Impaired AMPK control of alveolar epithelial cell metabolism promotes pulmonary fibrosis
Luis R. Rodríguez, Konstantinos-Dionysios Alysandratos, Jeremy Katzen, Aditi Murthy, Willy Roque Barboza, Yaniv Tomer, Sarah Bui, Rebeca Acín-Pérez, Anton Petcherski, Kasey Minakin, Paige Carson, Swati Iyer, Katrina Chavez, Charlotte H. Cooper, Apoorva Babu, Aaron I. Weiner, Andrew E. Vaughan, Zoltan Arany, Orian S. Shirihai, Darrell N. Kotton, Michael F. Beers
Luis R. Rodríguez, Konstantinos-Dionysios Alysandratos, Jeremy Katzen, Aditi Murthy, Willy Roque Barboza, Yaniv Tomer, Sarah Bui, Rebeca Acín-Pérez, Anton Petcherski, Kasey Minakin, Paige Carson, Swati Iyer, Katrina Chavez, Charlotte H. Cooper, Apoorva Babu, Aaron I. Weiner, Andrew E. Vaughan, Zoltan Arany, Orian S. Shirihai, Darrell N. Kotton, Michael F. Beers
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Research Article Metabolism Pulmonology

Impaired AMPK control of alveolar epithelial cell metabolism promotes pulmonary fibrosis

Abstract

Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously demonstrated that expression of an AT2 cell–exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell–derived (iPSC-derived) AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here, using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 cell self-renewal and accumulation of transitional epithelial cells. We identify deficient AMPK signaling as a critical component of AT2 cell dysfunction and demonstrate that targeting this druggable signaling hub can rescue the aberrant AT2 cell metabolic phenotype and mitigate lung fibrosis in vivo.

Authors

Luis R. Rodríguez, Konstantinos-Dionysios Alysandratos, Jeremy Katzen, Aditi Murthy, Willy Roque Barboza, Yaniv Tomer, Sarah Bui, Rebeca Acín-Pérez, Anton Petcherski, Kasey Minakin, Paige Carson, Swati Iyer, Katrina Chavez, Charlotte H. Cooper, Apoorva Babu, Aaron I. Weiner, Andrew E. Vaughan, Zoltan Arany, Orian S. Shirihai, Darrell N. Kotton, Michael F. Beers

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Figure 2

Defects in mitochondrial biogenesis and mitochondrial dynamics.

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Defects in mitochondrial biogenesis and mitochondrial dynamics. (A) Unsu...
(A) Unsupervised hierarchical clustering (Euclidean) heatmap of DEGs (FDR < 0.05) in the Hallmark PPAR signaling pathway and MitoCarta gene sets (row-normalized z-score) by popRNA-Seq demonstrates downregulation in AT2I73T cells at days 3 & 14 after tamoxifen induction (n = 4 mice per I73T time point, n = 8 WT mice). (B) Western blot of phosphorylated and total PGC1α (n = 3 biological replicates). (C) Time-dependent reduction in mtDNA copy number in AT2I73T (n = 3 mice for WT, n = 10 mice for day 7, n = 7 mice for day 14, n = 4 mice for day 28). (D) Unsupervised hierarchical clustering (Euclidean) heatmap of differentially expressed mtDNA genes by popRNA-Seq shows decreased expression in AT2I73T cells at day 14 (n = 4 mice per I73T time point and n = 8 WT mice). (E) Flow cytometry analysis of mitochondrial membrane potential (ΔΨm) demonstrates a time-dependent reduction in tetramethylrhodamine, methylester, fluorescence in AT2I73T cells (n = 4–5 mice per condition). Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). (F) Measurement of oxygen consumption rate (OCR) shows a time-dependent reduction in basal and maximal uncoupled mitochondrial respiration and spare respiratory capacity in AT2I73T cells (n = 4–9 mice per condition). (G) Representative transmission electron microscopy (TEM) images of murine AT2 cells from whole lung mounts. Scale bars: 600 nm. (H) MitoTracker staining of murine AT2 cells after 18 hours in culture shows altered mitochondrial network morphology in AT2I73T versus AT2WT cells. Scale bars: 10 nm. (I) Quantification of mitochondrial structure in AT2 cells using ImageJ (NIH) Mitochondrial Analyzer shows increased fragmentation, decreased branch length, and altered shape in AT2I73T cells (each point is an average of 1 field of view containing a minimum of 10 cells; cells were isolated and cultured from n = 4 mice per condition and 5–6 fields were quantified). (J) Western blot and densitometric quantification (n = 3 biological replicates) of mitochondrial dynamics proteins demonstrates altered regulation of dynamics and mitophagy in AT2I73T cells. All mean± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005 by ordinary 1-way ANOVA.