Pulmonary hypertension (PH) is a deadly vascular disease linked to an enigmatic repression of mitochondrial metabolism. Iron-sulfur (Fe-S) clusters are prosthetic groups that promote mitochondrial respiration and are regulated by the Fe-S assembly proteins ISCU and FXN (frataxin). Yet, the roles of Fe-S clusters in most human diseases including PH are unknown. We found that hypoxia-induced microRNA-210 represses ISCU, promoting Fe-S deficiency, pulmonary vascular metabolic dysregulation, and PH. We also found that FXN is down-regulated in PH and is controlled by the miR-130/301 family/PPAR? regulatory axis. We hypothesize that Fe-S deficiency, particularly in pulmonary vascular endothelium, is a critical pathogenic lynchpin of PH and is a common convergence point of genetic and acquired disease triggers. We plan to study both rodents and humans in vivo, delineating novel Fe-S-based origins of PH - namely, the coordinated microRNA-based regulation of ISCU/FXN by hypoxia and human genetic deficiencies of ISCU and FXN. Specific Aims: 1) Determine whether the miR-130/301 family represses FXN and Fe-S expression in order to control PH. In a hypoxic mouse model of PH and cultured pulmonary vascular endothelial cells from diseased mice coupled with novel biophysical assays to measure Fe-S levels, we will test the hypothesis that the miR-130/301 family down-regulates FXN in order to repress Fe-S biogenesis and mitochondrial respiration and thus promote PH. Such findings would identify miR-130/301-dependent control of FXN as a critical complement to the miR-210/ISCU axis in metabolic dysfunction and in the overall control of PH. 2) Determine whether up-regulation of miR-210 and miR-130/301 together promotes more robust down- regulation of Fe-S cluster expression and more severe PH manifestation than either miRNA alone. Using the model systems above, we will test the hypothesis that up-regulation of miR-210 and miR-130/301 together promote more robust down-regulation of Fe-S integrity and increased PH severity. Results would be invaluable for developing a roadmap for synergistic therapeutic targeting of microRNAs in PH. 3) Determine whether mutations of ISCU and FXN in humans directly promote PH. To assess for PH in human genetic deficiency of FXN or ISCU without hypoxia, we plan advanced cardiopulmonary exercise tests. We will also generate/study patient-specific inducible pluripotent stem cells to determine how the mutations control pulmonary vascular function. This rare combination of molecular study and patient testing should define PH risk in Fe-S deficiency, guiding clinical care and solidifying this paradigm's relevance in humans. Significance: This proposal incorporates rigorous expertise and new technological advancements in Fe-S biology coupled with a rare opportunity to translate mechanistic findings directly to humans. We aim to firmly establish Fe-S deficiency as a powerful and novel metabolic disease origin, a new therapeutic target for PH, and a foundation for discovery in other diseases that share similar hypoxic and metabolic underpinnings.