Cardiac bridging integrator 1 gene therapy rescues chronic non-ischemic heart failure in minipigs

Figures

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  Fig. 1. Myocardial cBIN1 levels in patients with DCM and HFrEF. a Myocardial cBIN1 levels in LV free wall samples are quantified using cBIN1 ELISA kit and normalized to total protein levels ([cBIN1]) in HF patients and non-HF donors (n = 10 per group). Data are presented as mean ± standard error of the mean (SEM). Unpaired Student’s t-test is used for two group comparison. *** indicates p < 0.001 for comparison vs. non-HF Donor. b–d Correlations between myocardial [cBIN1] and echocardiography measured left ventricular ejection fraction (LVEF) (b), left ventricular end-diastolic diameter (LVEDD) (c), and relative wall thickness (RWT) (d) in non-HF donors (black circles) and HF patients (red circles). Person’s correlation test was used for correlational analysis, followed by linear regression analysis.

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  Fig. 2. AAV9 transduced cBIN1 in myocardium rescues survival rates in minipigs with RVP-induced non-ischemic HFrEF. a Schematic illustration of experimental protocol (Created with BioRender.com). b Exogenous V5-labled transgene expression (RT-qPCR detected V5/HPRT1 and expressed as ΔΔCt when compared to PBS controls) in minipig left ventricular myocardium at termination (n = 5 sites across left ventricle in each pig). Data are presented as mean ± standard error of the mean (SEM). Kruskal-Wallis test is used for comparison to PBS group (15 samples from 3 PBS-treated animals). *, **, *** indicates p < 0.05, 0.01, 0.001. c Kaplan-Meier survival curves for all four groups (Sham, N = 3; GFP, N = 1; PBS, N = 4; cBIN1, N = 4). Significance was calculated using a Log-rank test.

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  Fig. 3. Myocardial cBIN1 is reduced in HFrEF minipigs and restored by AAV9-cBIN1 treatment. a Representative images of left ventricular myocardial cryosections labeled with WGA (red), V5 (green), and Dapi (blue) from each group (Scale bar: 50 μm) with quantification to the right for the percent of cardiomyocytes positive with V5 signal (images/hearts: n/N = 19/2; 16/2, 20/3 from sham, control, and cBIN1 groups). b Representative Western blots of cBIN1 detected by antibody against BIN1 exon 17 or SH3, as well as loading control GAPDH in LV samples from each group. Quantification of cBIN1 protein band (cBIN1/GAPDH and normalized to Sham group) is included in the bar graph to the right (LV samples/hearts: n/N = 9/3; 10/5, 11/4 from sham, control, and cBIN1 groups). c ELISA quantification of [cBIN1] (ng/mg protein) in LV (LV samples/hearts: n/N = 8/3; 11/5, 11/4 from sham, control, and cBIN1 groups). d RT-qPCR quantification of cBIN1 gene expression in LV (LV samples/hearts: n/N = 12/3; 20/5, 16/4 from sham, control, and cBIN1 groups). Data are presented as mean ± standard error of the mean (SEM). Ordinary one-way ANOVA followed by Fisher’s LSD test or non-parametric Kruskal-Wallis test…

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  Fig. 4. AAV9-cBIN1 improves LVEF with limited chamber dilation in minipigs with non-ischemic HFrEF. Echocardiography monitoring of LVEF (a) and LVEDV (b) in minipigs from baseline (RVP initiation timepoint) to terminal endpoint for each group (N = 3, 5, 4 from sham, control, and cBIN1 groups). Data are presented as mean ± standard error of the mean (SEM). Two-way ANOVA followed by Fisher’s LSD test for comparison among three groups was performed. *** indicates p < 0.001 for comparison vs. Sham. ### indicates p < 0.001 for comparison vs. Control.

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  Fig. 5. AAV9-cBIN1 rescues cardiac function in minipigs with non-ischemic HFrEF. a Representative four-chamber view images of the minipig heart at end diastolic (top) and systolic (bottom) phases on termination day (left). Quantification of ejection fraction (EF) and stroke volume (SV) from each group (N = 3, 5, 4 from sham, control, and cBIN1 groups), (right). b Representative images of M-mode short axis view of the heart at termination (left). Quantification of left ventricular end-diastolic diameter (LVEDD) and relative wall thickness (RWT) (N = 3, 5, 4 from sham, control, and cBIN1 groups) (right). Data are presented as mean ± standard error of the mean (SEM). Ordinary one-way ANOVA followed by uncorrected Fisher’s LSD test is used for comparison among three groups. *, **, *** indicates p < 0.05, 0.01, 0.001 for comparison vs. Sham. #, ##, ### indicates p < 0.05, 0.01, 0.001 for comparison vs. Control.

