ETI Corrector Therapy Suppresses Mitochondrial Respiration Independently of CFTR Mutational Status

Abstract

The relationship between CFTR mutational status and cellular energy metabolism remains unresolved. Using continuous long-term respirometry via the Resipher platform, we demonstrate that the Class II CFTR misfolding mutations ΔF508, G85E, and P67L all significantly increase cellular respiration in isogenic Fisher rat thyroid (FRT) and human bronchial epithelial (HBE) cells, consistent with the constitutive energy demands of misfolded-protein quality control. In contrast, Class I truncating mutations (G542X, W1282X), which produce no misfolded protein and no functional ion channels, reduced cellular respiration below wildtype levels. ΔF508-expressing HBE cells also exhibited impaired metabolic flexibility under culture conditions modeling either hypoglycemia or hyperglycemia. Strikingly, treatment of HBE cells with elexacaftor/tezacaftor/ivacaftor (ETI) significantly suppressed mitochondrial respiration to a comparable degree in both ΔF508 and wildtype HBE cells across all media glucose concentrations tested, establishing a CFTR-independent metabolic effect of the drugs. Despite reduced mitochondrial respiration, intracellular ATP was maintained under ETI treatment, paralleled by significantly increased lactate secretion, indicating a compensatory Warburg-like shift toward glycolytic ATP production. These findings have potential implications for the metabolic comorbidities increasingly observed in CF patients in the ETI era.

1. Introduction

Cystic fibrosis (CF) is associated with metabolic dysregulation that is poorly understood. Pancreatic insufficiency, present in approximately 85% of patients, causes severe malabsorption of fats, fat-soluble vitamins, and proteins [1]. This malabsorption, combined with elevated resting energy expenditure from chronic pulmonary infection and inflammation, produces the classic CF phenotype of failure to thrive and chronic undernutrition. Patients are accordingly prescribed high-fat, high-calorie diets exceeding 150% of typical caloric requirements [2]. Compounding these problems, CF patients exhibit impaired glucose handling, including defective GLUT4 trafficking and paracellular glucose leakage into the airway surface liquid, such that even absorbed calories are not efficiently used [3,4]. Whether the increased resting energy expenditure is primarily a consequence of systemic inflammation, poor nutrient absorption, or a cell-intrinsic consequence of CFTR misfolding has remained unresolved.

The clinical picture has shifted dramatically since the introduction of the elexacaftor/tezacaftor/ivacaftor (ETI) triple corrector therapy, which restores meaningful CFTR function in approximately 90% of patients [5]. While ETI has been transformative for lung function and quality of life, a new metabolic phenotype has emerged. Rather than undernutrition, many patients on ETI now display weight gain, obesity, dyslipidemia, hypertension, and frank metabolic syndrome [6–8]. This swing from a state of metabolic insufficiency to metabolic excess raises the important question of whether these changes simply reflect reduced disease burden or whether the ETI drugs themselves alter cellular metabolism. To date, few studies have interrogated the effects of either CFTR mutations or CFTR modulator drugs on cellular bioenergetics, much less the combination of CFTR mutations and modulator drugs. Jarosz-Griffiths et al. [9] demonstrated that cells expressing the most common CFTR mutation, ΔF508, exhibit elevated mitochondrial respiration, and that ETI treatment counters this hypermetabolic state. However, their study did not include wildtype CFTR-expressing cells. Thus, it remains unclear whether ETI’s metabolic effects are specific to correcting the misfolded-protein burden or represent a broader, CFTR-independent action of the drugs. The possibility of CFTR-independent effects on metabolism is supported by three recent studies. First, Braccia et al. [10] demonstrated that lumacaftor, a first-generation CFTR corrector therapy, induces mitochondrial and peroxisomal reorganization in CF bronchial epithelial cells. The following year, it was shown that ETI therapy suppresses mitochondrial respiration in monocyte-derived macrophages from both CF patients and healthy non CF controls [11]. Then, Dib et al. [12] corroborated the work of Braccia et al by showing that combined lumacaftor/ivacaftor treatment induces mitochondrial fragmentation in both CF cells and cells expressing wildtype CFTR.

Taken together, these few studies point to a substantial and underappreciated knowledge gap regarding the metabolic effects of CFTR mutations and the interaction of these mutations with ETI therapy. Here, we apply a new cellular respirometry platform, Resipher (Lucid Scientific), to characterize the cellular consequences of CFTR mutations and ETI treatment. Resipher monitors oxygen consumption continuously in cells maintained under normal culture conditions, which has heretofore not been possible with conventional extracellular flux analysis platforms. We demonstrate that Class II misfolding mutations increase, while Class I truncating mutations decrease, cellular respiration. We further show that ETI suppresses mitochondrial respiration and promotes glycolytic compensation in both CFTR mutant and normal control cells, providing further evidence that ETI remodels metabolism independently of CFTR status. These results have direct implications for managing the growing burden of CF and ETI-associated metabolic comorbidities.

