Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

• Keywords
• SIRT3, honokiol, mitochondria, nicotinamide adenine dinucleotide, pulmonary hypertension,
• MeSH
• Fibroblasts metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• NAD metabolism   MeSH
• Hypertension, Pulmonary * pathology   MeSH
• Sirtuin 3 * genetics   metabolism   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• honokiol MeSH Browser
• Mitochondrial Proteins MeSH
• NAD MeSH
• SIRT3 protein, human MeSH Browser
• Sirtuin 3 * MeSH

SIGNIFICANCE: The molecular events that promote the development of pulmonary hypertension (PH) are complex and incompletely understood. The complex interplay between the pulmonary vasculature and its immediate microenvironment involving cells of immune system (i.e., macrophages) promotes a persistent inflammatory state, pathological angiogenesis, and fibrosis that are driven by metabolic reprogramming of mesenchymal and immune cells. Recent Advancements: Consistent with previous findings in the field of cancer metabolism, increased glycolytic rates, incomplete glucose and glutamine oxidation to support anabolism and anaplerosis, altered lipid synthesis/oxidation ratios, increased one-carbon metabolism, and activation of the pentose phosphate pathway to support nucleoside synthesis are but some of the key metabolic signatures of vascular cells in PH. In addition, metabolic reprogramming of macrophages is observed in PH and is characterized by distinct features, such as the induction of specific activation or polarization states that enable their participation in the vascular remodeling process. CRITICAL ISSUES: Accumulation of reducing equivalents, such as NAD(P)H in PH cells, also contributes to their altered phenotype both directly and indirectly by regulating the activity of the transcriptional co-repressor C-terminal-binding protein 1 to control the proliferative/inflammatory gene expression in resident and immune cells. Further, similar to the role of anomalous metabolism in mitochondria in cancer, in PH short-term hypoxia-dependent and long-term hypoxia-independent alterations of mitochondrial activity, in the absence of genetic mutation of key mitochondrial enzymes, have been observed and explored as potential therapeutic targets. FUTURE DIRECTIONS: For the foreseeable future, short- and long-term metabolic reprogramming will become a candidate druggable target in the treatment of PH. Antioxid. Redox Signal. 28, 230-250.

• Keywords
• aerobic glycolysis, hypoxia, metabolism, mitochondria, pulmonary hypertension,
• MeSH
• Energy Metabolism MeSH
• Epigenesis, Genetic MeSH
• Glucosephosphate Dehydrogenase metabolism   MeSH
• Glucose metabolism   MeSH
• Glycolysis MeSH
• Hypoxia metabolism   MeSH
• Isoenzymes metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Macrophages MeSH
• Mitochondria metabolism   MeSH
• Oxidation-Reduction MeSH
• Pentose Phosphate Pathway MeSH
• Hypertension, Pulmonary etiology   metabolism   MeSH
• Gene Expression Regulation MeSH
• Superoxides metabolism   MeSH
• Carbon metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Glucosephosphate Dehydrogenase MeSH
• Glucose MeSH
• Isoenzymes MeSH
• Superoxides MeSH
• Carbon MeSH

BACKGROUND: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. METHODS: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. RESULTS: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. CONCLUSIONS: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

• Keywords
• TEEP-46, hypoxia, metabolism, mitochondria, pyruvate kinase, shikonin, splicing factors,
• MeSH
• Alternative Splicing MeSH
• Antagomirs metabolism   MeSH
• Endothelium, Vascular cytology   MeSH
• Fibroblasts cytology   drug effects   metabolism   MeSH
• Glycolysis MeSH
• Heterogeneous-Nuclear Ribonucleoproteins antagonists & inhibitors   genetics   metabolism   MeSH
• Histone Deacetylase Inhibitors pharmacology   MeSH
• Interleukin-1beta metabolism   MeSH
• Humans MeSH
• Macrophages cytology   immunology   metabolism   MeSH
• MicroRNAs antagonists & inhibitors   genetics   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Naphthoquinones pharmacology   MeSH
• Hypertension, Pulmonary metabolism   pathology   MeSH
• Cell Proliferation MeSH
• Protein Isoforms antagonists & inhibitors   genetics   metabolism   MeSH
• Polypyrimidine Tract-Binding Protein antagonists & inhibitors   genetics   metabolism   MeSH
• Pyruvate Kinase antagonists & inhibitors   genetics   metabolism   MeSH
• RNA Interference MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antagomirs MeSH
• Heterogeneous-Nuclear Ribonucleoproteins MeSH
• Histone Deacetylase Inhibitors MeSH
• Interleukin-1beta MeSH
• MicroRNAs MeSH
• MIRN124 microRNA, human MeSH Browser
• Naphthoquinones MeSH
• Protein Isoforms MeSH
• Polypyrimidine Tract-Binding Protein MeSH
• PTBP1 protein, human MeSH Browser
• Pyruvate Kinase MeSH
• shikonin MeSH Browser