Fig 1: 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 2: 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
Fig 3: Cardiotoxicity FXN Overexpression Is Independent of the Dose Administered and Vector Biodistribution and Compromised the Success of Cardiac Gene Therapy in Mck Mouse HeartMck mice were treated at 7 weeks of age with the non-optimized vector AAVRh.10-CAG-hFXN-HA (n = 6) or with the optimized vector AAVRh.10-hFXN (n = 8) at 2.5 × 1013 vg/kg, followed up by echocardiography, and then sacrificed at 15 weeks of age to perform molecular analysis. NaCl-injected WT (n = 8–10) mice were used as control. (A and B) Schematic description of the vector construct for (A) the non-optimized AAVRh10-CAG-hFXN-HA vector, and (B) the optimized AAVRh.10-hFXN vector. (C) qPCR quantification of the number of VCN in the heart. Individual data points are reported, with mean and SD. Welch’s t test, n.s. for p > 0.05. (D) ELISA assay quantification of hFXN protein concentration in the heart, normalized to mg of total heart protein. Black dotted line represents the endogenous mouse FXN level in untreated C57/B6J WT mice (i.e., 147 ± 42 ng/mg) (n = 6). Individual data points are reported, with mean and SD. Welch’s t test, p values are reported. (E) Western blot (WB) analysis of total heart protein extract from Mck mice treated with the non-optimized vector (n = 3) or the optimized vector (n = 5). Immunoblotting against FXN, SDHb, GAPDH, and beta-tubulin (β-Tub). (F and G) WB quantification of the relative protein levels of FXN (F) and SDHb (G), normalized to GAPDH. Welch’s t test, p values are reported. (H) WB analysis of FXN protein maturation, in the heart of Mck mice treated with the optimized vector (n = 5) and expressing up to 179-fold the normal level of FXN. The dose of vector, VCN, and [hFXN] corresponding to each sample are reported. (I–L) Longitudinal echocardiography analysis. For control, WT mice were injected with NaCl (n = 8), and historical data are plotted for NaCl-injected Mck mice (n = 10). Data are reported as mean ± SD. Statistical analyses are presented in Table S4. (I) LV SF. (J) CO normalized to BW. (K) LV mass normalized to BW. (L) LV end-systole diameter (LV-ESD) reported as individual kinetic for treated Mck mice. See also Figure S4 for LV end-diastole measurement and BW. (M) qRT-PCR quantification of the cardiac gene expression of Nppa and Nppb, normalized to 18S and reported as percentage of NaCl-injected WT mice. Brown-Forsythe and Welch ANOVA test, p values are reported with n.s. p > 0.05.
Fig 4: FXN Cardiotoxic Overexpression Is Associated with Acute Heart Fibrosis and Inflammation(A) Histological analysis of fibrosis and cell infiltrates following H&E staining of heart tissue sections. Analysis of Mck mice treated with the AAVRh.10-hFXN (n = 8) or the AAVRh.10-hFXN-HA vector (n = 6) and WT mice treated with the AAVRh.10-hFXN vector at 2.5 × 1013 (n = 2) or 5 × 1012 (n = 1) vg/kg. The VCN and [FXN] in ng/mg are reported for each animal. (B) WGA staining of the extracellular matrix on heart tissue sections. Analysis of Mck mice injected with AAVRh.10-hFXN (n = 3) or AAVRh.10-CAG-hFXN-HA (n = 6) at 2.5 × 1013 vg/kg, WT mice injected with NaCl (n = 11) and untreated 9-week-old Mck mice (n = 6). (C) Quantification of heart surface labeled with WGA. Brown-Forsythe and Welch ANOVA test, p values are reported with n.s. p > 0.05. (D and E) qRT-PCR analysis of the heart mRNA level normalized to 18S and reported as percentage of NaCl-WT level. Brown-Forsythe and Welch ANOVA test, p values are reported with n.s. p > 0.05. (D) Col1a1, Col3a1, and Tgfb1 mRNA levels. (E) Il1b, Il6, and Tnfα. (F) Representative observations at low and high magnification of heart tissue sections immunolabeled for the monocyte cell marker CD14 (same exposure time). Analysis of 15-week-old NaCl-injected mice (n = 3) and Mck mice treated with AAVRh.10-hFXN (n = 3) or AAVRh.10-CAG-hFXN-HA (n = 3) at 2.5 × 1013 vg/kg. See also Figure S5 for supplementary histological analysis of CD14, CD45, and CD3.
Fig 5: HSP60 processing in frataxin‐deficient neurons. Western blot detection of HSP60 precursor (p) and mature (m) forms in crude extracts from Scr, Fxn1 and Fxn2 cells after 5 days of culture without treatment (lanes 1–3) or treated with TAT‐MTScs‐frataxin at 7 μg/ml (lanes 4–6). Mw markers are indicated. Note that precursor form increases in untreated Fxn1 and Fxn2 cultures and the processing is restored by TAT‐MTScs‐FXN addition.
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