markers of EC coupling—in HASM cells. Decreased EC coupling manifests as attenuated CCh-induced bronchoconstriction in human small airways. Horizontal arrows: no change; downward arrows: decrease. GPCR = G Protein-Coupled Receptor; IP3 = inositol trisphosphate; MLCK = myosin light chain kinase; PLC = phospholipase C; RyR = ryanodine receptors.
[More] [Minimize]Intervention studies in obese patients with asthma suggest that weight loss, even by 5–10%, using caloric restriction and physical exercise improves asthma control and quality of life (21, 22). Furthermore, a clinical trial on a smaller cohort of nonobese patients with mild asthma showed that fasting attenuated the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome (23). Caloric restriction certainly regulates functions of a variety of cells, but our findings demonstrate for the first time that reduced energy level in HASM cell attenuates EC coupling and bronchoconstriction.
Glycolysis induces a rapid, albeit inefficient, route to generate ATP, which is critical for phosphorylation of various signaling proteins (24). Because ASM acts as the pivotal tissue regulating bronchomotor tone in health and disease, we sought to identify whether glycolysis modulates EC coupling in ASM cells. Attenuation of phosphorylation by PFK15, of multiple proteins in EC coupling—MLC, MYPT1, and Akt—suggests that lack of ATP is the central mechanism driving these bronchoprotective effects. Reduced cAMP (which originates from ATP) and elevated AMPK activation by PFK15 confirm ATP depletion and cellular homeostatic response. AMPK-mediated phosphorylation of ACC typically leads to increased fatty acid oxidation (25). However, cell lineage and species differences may have a role in AMPK/ACC axis–mediated regulation of β-oxidation. For instance, studies on ACC1/2 transgenic mice showed that myocardial fatty acid oxidation was not reliant on AMPK-dependent inhibition of ACC (26). Little is known about regulation of fatty acid oxidation in ASM cells. Fatty acid oxidation is reportedly elevated in ASM cells derived from donors with asthma to cater for higher energy needs for proliferation and airway remodeling (27). Some murine studies suggest a causative link between elevated fatty acid oxidation and airway inflammation and AHR (28). It is suggested that cells shift to fatty acid oxidation, aerobic glycolysis, and creatine phosphate usage as the immediate sources of ATP during anabolic processes such as proliferation (reviewed in Reference 29). Our findings lend support to this interpretation, by linking glycolysis inhibition and ATP depletion to attenuated contractile function of ASM. In addition, ATP can serve as a signaling molecule, acting through purinergic (P2) receptors in airway structural cells to regulate important functions such as mucociliary clearance and bronchoconstriction (30). ATP released from injured airway epithelium acts on ASM via the P2X receptor to amplify agonist-induced bronchoconstriction, in a RhoA-dependent manner (31, 32).
The regulatory role of glycolysis has also been reported in other cell types. In mouse models of diet-induced obesity, adipose tissue macrophages switched to proinflammatory M1 phenotype in response to enhanced glycolysis and increased ATP generation, resulting in localized and systemic inflammation (33). It remains to be determined whether ATP secretion and paracrine signaling contribute to hyperreactivity in obese donor–derived ASM.
Partial downregulation of PFKFB3 expression by siRNA failed to elicit a bronchoprotective effect similar to that seen in PFK15 treatment, perhaps due to 1) sufficient enzymatic activity in the residual protein; or 2) recruitment of redundant pathways to compensate for reduced PFKFB3 activity. The former is the likely mechanism, because overnight exposure to PFK15 attenuated agonist-induced MLC phosphorylation. In the short-term treatment, PFK15-mediated inhibition of MLC phosphorylation appears to be partially reversed at 4 hours, probably due to an early compensatory response by the cells to restore energy metabolism. This interpretation is supported by elevated AMPK phosphorylation at the 4-hour exposure to PFK15. However, overnight exposure to PFK15 elicited a sustained inhibition of EC coupling, suggesting the early compensatory mechanism is rendered inefficient. An alternate hypothesis is that overnight exposure to PFK15 inhibits EC coupling by a mechanism independent of ATP depletion.
