Fig 1: High Level of FXN-HA Overexpression in WT Mice Cardiomyocytes Leads to Mitochondrial Function and Structure ImpairmentRepresentative histological observations from the correlative analysis of adjacent heart tissue sections/sample from WT mice treated at 7 weeks of age with AAVRh.10-CAG-hFXN-HA vector (5 × 1014 vg/kg, n = 4; 5 × 1013 vg/kg, n = 3) and sacrificed at 21 weeks. For controls, 21-week-old NaCl-injected WT mouse (n = 1) and 9-week-old untreated Mck mice (n = 2) were also analyzed. Each image series corresponds to the same individual for which dose of vector, VCN, and [hFXN] are reported. From left to right, co-staining and co-localization analysis of hFXN-HA protein expression and SDH enzymatic activity, cytochrome C oxidase (COX), and NADH-ubiquinone oxidoreductase (NADH) enzymatic activities assessed by in situ histoenzymatic assay are shown. Transmission electron microscopy (TEM) observations at low and high magnification of the LV myocardium is shown, following negative stain performed to assess cardiomyocyte and mitochondria ultrastructure. Finally, TEM observations of adjacent ultrathin sections at high magnification following bismuth sodium tartrate labeling of iron-ferritin complexes are shown. White arrows indicate non-iron mitochondrial electron-dense bodies, blue arrowheads indicate collapsed mitochondrial cristae, orange arrowheads indicate mitochondrial iron deposits, yellow arrowheads indicate mitochondrial iron-ferritin complex accumulation in mitochondria, and red arrowheads indicate ferritin sequestrated in the lysosome. myo, myofibrils; mito, mitochondria; nuc, nucleus. See also Figure S3 for extended histological analysis.
Fig 2: Cardiotoxic Overexpression of FXN-HA Is Associated with Increased Mitochondria Biomass and the Induction of the Integrated Stress Response(A) Co-labeling and co-localization observation for prohibitin (Phb) and the enzymatic activity of SDH, on heart tissue sections from WT mice treated with AAVRh.10-hFXN-HA (5 × 1014, n = 4; 5 × 1013, n = 3). For controls, NaCl-injected WT and untreated Mck mice are represented. For each image series, the dose of vector, VCN, and [hFXN] are reported. (B-D) qRT-PCR analysis of heart mRNA levels reported to 18S or Hprt (depending on the abundance of the target gene) and reported as percentage of NaCl-injected WT mice level. Individual data points are reported, with mean and SD. Brown-Forsythe and Welch ANOVA test, p values are reported with n.s. p > 0.05. (B) Integrated stress response (ISR) hallmark genes. (C and D) Downstream ISR-target genes encoding for secreted proteins (C) Fgf21 and (D) Gdf15.
Fig 3: High Level of FXN-HA Overexpression in the Heart of WT Mice Treated with AAVRh.10-CAG-hFXN-HA Vector Is Associated with Cardiac Fibrosis and Subclinical Impairment of Heart Function and MorphologyWT C57/B6J mice were treated at 7 weeks of age at 5 × 1014 (n = 4, red) or 5 × 1013 (n = 3, blue) vg/kg and sacrificed at 21 weeks. Unless stated otherwise, individual data points are plotted, with mean and SD. (A) qPCR quantification of the VCN in the heart. Light orange area represents the VCN range in Mck mice treated at 5 × 1013 vg/kg (n = 3) with the same vector from a previous study.15 (B) qRT-PCR analysis of the transgene mRNA level normalized to mouse Fxn mRNA level. (C) ELISA assay quantification of hFXN-HA protein concentration ([hFXN]) in the heart, normalized to mg of total heart protein. Light orange area represents the [hFXN] range in Mck mice treated at 5 × 1013 vg/kg (n = 3) from a previous study and reported in Figure 1.15 Black dotted line represents the endogenous level of FXN in untreated WT mice. (D) BW reported as individual, with males in blue and females in red. For untreated WT mice, historical data were plotted as mean ± SD. (E) Representative images from the histological analysis of adjacent heart tissue section collected from WT mice (5 × 1014, n = 4; 5 × 1013, n = 3) and stained with HA or WGA. Red arrows indicate fibrosis and cell infiltrates and yellow arrows indicate cardiomyocytes displaying subcellular disorganization. VCN and [hFXN] values are reported above for each animal. See also Figure S3 for the extended histological analysis. (F) Quantification of heart surface labeled with WGA. NaCl-injected WT mice sacrificed at 15 or 22 weeks of age (n = 11) were used as control. Brown-Forsythe and Welch one-way ANOVA statistical test, p values are reported with n.s. p > 0.05. (G–H) qRT-PCR quantification of cardiac gene expression normalized to 18S and reported as percentage of NaCl-treated WT mice (n = 9). (G) Col1a1, Col3a1, and Tgfβ1 mRNA level. Two-way ANOVA and FDR 5% statistical test, p values are reported with n.s. p > 0.05. (H) Nppa mRNA level. Brown-Forsythe and Welch one-way ANOVA test, p values are reported with n.s. p > 0.05. (I and J) Echocardiography at 21 weeks of age. For control, historical data from 21-week-old WT mice treated with NaCl are reported. Brown-Forsythe and Welch one-way ANOVA test, p values are reported with n.s. p > 0.05. (I) LV SF. (J) LV mass normalized to BW. (K) Heart ventricle weight measured upon necropsy at 21 weeks of age, normalized to BW. (L) Correlation analysis between [hFXN] and LV SF. Spearman non-parametric correlation coefficient and p value are reported.