2. Materials and Methods

2.1. Cell Culture

Fischer rat thyroid (FRT) epithelial cells were obtained from Dr. E. Sorscher (University of Alabama at Birmingham). Cells were maintained in Coon’s F12 modified medium (Millipore-Sigma, F6636) supplemented with 5% fetal bovine serum (FBS; Hyclone, USA), 1% Sodium Bicarbonate, and 1% Penicillin–Streptomycin (Gibco). For selection and maintenance, FRT-Flp4 base cells were cultured in 100–200 μg/mL hygromycin (Thermo Fisher, R250-1). All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO₂, and the medium was replaced every 2–3 days. The Cystic Fibrosis Foundation Therapeutics Lab (CFFT) kindly provided the gene-edited 16HBE14o⁻ CFTR cell lines. Cells were expanded in T-75 flasks coated with LHC-8 basal medium supplemented with 7.5% bovine serum albumin (1.34 μL/mL), 10 μL/mL bovine collagen type I (Advanced BioMatrix), and 10 μL/mL human plasma fibronectin (Thermo Fisher Scientific). Cultures were maintained at 37 °C and 5% CO₂ in MEM (Gibco) containing 10% FBS and 1% Penicillin–Streptomycin, with medium refreshed every 2–3 days.

2.2. Oxygen Consumption Rate (OCR) Measurements

OCR was assessed using the Resipher real-time metabolic analysis platform (Lucid Scientific). For both FRT-Flp cells expressing various CFTR constructs (WT, F508del, N1303K, P67L, and G85E) and 16HBE14o⁻ cells bearing CFTR variants (WT, ΔF508, G542X, W1282X), approximately 15,000 cells per well were seeded in 96-well plates and cultured under standard conditions. After 20 hours of equilibration, OCR monitoring was initiated and followed continuously for 48 hours. For the glucose-dependence and ETI experiment, approximately 20,000 16HBE14o⁻ cells per well were plated in coated 96-well plates. After 20 hours, the medium was replaced with glucose-free MEM supplemented with 2.5, 5.5-, or 25.5-mM glucose, 10% FBS, and 1% Penicillin–Streptomycin. Within each condition, cells were treated with either DMSO or a combination of CFTR modulators (ETI: 3 μM Elexacaftor, 3 μM Tezacaftor, and 1 μM Ivacaftor). OCR was monitored for 48 hours.

2.3. Lactate and ATP Assays

Approximately 20,000 16HBE14o⁻ CFTR WT cells per well were plated in coated 96-well plates. After 20 hours, the medium was replaced with glucose-free MEM supplemented with 2.5, 5.5, or 25.5 mM glucose, 10% FBS, and 1% Penicillin–Streptomycin. Cells were treated with either DMSO or ETI (3 μM Elexacaftor, 3 μM Tezacaftor, and 1 μM Ivacaftor). After 24 hours of treatment, lactate production was quantified using the Lactate-Glo Assay (Promega) according to the manufacturer’s protocol. Cellular ATP content was measured using CellTiter-Glo 2.0 (Promega). Luminescence was recorded using a BioTek Synergy HTX plate reader.

2.4. Immunoblotting

Protein lysates were resolved by SDS–PAGE and transferred to PVDF-FL membranes. Membranes were probed with the following primary antibodies: anti-CFTR monoclonal antibody 596 (CFFT; 1:1000) and anti-HSP90 rabbit monoclonal antibody (Cell Signaling Technology; 1:5000). Signals were detected using an Odyssey imaging system (Licor).

