Dataset Information

Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells.

ABSTRACT:

Objective

NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1^G208C and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1^G206C rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 (cluster of differentiation 36) and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation.

Conclusions

The alteration in iron-sulfur metabolism completely transforms the proteomic landscape of the mitochondria, leading toward metabolic plasticity and redistribution of energy sources to the acquisition of a proliferative phenotype by the PASMC.

SUBMITTER: James J

PROVIDER: S-EPMC7837686 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:NFU1 is an iron-sulfur (Fe-S) scaffold protein, involved in Fe-S assembly and transfer to a range of mitochondrial metalloproteins. Patients with the NFU1G208C mutation develop pulmonary arterial hypertension (PAH) with 70% penetrance. Rats with NFU1G206C homozygous mutation demonstrated the PAH phenotype showing increased pulmonary vasculature remodeling, right ventricular (RV) hypertrophy, and RV pressure. In the present study, we discovered phenotypic metabolic changes in pulmonary arterial smooth muscle cells (PASMC) from NFU1G206C homozygous mutant rats associated with alterations in the mitochondrial proteome. Quantitative analysis of the mitochondrial proteome showed significant changes in the abundance of 208 proteins involved in various metabolic and antioxidant functions in response to the NFU1 mutation. Our data indicates that the NFU1G206C homozygous mutant rats have decreased expression of complex I and II, which are known to depend on iron-sulfur clusters, and increased expression of complexes III to V, accompanied with a significant decrease in mitochondrial function, pyruvate dehydrogenase (PDH) activity and amplified glycolysis and anabolism in PASMC. The NFU1 mutation produced a dysregulated antioxidant system in the mitochondria which leads to increased levels of reactive oxygen species. Due to alterations in apoptosis regulating proteins, the NFU1G206C cells were found to exhibit high proliferation rates and resistance to apoptosis as compared with the wild type (WT). Finally, the NFU1G206C mitochondrial proteome showed significant abundance changes in proteins that regulate fatty acid (FA) metabolism and exhibited increased FA oxidation compared to WT, suggesting increased FA oxidation compensates for the decreased PDH activity. In conclusion, our data indicates that the NFU1G206C mutation induces a metabolic shift in the PASMC, which results in a hyper-proliferative and apoptosis resistant phenotype and presents a novel cellular model to study PAH.

Project description:The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.

Dataset Information

Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells.

Objective

Conclusions

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