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  Fig. 6. AAV9-cBIN1 rescues gross heart anatomy in minipigs with non-ischemic HFrEF. a Frontal and transverse view of gross morphology of the minipig heart at termination. Scale bar, 5 cm. b Quantification of heart weight normalized to body weight. c Quantification of left ventricular (LV) cross area from transverse view (N = 3, 5, 4 from sham, control, and cBIN1 groups). Data are presented as mean ± standard error of the mean (SEM). Ordinary one-way ANOVA followed by uncorrected Fisher’s LSD test is used for comparison among three groups. *** indicates p < 0.001 for comparison vs. Sham. ## indicates p < 0.01 for comparison vs. Control.

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  Fig. 7. AAV9-cBIN1 ameliorates fluid retention in minipigs with non-ischemic HFrEF. a Lung weight (LW) measured as the ratio of wet over dry lung weight of the minipigs at termination. b Thoracic cavity and abdominal fluid volume measured during termination procedure. c The mRNA level of Natriuretic Peptide B (NPPB) and Natriuretic Peptide A (NPPA) normalized to the housekeeping gene HPRT1 (quantified by ΔCt of NPPB/HPRT1 or NPPA/HPRT1) (LV samples/hearts: n/N = 15/3; 19/5, 20/4 from sham, control, and cBIN1 groups). Data are presented as mean ± standard error of the mean (SEM). Ordinary one-way ANOVA followed by uncorrected Fisher’s LSD test or non-parametric Kruskal-Wallis test followed by uncorrected Dunn’s test is used for comparison among three groups. *, *** indicates p < 0.05, 0.001 for comparison vs. Sham. #, ### indicates p < 0.05, 0.001 for comparison vs. Control.

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  Fig. 8. AAV9-cBIN1 rescues transverse-tubule luminal dilation and organization in minipigs with non-ischemic HFrEF. a Representative transmission electron microscopy images of left ventricular free wall of minipig hearts with zoomed-in images of the boxed area (left). Scale bar, 2 µm. b Quantification of transverse-tubule (TT) cross-sectional area in each group (TT/hearts: n/N = 221/2; 203/3, 306/3 from sham, control, and cBIN1 groups) (right). c–f Representative images with power spectrum analysis for BIN1 (c, d) and wheat germ agglutinin (WGA) (e, f) in myocardial sections from each group. (cells/hearts: n/N = 47/2; 51/3, 36/3 for BIN1; 46/2; 45/3, 34/3 for WGA from sham, control, and cBIN1 groups). Scale bar, 10 µm. Data are presented as mean ± standard error of the mean (SEM). Non-parametric Kruskal-Wallis test followed by uncorrected Dunn’s test is used for comparison among groups. *** indicates p < 0.001 for comparison vs. Sham. ### indicates p < 0.001 for comparison vs. Control. g Cross-sectional views of cardiomyocyte images indicate colocalization between BIN1 (green) and WGA (magenta). Group labels are included to the left of the images. Scale bar, 20 µm.

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  Fig. 9. AAV9-cBIN1 reorganizes intracellular distribution of ryanodine receptors and L-type calcium channels in HFrEF minipig cardiomyocytes. a Representative confocal images of ryanodine receptor 2 (RyR2, green) and L-type calcium channels (Cav1.2, magenta) labeled myocardial sections from each group. Scale bar, 10 µm. b–c Quantification of RyR2 (b) (cells/hearts: n/N = 52/2; 47/3, 55/3 from sham, control, and cBIN1 groups) and Cav1.2 (c) (cells/hearts: n/N = 45/2; 45/3, 36/3 from sham, control, and cBIN1 groups) peak power density in cardiomyocytes from each group. Data are presented as mean ± standard error of the mean (SEM). Non-parametric Kruskal-Wallis test followed by Dunn’s test is used for comparison among three groups. *** indicates p < 0.001 for comparison vs. Sham. ### indicates p < 0.001 for comparison vs. Control.