3. Results

3.1. Class II CFTR mutations increase cellular respiration in Fisher rat thyroid (FRT) cells

To date, more than 2100 mutations have been identified in the CFTR gene [13]. The most common class of mutation, affecting ~85% of patients, are the so-called Class II mutations, such as ΔF508, which cause defective protein processing. Class II mutant CFTRs misfold and are degraded by the cell’s quality control system before reaching the cell membrane. Misfolded CFTR triggers energy-intensive quality control processes where ATP-dependent chaperones and the p97 ATPase expend energy to attempt refolding or to extract the protein for proteasomal degradation. We hypothesized that the constitutive expression of Class II CFTR mutations would result in increased mitochondrial utilization to meet this metabolic demand. To test this, we used a new technology called Resipher (Lucid Scientific) to monitor mitochondrial oxygen consumption long-term in Fisher rat thyroid (FRT) isogenic cell lines engineered to stably express wildtype CFTR or the Class II patient mutations ΔF508, G85E, P67L, and N1303K. The rate of oxygen consumption was followed over a 48 hr period in these cell lines. Three of the four Class II mutations, ΔF508, G85E, P67L, were associated with increased cellular respiration relative to FRT cells expressing wildtype CFTR over the entire time course of monitoring (Figure 1). N1303K exhibited a trend toward higher respiration but this was not statistically significant (Figure 1B). Upon immunoblotting, it was observed that N1303K-expressing cells had more CFTR protein (Figure 1E), as both the “b” band (core-glycosylated, ER-retained immature protein) and some apparent “c” band (fully glycosylated, mature CFTR that has trafficked through the Golgi to the cell surface). The presence of some cell-surface, functional CFTR in N1303K expressing cells may be associated with more moderate demands on metabolism.

Figure 1. Class II CFTR mutations increase cellular respiration in Fisher rat thyroid (FRT) cells.

FRT cells stably expressing wildtype (WT) CFTR or the Class II patient mutations ΔF508 (A), N1303K (B), G85E (C), or P67L (D) were seeded at approximately 15,000 cells per well and the mitochondrial oxygen consumption rate (OCR) was monitored continuously for 48 hours using the Resipher real-time metabolic analysis platform (Lucid Scientific). Data are expressed as oxygen flux (fmols/mm²/s) and plotted as mean ± standard deviation. Asterisks indicate timepoints at which the mutant CFTR-expressing cell line differed significantly from WT CFTR-expressing cells (p < 0.05). N = 4 wells per condition. (E) Representative immunoblot of total cell lysates from all six FRT cell lines probed with anti-CFTR antibody and anti-HSP90 as a loading control. The mature, complex-glycosylated form of CFTR (C band) and the immature, core-glycosylated ER-retained form (B band) are indicated.

3.2. Class II, but not Class I CFTR mutations, also increase cellular respiration in human bronchial epithelial (HBE) cells

We next sought to replicate our results in a more physiologically relevant cell type, human bronchial epithelial cells (HBE). First, we repeated the Resipher assay using HBE cells engineered to express the Class II common CFTR mutation, ΔF508. As observed in FRT cells, HBE cells expressing ΔF508 exhibited significantly increased cellular respiration compared to HBE cells expressing wildtype CFTR (Figure 2A). Then, we asked whether truncating Class I mutations would also affect respiration. HBE cells expressing two different truncating mutations, G542X and W1282X, were compared to cells expressing wildtype CFTR. Interestingly, both truncating mutations were found to reduce, rather than increase, cellular respiration (Figure 2B,C).

Figure 2. Class II but not Class I CFTR mutations increase cellular respiration in human bronchial epithelial (HBE) cells.

Gene-edited 16HBE14o⁻ cells expressing WT CFTR, ΔF508 (A), G542X (B), or W1282X (C) were seeded at approximately 15,000 cells per well and OCR was monitored continuously for 48 hours using the Resipher platform. Data are expressed as oxygen flux (fmols/mm²/s) and plotted as mean ± standard deviation. Asterisks indicate timepoints at which the indicated CFTR variant-expressing cells differed significantly from WT CFTR-expressing cells (p < 0.05). N = 4 wells per condition.

3.3. Respiration is responsive to glucose availability in ΔF508 HBE cells

CF patients frequently display aberrant glucose metabolism [14,15]. We asked whether modulating glucose availability would affect HBE cellular respiration, and second, whether the presence of ΔF508 CFTR would alter this response. To that end, HBE cells expressing wildtype CFTR and HBE cells expressing ΔF508 CFTR were grown under normoglycemia (5.5 mM) and then either remained in normoglycemia for respiration monitoring or were switched into media representing hypoglycemia (2.5 mM) or hyperglycemia (25 mM). In wildtype HBEs, cells maintained under hypoglycemia adapted by increasing mitochondrial respiration to meet metabolic needs, with respiration significantly increasing by 15 hrs after switching into low glucose media (Figure 3A). In contrast, ΔF508 HBE cells reacted to the introduction of low glucose media with an initial moderate suppression of respiration over the first 15 hrs. This suppression period was followed by a compensatory induction of respiration as observed in wildtype, but with a time delay, such that statistical significance was not reached until the 27 hr mark (Figure 3B). ΔF508 HBE cells also exhibited an altered response to hyperglycemia. Whereas wildtype HBE cells showed suppressed respiration in 25 mM glucose only at the very end of the monitoring period (48 hr mark), ΔF508 HBE cells shifted into 25 mM glucose immediately suppressed respiration and it remained significantly suppressed throughout the 48 hr period. These results indicate that CFTR mutations may indeed alter the metabolism of glucose by the bronchial epithelium.

