The role of mitochondrial dysfunction in reticulocytes and hemolysis. (A) ATP production: mitochondrial ATP production, as measured by the Seahorse XF assay, is significantly decreased in NFU1 reticulocytes. ATP production is measured as the difference in the OCR after the addition of oligomycin, a complex V inhibitor (n = 5-7). (B-C) Immature reticulocytes: the number of immature reticulocytes, identified by the persistence of mitochondria under microscopy, is markedly increased in the blood of NFU1 rats. Scale bar, 50 μm (n = 8). (D) Circulating reticulocytes: reticulocyte counts are significantly elevated in NFU1 rats, indicating increased production, decreased clearance, and maturation of reticulocytes (n = 10). (E) EPO levels: EPO levels, which control reticulocyte and RBC production, determined by enzyme-linked immunosorbent assay are significantly elevated in the plasma of NFU1 rats (n = 10). (F-G) Carbonic anhydrase 9 (CA9) expression: immunohistochemical staining shows increased CA9 expression in the lungs of NFU1 rats, indicating a potential hypoxic environment. Scale bar, 200 μm (n = 4). (H) Circulating free heme: free heme levels in the plasma are significantly elevated in NFU1 rats, indicating hemolysis (n = 10). (I-J) Barrier disruption: FITC-dextran extravasation assays reveal significant barrier disruption in the lungs of NFU1 rats. Scale bar, 5 mm (n = 7-8). (K) RVSP: RVSP is significantly increased in NFU1 rats, indicating significant PH (n = 8). (L) Fulton index: the Fulton index, indicative of right ventricular remodeling (calculated as follows: right ventricle/left ventricle + septum), is significantly elevated in NFU1 rats (n = 8). (M-N) Vascular occlusion score: the vascular occlusion score, a measure of vascular remodeling, is markedly increased in NFU1 lungs. Scale bar, 100 μm (n = 5). (O) LA treatment: LA supplementation in NFU1 rats significantly improves mitochondrial ATP production (n = 5). LA is a cofactor of PDH, a mitochondrial enzyme. LAS synthesizes LA. The NFU1 protein inserts Fe-S clusters in LAS, enabling LA synthesis. The NFU1 mutation impairs LA synthesis because of LAS dysfunction. LA supplementation directly circumvents the LAS dysfunction. (P) Reticulocyte count posttreatment: reticulocyte numbers are normalized in NFU1 rats after LA treatment (n = 10-12). (Q-R) Immature reticulocytes posttreatment: LA treatment significantly reduces the number of immature reticulocytes in NFU1 rats, indicating improved reticulocyte maturation (n = 10-12). (S) Free heme posttreatment: free heme levels are significantly reduced in NFU1 rats after LA treatment, indicating decreased hemolysis (n = 9-10). (T-U) Barrier protection posttreatment: LA treatment significantly protects NFU1 rats from barrier disruption, as shown by reduced FITC-dextran extravasation in the lungs. Scale bar, 10 mm (n = 8). (V) RVSP posttreatment: LA treatment significantly protects NFU1 rats from increased RVSP, indicating a protection from the induction of PH (n = 8). (W) Fulton index posttreatment: LA treatment prevents the rise in the Fulton index in NFU1 rats (n = 8). (X-Y) Vascular remodeling posttreatment: LA treatment in NFU1 rats significantly reverses pulmonary vascular remodeling. Scale bar, 100 μm (n = 5-6). For panels A-R, a P value of <.05 was considered significant as determined by an unpaired t test. For panel S, significance was determined using 1-way analysis of variance with selected comparisons between NFU1 vs NFU1-LA and NFU1 vs wild-type (WT) groups, followed by a Holm-Sidak post hoc test. (T-Y) used an unpaired t test to determine significance, with a P value of < .05 considered significant. ATP, adenosine triphosphate; FITC, fluorescein isothiocyanate; OCR, oxygen consumption rate.
Figure 1.

