Rats with a Human Mutation of NFU1 Develop Pulmonary Hypertension

Abstract

NFU1 is a mitochondrial protein that is involved in the biosynthesis of iron-sulfur clusters, and its genetic modification is associated with disorders of mitochondrial energy metabolism. Patients with autosomal-recessive inheritance of the NFU1 mutation G208C have reduced activity of the respiratory chain Complex II and decreased levels of lipoic-acid-dependent enzymes, and develop pulmonary arterial hypertension (PAH) in ∼70% of cases. We investigated whether rats with a human mutation in NFU1 are also predisposed to PAH development. A point mutation in rat NFU1^G206C (human G208C) was introduced through CRISPR/Cas9 genome editing. Hemodynamic data, tissue samples, and fresh mitochondria were collected and analyzed. NFU1^G206C rats showed increased right ventricular pressure, right ventricular hypertrophy, and high levels of pulmonary artery remodeling. Computed tomography and angiography of the pulmonary vasculature indicated severe angioobliterative changes in NFU1^G206C rats. Importantly, the penetrance of the PAH phenotype was found to be more prevalent in females than in males, replicating the established sex difference among patients with PAH. Male and female homozygote rats exhibited decreased expression and activity of mitochondrial Complex II, and markedly decreased pyruvate dehydrogenase activity and lipoate binding. The limited development of PAH in males correlated with the preserved levels of oligomeric NFU1, increased expression of ISCU (an alternative branch of the iron-sulfur assembly system), and increased complex IV activity. Thus, the male sex has additional plasticity to overcome the iron-sulfur cluster deficiency. Our work describes a novel, humanized rat model of NFU1 deficiency that showed mitochondrial dysfunction similar to that observed in patients and developed PAH with the same sex dimorphism.

Keywords: Complex II; iron-sulfur cluster; mitochondrial dysfunction; pulmonary arterial hypertension; sex difference.

Figure 1.

Hemodynamic changes in NFU1 mutant rats. (A) Right ventricular systolic pressure (RVSP) was slightly increased in heterozygote female rats (HZ-F) and significantly elevated in homozygote female rats (HM-F). (B) RVSP was only slightly elevated in HM males (HM-M). (C–F) RV dP/dt max (C) as a measure of RV contractility and RV dP/dt min (E) as a measure of RV contractility was significantly elevated in HM-F, but not HM-M, rats (D and F). (G) The Fulton index, the ratio of the RV to the left ventricle plus septum (RV/[LV+S]), was insignificantly increased in HZ-F and markedly increased in HM-F. (H) In males, the Fulton index was significantly higher in the HM group than in the HZ group. (I and J) The RV weight to body weight ratio (RV/BW) showed a trend similar to that observed for the Fulton index in females and males. (K and L) The average dP/dt during the isovolumetric relaxation period (IRP) as a measure of RV diastolic relaxation was significantly decreased in the HM rats of either sex. (M and N) Although the end-diastolic pressure (EDP) showed no changes in females (M), it was found to be significantly elevated in the HM-M group (N), confirming a more pronounced RV diastolic dysfunction in males. Data are presented as mean ± SEM, n = 7–10, *P < 0.05 versus WT, ANOVA. ^†P < 0.05 versus HZ. dP/dt = a mathematical expression meaning derivative of pressure over time; F = female; M = male; WT = wild type.

Figure 2.

(A) Histological changes in the lungs. Representative images of Verhoeff–van Gieson-stained lungs show a severe remodeling of pulmonary arteries. Scale bars: 100 μm. (B) Quantitative analysis of pulmonary artery remodeling. The vascular occlusion score was significantly increased in both male and female HZ and HM groups compared with control (mean ± SEM, n = 5–7, *P < 0.05 versus control, ANOVA; for each animal, a random 10 vessels were averaged). (C) Representative three-dimensional micro–computed tomography images of the pulmonary vasculature in WT and HM female rats. Three different views of each lung are shown. (D) Representative microangiogram images show the vascular morphology of the right middle lobe of female WT and HM rats (scale bars: 5 mm) and the morphology of small pulmonary arteries (scale bars: 1 mm). Double-sided arrows show that HM rats have an increase in the area of low vascular density. This decrease in the complexity and number of the small pulmonary arteries could be due to a combination of pulmonary arterial hypertension–associated microvascular rarefaction and vasoobliterative disease. The arrowheads point to multiple occlusions of the small pulmonary arteries that appear as a break in pulmonary artery integrity or as an extraslim section.