Figure 3. ΔF508-expressing HBE cells exhibit dysregulated metabolic responses to glucose availability.

16HBE14o⁻ cells expressing WT CFTR (A) or ΔF508 CFTR (B) were seeded at approximately 20,000 cells per well and equilibrated for 20 hours in medium containing 5.5 mM glucose. Medium was then replaced with MEM containing either 2.5 mM (hypoglycemia, blue squares), 5.5 mM (normoglycemia, gold circles), or 25 mM (hyperglycemia, gray triangles) glucose, and OCR was monitored continuously for 48 hours using the Resipher platform. Data are expressed as oxygen flux (fmols/mm²/s) and plotted as mean ± standard deviation. Asterisks indicate timepoints at which the 2.5 mM or 25 mM glucose condition differed significantly from the 5.5 mM normoglycemic condition within each cell line (p < 0.05). N = 4 wells per condition.

3.4. ETI combination therapy suppresses cellular respiration and promotes lactate secretion in HBE cells

Our data above indicate that CFTR patient variants such as ΔF508 alter cellular bioenergetics as well as glucose handling, which leads to the important question of whether ETI combination therapy also affects metabolism, and if so, whether it interacts with CFTR status. To interrogate this, we repeated the experiment described above wherein cellular respiration was monitored in HBE cells under three glucose concentrations, but this time, half the cells received ETI drugs (3 μM Elexacaftor, 3 μM Tezacaftor, and 1 μM Ivacaftor) and half received vehicle only. Figure 4 shows the cellular respiration rates at the 48 hr mark. Importantly, we observed that ETI significantly reduced cellular respiration and did so to a similar degree in both wildtype and ΔF508 HBE cells (Figure 4A,B). The suppression of respiration occurred at all three glucose concentrations tested. The suppression of mitochondrial respiration even in wildtype cells indicates that ETI does not merely correct the hypermetabolic phenotype associated with mutant CFTR expression, as has been previously suggested [9], but rather, may have CFTR-independent effects on metabolism. We reasoned that if ETI suppresses mitochondrial respiration, the cells may compensate by increasing ATP production via glycolysis. We therefore measured lactate secretion into the media by wildtype HBEs ± ETI drugs. Indeed, under either hypoglycemic (2.5 mM) or hyperglycemic (25 mM) conditions, ETI was associated with significantly increased secretion of lactate (Figure 4C). The ability of glycolysis to compensate for reduced respiration was indicated by the intracellular concentration of ATP, which did not significantly vary by either glucose availability or by ETI treatment (Figure 4D).

Figure 4. ETI combination therapy suppresses cellular respiration and increases glycolytic lactate secretion independently of CFTR mutation status.

16HBE14o⁻ cells expressing ΔF508 CFTR (A) or WT CFTR (B, C, D) were seeded at approximately 20,000 cells per well and equilibrated for 20 hours. Medium was then replaced with MEM supplemented with either 2.5, 5.5, or 25 mM glucose and containing either vehicle (DMSO, gold bars) or ETI modulators (3 μM elexacaftor, 3 μM tezacaftor, 1 μM ivacaftor; blue bars). OCR was monitored for 48 hours using the Resipher platform, and the values shown represent flux measurements at the 48-hour endpoint. (A, B) Bar graphs show the OCR (fmols/mm²/s) in ΔF508 (A) and WT (B) HBE cells treated with DMSO or ETI across the three glucose concentrations. ETI significantly reduced cellular respiration at all glucose concentrations in both ΔF508 and WT HBE cells. (C) Lactate secreted into conditioned medium was quantified in WT HBE cells using the Lactate-Glo assay (Promega) after 24 hours of treatment. ETI significantly increased lactate secretion under hypoglycemic (2.5 mM) and hyperglycemic (25 mM) glucose conditions, indicating a compensatory shift toward glycolysis. (D) Intracellular ATP content in WT HBE cells was measured using CellTiter-Glo 2.0 (Promega). ATP levels were not significantly altered by glucose concentration or ETI treatment, indicating that glycolytic compensation maintained cellular energy charge despite reduced mitochondrial respiration. All bars represent mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 for ETI vs. DMSO-treated cells within the same glucose condition.