The role of mitochondrial dysfunction in reticulocytes and hemolysis. (A) ATP production: mitochondrial ATP production, as measured by the Seahorse XF assay, is significantly decreased in NFU1 reticulocytes. ATP production is measured as the difference in the OCR after the addition of oligomycin, a complex V inhibitor (n = 5-7). (B-C) Immature reticulocytes: the number of immature reticulocytes, identified by the persistence of mitochondria under microscopy, is markedly increased in the blood of NFU1 rats. Scale bar, 50 μm (n = 8). (D) Circulating reticulocytes: reticulocyte counts are significantly elevated in NFU1 rats, indicating increased production, decreased clearance, and maturation of reticulocytes (n = 10). (E) EPO levels: EPO levels, which control reticulocyte and RBC production, determined by enzyme-linked immunosorbent assay are significantly elevated in the plasma of NFU1 rats (n = 10). (F-G) Carbonic anhydrase 9 (CA9) expression: immunohistochemical staining shows increased CA9 expression in the lungs of NFU1 rats, indicating a potential hypoxic environment. Scale bar, 200 μm (n = 4). (H) Circulating free heme: free heme levels in the plasma are significantly elevated in NFU1 rats, indicating hemolysis (n = 10). (I-J) Barrier disruption: FITC-dextran extravasation assays reveal significant barrier disruption in the lungs of NFU1 rats. Scale bar, 5 mm (n = 7-8). (K) RVSP: RVSP is significantly increased in NFU1 rats, indicating significant PH (n = 8). (L) Fulton index: the Fulton index, indicative of right ventricular remodeling (calculated as follows: right ventricle/left ventricle + septum), is significantly elevated in NFU1 rats (n = 8). (M-N) Vascular occlusion score: the vascular occlusion score, a measure of vascular remodeling, is markedly increased in NFU1 lungs. Scale bar, 100 μm (n = 5). (O) LA treatment: LA supplementation in NFU1 rats significantly improves mitochondrial ATP production (n = 5). LA is a cofactor of PDH, a mitochondrial enzyme. LAS synthesizes LA. The NFU1 protein inserts Fe-S clusters in LAS, enabling LA synthesis. The NFU1 mutation impairs LA synthesis because of LAS dysfunction. LA supplementation directly circumvents the LAS dysfunction. (P) Reticulocyte count posttreatment: reticulocyte numbers are normalized in NFU1 rats after LA treatment (n = 10-12). (Q-R) Immature reticulocytes posttreatment: LA treatment significantly reduces the number of immature reticulocytes in NFU1 rats, indicating improved reticulocyte maturation (n = 10-12). (S) Free heme posttreatment: free heme levels are significantly reduced in NFU1 rats after LA treatment, indicating decreased hemolysis (n = 9-10). (T-U) Barrier protection posttreatment: LA treatment significantly protects NFU1 rats from barrier disruption, as shown by reduced FITC-dextran extravasation in the lungs. Scale bar, 10 mm (n = 8). (V) RVSP posttreatment: LA treatment significantly protects NFU1 rats from increased RVSP, indicating a protection from the induction of PH (n = 8). (W) Fulton index posttreatment: LA treatment prevents the rise in the Fulton index in NFU1 rats (n = 8). (X-Y) Vascular remodeling posttreatment: LA treatment in NFU1 rats significantly reverses pulmonary vascular remodeling. Scale bar, 100 μm (n = 5-6). For panels A-R, a P value of <.05 was considered significant as determined by an unpaired t test. For panel S, significance was determined using 1-way analysis of variance with selected comparisons between NFU1 vs NFU1-LA and NFU1 vs wild-type (WT) groups, followed by a Holm-Sidak post hoc test. (T-Y) used an unpaired t test to determine significance, with a P value of < .05 considered significant. ATP, adenosine triphosphate; FITC, fluorescein isothiocyanate; OCR, oxygen consumption rate.

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