Figure 2.

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 Morphology

WT 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.