Figure 3.

Alteration in the activities of electron transport chain complexes. The enzymatic activities of individual complexes were measured in mitochondria isolated from liver. (A–C) We found that in the female HM group, Complex I (A) and Complex II (B) activity was significantly decreased, whereas Complex IV activity was unchanged (C). (D–F) In males, Complex I activity was unchanged, with a downward trend in the HM group (D); Complex II activity was significantly decreased (E); and Complex IV activity was significantly elevated in the HM group (F). Data are presented as mean ± SEM, n = 5–10, *P < 0.05 versus control, ANOVA.

Figure 4.

Expression levels of oxidative phosphorylation complexes. (A–D) Using an oxidative phosphorylation antibody cocktail, we found that in females (A), HM rats with the NFU1 mutation showed a significant decrease in the Complex I subunit (NDUFB8) (B) and both Complex II subunits (SDHA [Fp]) [C] and SDHB [Ip] [D]). (E–G) There were no changes in expression of the Complex III subunit (UQCRC2) (E), Complex IV subunit (MCTO1) (F), or Complex V subunit (ATP5A) (G). (H–J) In males (H), the HM group showed a decrease only in expression of Complex I (I) and the Complex II SDHA subunit (J). (K and L) The SDHB subunit (K) was unaltered, whereas Complex III expression was increased, in both the HZ and HM groups (L). (M and N) Complex IV (M) and Complex V (N) subunits were similar to control. Data are presented as mean ± SEM, n = 7–9, *P < 0.05 versus control, ANOVA.

Figure 5.

The oligomeric state of NFU1. (A–C) Mitochondrial separation of NFU1 oligomers on a seminative gel showed increased hexameric form in the HZ group and decreased hexameric form in the female HM group (A and B); a dimeric form of NFU1 was unchanged (A and C). (D–F) In males, mitochondria NFU1 oligomers showed unchanged levels of the hexameric form (D and E) and significantly reduced dimers in the HM group (D and F). Data are presented as mean ± SEM, n = 6–9, *P < 0.05 versus control, ANOVA.

Figure 6.

Pyruvate dehydrogenase (PDH) activity and expression. (A and B) Western blot showed increased expression levels of PDH in the lung lysates of female HM rats. (C) However, PDH activity in mitochondria lysate was significantly decreased. (D–F) In males, Western blot analysis showed unchanged levels of PDH expression (D and E), but activity was decreased in the HM group (F). Changes in PDH activity were strongly correlated with the level of PDH lipoylation. (G–J) In HM groups of either sex, the amount of lipoate-containing PDH was severely downregulated, confirming that a mutation in the NFU1 protein mediates impaired lipoic acid synthesis. Data are presented as mean ± SEM, n = 5–7, *P < 0.05 versus control, ANOVA. ^†P < 0.05 versus HZ.

Figure 7.

Iron-sulfur cluster scaffold protein ISCU. (A and B) Western blot analysis of female lung lysates (A) showed unaltered protein levels of ISCU (B). (C and D) In contrast, in males, the levels of ISCU protein were found to be significantly increased in the HM group. Data are presented as mean ± SEM, n = 6–7, *P < 0.05 versus control, ANOVA.

Figure 8.

Aconitase (ACO) and ferritin levels in lung lysates. (A–C) Western blot analysis indicates a slight increase in ferritin levels (A and B) in the HZ female group and unchanged levels of aconitase (A and C). (D–F) In males, both ferritin (D and E) and aconitase (D and F) levels were similar to control. Data are presented as mean ± SEM, n = 6–7, *P < 0.05 versus control, ANOVA.