Fig 4: FXN Cardiotoxic Overexpression Is Associated with Impaired Mitochondrial Function and Ultastructure, but Not with Iron OverloadRepresentative histological observations from the analysis of adjacent heart tissue sections/sample collected from mice treated at 7 weeks of age with the AAVRh.10-hFXN vector and sacrificed at 15 weeks of age. Analysis of Mck mice treated at 2.5 × 1013 vg/kg (n = 3), WT mice treated at 2.5 × 1013 (n = 2) or 5 × 1012 vg/kg (n = 1), NaCl-injected WT mice (n = 2) and untreated 9-week-old Mck mice (n = 2). See also Figure S6 for low-magnification imaging. (A) Co-staining and co-localization analysis of hFXN-HA protein expression and SDH enzymatic activity, respectively, labeled by immunofluorescence and in situ histoenzymatic assay. (B) NADH enzymatic activities assessed by in situ histoenzymatic assay. (C) TEM observations at low and high magnification of the LV myocardium, following negative stain. White arrows indicate non-iron mitochondrial electron-dense bodies; myo, myofibrils; mito, mitochondria; nuc, nucleus. (D) DAB-enhanced Perls labeling of iron deposits.
Fig 5: High Expression of FXN-HA in Cardiomyocytes Is Associated with Impaired Succinate Dehydrogenase (SDH) Enzymatic Activity despite Functional Rescue of Mck Mice Treated with AAVRh.10-CAG-hFXN-HA(A–C) Histological analysis of heart tissue sections collected from the Mck mice treated at 5 weeks of age with the AAVRh.10-CAG-hFXN-HA vector and sacrificed at 12 weeks. Representative images are from the Mck mouse treated with 5 × 1013 vg/kg and expressing the highest level of hFXN-HA (10,927 ng/mg) and from another Mck mouse treated with 2.5 × 1013 vg/kg and with lower hFXN-HA level (695 ng/mg). For controls, heart tissue sections from 12-week-old NaCl-injected wild-type (WT) mice and 9-week-old untreated Mck mice were also analyzed. The corresponding vector copies per diploid genome (VCN) and tissue concentration in human FXN ([hFXN] in ng per mg of total protein) are reported. The same time exposure was used for all animals. (A) Co-staining and co-localization analysis of hFXN-HA overexpression and SDH enzymatic activity. Acquisitions of a single microscopic field at low and high magnification are shown, to compare the distribution of hFXN-HA expression hotspots and SDH activity impairment. (B) Hematoxylin and eosin (H&E) and wheat germ agglutinin (WGA) staining. (C) DAB-enhanced Perls labeling of iron deposits with methyl green counterstaining. (D–F) Longitudinal echocardiography analysis of the same Mck mice, represented here as individual kinetics. Data are represented as mean ± SD for WT control mice (n = 7) and untreated Mck mice (n = 10). For untreated Mck mice, historical data were plotted. Statistical analyses are reported in Table S2. (D) Left ventricle (LV) shortening fraction (SF). (E) Cardiac blood output (CO) measured at the aorta and normalized to body weight (BW). (F) LV mass normalized to BW. See also Figures S1A–S1C for the extended echocardiography analysis of this cohort of mice. See also Figures S1D–S1H for the echocardiography follow-up, until 25 weeks of age, of a second cohort of Mck mice treated at 5 weeks of age at 5 × 1013 vg/kg. Figure partially adapted from Belbellaa et al.15
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