We previously reported that the Gα12-RhoA (Rho-associated coiled-coil containing protein 1)/ROCK1 signaling axis mediates carbachol-induced EC coupling and ASM cell shortening in HASM cells (34). A recent study showed that glycolysis inhibition with a hexokinase 2 inhibitor reduced ROCK2-MLC2–mediated contractility in neoplastic pericytes (35), aligning with our findings that glycolysis inhibition attenuated the RhoA-MYPT1-MLC2 pathway in HASM cells. On the other hand, RhoA/ROCK1 activation was also shown to trigger glycolysis to induce cofilin and MLC phosphorylation in human aortic endothelial cells to regulate the contractile machinery (36). These observations suggest a positive feedback interaction between glycolysis and cellular contractile machinery. Taken together, these data suggest that glycolysis inhibition and subsequent ATP depletion might also elicit a direct inhibitory effect on Gα12-mediated bronchoconstriction.
hPCLS provide an integrated platform to assess functions of airway structural cells and certain transient/inflammatory cells in situ (37, 38). We pioneered the use of hPCLS in a variety of experimental paradigms, including toxic injury, microbial infections, and β2AR desensitization (3, 39–41). In the current study, we have expanded the physiological applications of the PCLS model to include assessment of metabolic functions, allowing us to bridge metabolism and bronchoconstriction at the lung tissue level. In spite of attenuating isoprenaline-induced cAMP, PFK15 bronchoprotects small airways with an efficacy equivalent to that of the β2AR agonist formoterol. This is a significant finding, because bronchoprotection is central to therapeutic management of asthma exacerbation. Although β2AR agonists serve as bronchodilators and represent the mainstay in asthma therapy, prolonged exposure to the β2AR agonists can desensitize the receptors and limit their efficacy. Surprisingly, there have been no novel bronchodilators with unique mechanisms of action over the last 50 years. Our findings suggest that reversible ATP depletion by glycolysis-targeting small molecules and approaches could be feasible candidates for attenuating EC coupling and bronchoprotection of human airways.
We acknowledge the limitations of using a pharmacological inhibitor as the main experimental tool to test our central hypothesis. However, parallel readouts on metabolic activities and EC coupling markers in most of the experiments reasonably mitigate these limitations. Our findings suggest that glycolysis inhibition, via abolishing cellular cAMP concentrations, may interfere with the bronchodilator responses to β2AR agonists. As a bronchoprotective agent, PFK15 is equieffective to formoterol, although the potency of PFK15 is much lower than that of formoterol. The lower potency of PFK15 in bronchoprotection may be the net result of attenuated cAMP concentrations and depleted cellular ATP. We also recognize that asthma may engender a unique metabolic profile in ASM cells to recruit redundant metabolic (and nonmetabolic) pathways to drive AHR. Therefore, comprehensive metabolomic analyses of ASM cells from donors with asthma will be essential to predict the role of glycolysis in AHR.
In summary, our study identifies glycolysis as a targetable metabolic pathway to transiently starve HASM cells of ATP to inhibit EC coupling and elicit bronchoprotection.
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Supported by National Heart, Lung, and Blood Institute grant P01 HL114471 and National Center for Advancing Translational Sciences grant UL1TR003017.
Author Contributions: Conception and design of experiments: S.X., J.A.J., R.A.P., G.C., and A.G. Execution of experiments: S.X., N.K., J.W., J.A.J., and C.G. Interpretation of results: S.X., J.A.J., R.A.P., A.G., G.C., and J.W. Drafting and editing of manuscript: S.X., J.A.J., R.A.P., and A.G.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2021-0495OC on October 13, 2022
Author disclosures are available with the text of this article at www.atsjournals.org.
