HRAS germline mutations impair LKB1/AMPK signaling and mitochondrial homeostasis in Costello syndrome models
Laetitia Dard, … , Didier Lacombe, Rodrigue Rossignol
Laetitia Dard, … , Didier Lacombe, Rodrigue Rossignol
Published March 1, 2022
Citation Information: J Clin Invest. 2022;132(8):e131053. https://doi.org/10.1172/JCI131053.
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HRAS germline mutations impair LKB1/AMPK signaling and mitochondrial homeostasis in Costello syndrome models

Abstract

Germline mutations that activate genes in the canonical RAS/MAPK signaling pathway are responsible for rare human developmental disorders known as RASopathies. Here, we analyzed the molecular determinants of Costello syndrome (CS) using a mouse model expressing HRAS p.G12S, patient skin fibroblasts, hiPSC-derived human cardiomyocytes, a HRAS p.G12V zebrafish model, and human fibroblasts expressing lentiviral constructs carrying HRAS p.G12S or HRAS p.G12A mutations. The findings revealed alteration of mitochondrial proteostasis and defective oxidative phosphorylation in the heart and skeletal muscle of CS mice that were also found in the cell models of the disease. The underpinning mechanisms involved the inhibition of the AMPK signaling pathway by mutant forms of HRAS, leading to alteration of mitochondrial proteostasis and bioenergetics. Pharmacological activation of mitochondrial bioenergetics and quality control restored organelle function in HRAS p.G12A and p.G12S cell models, reduced left ventricle hypertrophy in CS mice, and diminished the occurrence of developmental defects in the CS zebrafish model. Collectively, these findings highlight the importance of mitochondrial proteostasis and bioenergetics in the pathophysiology of RASopathies and suggest that patients with CS may benefit from treatment with mitochondrial modulators.

Authors

Laetitia Dard, Christophe Hubert, Pauline Esteves, Wendy Blanchard, Ghina Bou About, Lyla Baldasseroni, Elodie Dumon, Chloe Angelini, Mégane Delourme, Véronique Guyonnet-Dupérat, Stéphane Claverol, Laura Fontenille, Karima Kissa, Pierre-Emmanuel Séguéla, Jean-Benoît Thambo, Lévy Nicolas, Yann Herault, Nadège Bellance, Nivea Dias Amoedo, Frédérique Magdinier, Tania Sorg, Didier Lacombe, Rodrigue Rossignol

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Figure 6

Mitochondrial bioenergetics is impaired in CS hiPSC-derived cardiomyocytes.

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Mitochondrial bioenergetics is impaired in CS hiPSC-derived cardiomyocyt...
(A) Human induced pluripotent stem cells (hiPSCs) were established by electroporation of different primary fibroblasts cell lines. Characterization of the hiPSC-derived cardiomyocytes using cardiac troponin T staining; 2 examples are shown for (B) the control AG08H and (C) the Costello G12S hiPSC-derived cardiomyocytes. Original magnification, ×40. (D) The percentage of Troponin T–positive cells determined using immunocytochemistry is shown for the 4 lineages of hiPSC-derived cardiomyocytes. (E) Expression of the keratin sulfate antigens Tra1-60 and Tra1-81 and the glycolipid antigen SSEA4 was verified by flow cytometry. (F) Determination of AMPKα2 (PRKAA2) mRNA expression level by Q-PCR in hiPSC-derived cardiomyocytes, obtained from 2 patients with CS and 2 controls (n = 3). (G) Measurement of the total cellular steady-state ATP content in hiPSC-derived cardiomyocytes, obtained from 2 patients with CS and 2 controls (n = 3). Evaluation of mitochondrial ATP synthesis was performed using inhibitors of oxidative phosphorylation: antimycin A, oligomycin, and rotenone. (H) Survival of the hiPSC-derived cardiomyocytes, obtained from 2 patients with CS and 2 controls, in an obligatory oxidative growth medium (n = 3). Data are expressed as percentage of the cell number in glucose medium. (I) Western blot evaluation of the expression level of various respiratory chain proteins on hiPSC-derived cardiomyocytes, obtained from 2 patients with CS and 2 controls, using the Oxphos kit from Abcam. (J) Determination of the protein expression level of AMPK, T172-P-AMPK and ser428P-LKB1. Protein loading was verified using the GAPDH marker. Data are expressed as the mean ± SEM. Unpaired t test was used to compare the 2 groups of hiPSC-CMs (controls and patients). For G, 1-way ANOVA with Dunnett’s correction was used to compare the 3 groups of cells. **P < 0.01, ***P < 0.001.