Dysfunctional Mitochondria and Hemolysis are Linked by Impaired Maturation of Reticulocytes: Possible Role in Pulmonary Hypertension

Abstract

IntroductionWe previously demonstrated that significantly elevated levels of circulating free heme (CFH) is associated with preclinical (Sugen/Hypoxia in rats) pulmonary hypertension (PH) and clinical pulmonary arterial hypertension (PAH). However, the pathogenic mechanisms of free heme release in PAH patients and animal models in the absence of hemolytic disease are yet to be elucidated.HypothesisThe mitochondria play a crucial role in reticulocyte maturation (precursors to red blood cells). Our hypothesis tests if mitochondrial dysfunction (MD) impaired reticulocyte maturation increases its fragility and causes rupture, elevating CFH levels that promote PAH.MethodsRecently reported genetic PH rat models carrying the NFU1G206C mutation spontaneously develop PH with severe MD. Erythropoiesis activation, reticulocyte counts, and CFH levels were monitored in this model. ChemiDoc imaging system was used to visualize FITC‐dextran extravasation in fresh transversely cut pulmonary slices 24 hours after FITC‐dextran injection (60 mg/kg. i.v.). Changes in right ventricle (RV) hemodynamics were used to determine the level of disease severity. Pulmonary artery smooth muscle cells (PASMCs) isolated from wild‐type (WT) and NFU1G206C rats were used to evaluate effects of free heme in vitro. Matrigel assay was used to evaluate the tube formation potential of endothelial cells (EC’s).ResultsThe NFU1G206C rats showed significantly higher erythropoietin levels and immature reticulocyte counts compared to WTs. Correspondingly, significantly elevated plasma free heme and extravasation of FITC‐dextran into lung parenchyma were observed, indicating pulmonary endothelial barrier dysfunction, previously demonstrated as an essential contributor to PH pathobiology. Notably, treatment of human pulmonary artery EC’s with 50µM heme completely abolished EC tube formation, indicating that CFH could trigger a cellular environment leading to PH. Additionally, exposing isolated PASMC from NFU1G206C to 50µM heme increased the proliferation rate by ~1.5 and ~2.9 folds compared to untreated NFU1G206C or WT PASMC. Chronic lipoic acid (LA) supplementation to restore mitochondrial function (1.7mM orally), normalized total/immature reticulocyte counts in NFU1G206C rats (WT/NFU1G206C/NFU1G206C+LA for total and immature reticulocyte, p<0.002 and p<0.001) and fully reversed the PAH phenotype. Alternatively, the RV systolic pressure of NFU1G206C rats was significantly (p<0.0001) attenuated by blocking the heme transporter (HCP1) with sulfasalazine (SL, 20mg/ml/kg, i.p.).ConclusionWe show that mitochondrial dysfunction impaired reticulocyte maturation promotes hemolysis resulting in increased CFH. Elevated CFH can trigger pathogenic pathways leading to PH/PAH via EC barrier dysfunction and increased PASMC proliferation. Treatment approaches that improve mitochondrial metabolism (LA) or inhibit free heme entrance to the cell (SL) abrogate the PH phenotype, highlighting the critical role of free heme‐induced signaling in disease pathobiology.