4. Discussion

The data presented here help to fill the knowledge gap regarding CF, ETI, and metabolism. First, we showed that CFTR mutations alter cellular bioenergetics through a cell-intrinsic mechanism. However, not all CFTR mutations have the same effect. Class II CFTR mutations causing constitutive misfolded protein stress are sufficient to increase cellular respiration, while Class I truncating mutations that produce no misfolded protein have the opposite effect, reducing cellular respiration. The hypermetabolic phenotype of Class II mutations is consistent with the substantial energy demands of the proteostasis network. ΔF508 and related misfolding variants constitutively engage ATP-dependent chaperones, the ubiquitin conjugation machinery, and the p97 ATPase complex in a futile cycle of attempted refolding and degradation [16,17]. That simply expressing ΔF508, G85E, or P67L in FRT cells, which normally lack CFTR entirely, is sufficient to elevate oxygen consumption argues strongly that this is a direct consequence of misfolded protein stress, not a secondary systemic effect. If true, this would suggest that tissues harboring the greatest burden of misfolded CFTR, i.e. the lungs and gut, are the primary drivers of the hypermetabolic phenotype so commonly observed in patients during the pre-ETI era. Indeed, resting energy expenditure (REE) drops ~10% in CF patients following double lung transplant, and in ΔF508 mice, where CF is almost exclusively a gut disease, restoring intestinal CFTR normalizes their elevated REE [18,19].

In contrast to the common Class II mutations, our cellular respirometry showed that Class I truncating mutations reduce cellular respiration compared to wildtype CFTR when expressed in HBE cells. Another interpretation of this data is that it unveils the metabolic cost of expressing wildtype CFTR. While not as energetically demanding as Class II misfolding mutants, the folding and trafficking of wildtype CFTR is still an energy-intensive process [20]. Further, active CFTR ion channels on the cell membrane hydrolyze ATP [21]. The truncating mutants G542X and W1282X, on the other hand, produce no misfolded protein and therefore impose no constitutive proteostatic energy demand, and since no active ion channels are formed, the demand for ATP to fuel channel gating is also absent. Future work is needed to understand the in vivo ramifications of the hypometabolic effect of Class I mutations. If confirmed in vivo, this phenomenon could have implications for nutritional management strategies that have historically been developed around the Class II hypermetabolic phenotype.

The dysregulated response of ΔF508 HBE cells to changes in glucose availability adds another dimension to the CF metabolic phenotype. Whereas wildtype HBE cells respond to hypoglycemia with a prompt compensatory increase in mitochondrial respiration, ΔF508 cells exhibit a blunted and delayed version of this adaptation, as well as an exaggerated suppression under hyperglycemia. This metabolic inflexibility may be clinically relevant given that CF patients experience frequent glucose dysregulation, including hypoglycemic episodes after initiating modulator therapy and CF-related diabetes in adulthood [22]. Impaired ability to mount a rapid metabolic response to falling glucose could render ΔF508-expressing airway cells transiently energy-deficient under conditions that normal cells readily tolerate.

Perhaps the most important finding of our study is that ETI suppresses mitochondrial respiration equally in wildtype and ΔF508 HBE cells. Our findings implicate a CFTR-independent mechanism, potentially involving direct interaction between one or more ETI component drugs and the mitochondrial electron transport chain. This interpretation is reinforced by Aridgides et al. [11], who showed that ETI suppresses respiration in macrophages from both CF patients and healthy controls, and by Dib et al. [12], who reported that lumacaftor/ivacaftor induces mitochondrial network fragmentation, generally associated with reduced respiratory capacity. The convergence of these independent datasets makes a compelling case that metabolic suppression is an intrinsic property of CFTR corrector drugs. Despite reduced mitochondrial respiration, intracellular ATP was maintained under ETI treatment, paralleled by significantly increased lactate secretion, indicating a compensatory shift toward glycolytic ATP production. This Warburg-like reprogramming warrants attention in the context of long-term ETI exposure, particularly given the elevated colorectal cancer risk already documented in CF patients [23]. Whether chronic glycolytic reprogramming in ETI-treated tissues could create a permissive environment for tumorigenesis is an important open question.

In summary, these findings reframe our understanding of CF metabolism and raise important questions about the systemic consequences of lifelong ETI exposure, particularly as the drug is now approved beginning at two years of age. An important caveat of our work is that it was limited to cell culture models. Whether these metabolic effects translate in vivo remains to be established. Furthermore, the relative contributions of elexacaftor, tezacaftor, and ivacaftor individually to the observed metabolic suppression are unknown. Dissecting drug-specific effects is essential for the rational development of next-generation modulators with improved metabolic profiles and will be a focus of future work.