Gene therapy prevents onset of mitochondrial cardiomyopathy in neonatal mice with Ndufs6 deficiency

Abstract

Mutations in genes affecting mitochondrial complex I (CI) can lead to mitochondrial cardiomyopathy (MCM) yet no effective treatment. This study sought to determine whether adeno-associated virus 9 (AAV9)-based gene therapy could prevent or rescue Ndufs6 deficiency-induced MCM at different disease stages. Using Ndufs6^gt/gt mice to mimic MCM, cardiac dysfunction was evident at week 4 post-birth, showing reduced ejection fraction, CI activity, increased fibrosis, mitochondrial fission, and disrupted cristae. Neonatal and adult mice were intravenously given AAV9-hNdufs6 (1e14 vg kg^-1). AAV9-hNdufs6 therapy effectively prevented neonatal mice's cardiac dysfunction onset, preserving CI activity and cristae structure for 11 months. In contrast, therapy in adult mice post-disease onset failed to reverse or halt progression of heart dilation and failure after 3 months, showing mitochondrial abnormalities and cardiomyocyte apoptosis. Mechanistically, adult mouse Kupffer cells demonstrated enhanced phagocytic capabilities compared to neonatal mice, with higher expression levels of AAV9 cell surface receptors observed in neonatal mouse hearts, rendering neonatal mice more responsive to AAV9-mediated gene therapy for heart tissue. Additionally, AAV9-hNdufs6 gene therapy initiated at an early stage increased Ndufs6 expression in cardiac tissue, preserved mitochondrial structure and function, prevented cardiomyocyte fibrosis through modulation of the AMPK/Drp1 signaling pathway. In conclusion, early intervention with AAV9-hNdufs6 gene therapy can effectively prevent the onset of MCM, but intervention after disease onset has limited efficacy.

Fig. 1. Ndufs6 knock out in heart tissue could develop cardiomyocyte in mice.

A Procedure for identification of Ndufs6^gt/gt mice. B Representative images of left ventricular echocardiograph in WT and Ndufs6^gt/gt mice and M-mode echocardiograph data (EF% and FS%) of left ventricle in WT and Ndufs6^gt/gt mice (n = 6). C Representative images of heart and cardiac cross section with H&E staining from WT and Ndufs6^gt/gt mice, followed by statistical analysis of LVEDD and HW/BW (n = 6). Scale bar = 1 mm. D Representative images of Masson’s trichrome staining and quantitative measurement of heart fibrosis (n = 6). Scale bar = 100 μm. E Representative images of mitochondrial ultrastructure and quantification of mitochondrial fragmentation (n = 6). Scale bar = 2 μm. F Representative images of mitochondrial cristae and the quantification of the degree of cristae damage (n = 6). Scale bar = 500 nm. G Mitochondrial CI activity of WT and Ndufs6^gt/gt group (n = 6). H Survival rate of WT and Ndufs6^gt/gt groups (n = 15).

Fig. 2. Administration of AAV9-hNdufs6 prevented cardiac dysfunction in newborn Ndufs6^gt/gt mice.

A Schematic diagram of prophylactic gene therapy for neonatal Ndufs6^gt/gt mice. B Representative M-mode echocardiograms and quantification of left ventricular EF% and FS% for 3-month-old AAV neonatal therapy mice (n = 7). C The quantification of M-mode echocardiograms of left ventricular EF% (WT vs. Ndufs6^gt/gt, **p = 0.0033; Ndufs6^gt/gt vs. AAV-Neonatal, **p = 0.0074) and FS% (WT vs. Ndufs6^gt/gt, **p = 0.0021; Ndufs6^gt/gt vs. AAV-Neonatal, **p = 0.0055) for 11-month-old WT, Ndufs6^gt/gt and AAV-Neonatal mice (n = 3). D Treadmill exercise tolerance test in 3-month-old AAV neonatal treatment group (n = 7). E Survival rate of Ndufs6^gt/gt and AAV-Neonatal group (n = 15). (*p = 0.0194).

Fig. 3. Administration of AAV9-hNdufs6 prevented pathological changes in newborn Ndufs6^gt/gt mice.

A Representative images illustrating the morphology of heart. Scale bar = 1 mm. B Quantitative analysis of LVEDD of WT, Ndufs6^gt/gt and AAV-Neonatal mice at 3 months old (n = 6). C Quantitative analysis of HW/BW ratio of WT, Ndufs6^gt/gt and AAV-Neonatal mice at 3-months old (n = 6). D Representative images of Masson’s trichrome staining and quantitative measurement of heart fibrosis (n = 4). Scale bar = 100 μm. E Representative images of TUNEL staining and quantification analysis of the heart tissue from WT, Ndufs6^gt/gt and AAV-Neonatal mice (n = 4). Scale bar = 50 μm. The tissue slides utilized are consecutive sections from the same specimen stained with Masson’s trichrome on (D). F Representative mitochondrial ultrastructure and quantitative measurement of mitochondrial fragmentation in different experimental groups (n = 4). Scale bar = 1 μm. G Representative images of mitochondrial cristae morphology and the quantification of the degree of cristae damage, scale bar = 500 nm in WT, Ndufs6^gt/gt and AAV-Neonatal groups. The results were calculated based on mitochondrial classification system (n = 4, with at least 100 mitochondria averaged for each replicate).

Fig. 4. Administration of AAV9-hNdufs6 prevented cardiomyopathy in Ndufs6^gt/gt mice via the AMPK pathway.

A Cardiac mitochondrial CI activity results of WT, Ndufs6^gt/gt, and AAV-Neonatal groups (n = 6). B Protein expression levels of p-Drp1, Drp1, p-AMPK and AMPK in cardiac tissue (expressed as relative fold, normalized to β-Actin and followed by normalization to WT gray scale/ β-Actin gray scale) were analyzed in the WT, Ndufs6^gt/gt, and AAV-Neonatal groups (B, C). Quantitative assessment of p-Drp1/Drp1 (B) and p-AMPK/AMPK (C) expressions in WT mice, Ndufs6^gt/gt and AAV-Neonatal mice is presented (n = 4). D DHE staining for ROS and quantification are illustrated (n = 4). Scale bar = 50 μm.

Fig. 5. Administration of AAV9-hNdufs6 could not reverse cardiac dysfunction in adult Ndufs6^gt/gt cardiomyopathy mice.

A Schematic diagram of gene therapy for adult Ndufs6^gt/gt mice. B Representative M-mode echocardiograms and quantification of left ventricular EF% (*p = 0.0134) and FS% (*p = 0.0222) (n = 7). C Running time of treadmill exercise tolerance test for WT, Ndufs6^gt/gt, and AAV-Adult groups (n = 7). D Quantification of LVEDD for WT, Ndufs6^gt/gt, and AAV-Adult groups (***p = 0.0001) (n = 6). E Quantification of HW/BW for WT, Ndufs6^gt/gt, and AAV-Adult groups (*p = 0.0377) (n = 6). F Representative images of Masson’s trichrome staining and measurement of fibrosis (n = 4). Scale bar = 100 μm. G Representative images of TUNEL staining for apoptosis and quantification analysis of cardiomyocytes from WT, Ndufs6^gt/gt, and AAV-Adult mice (n = 4). Scale bar = 50 μm. The tissue slides utilized are consecutive sections from the same specimen stained with Masson’s trichrome on (F).

Fig. 6. The impact of phagocytosis activity of KCs on the effect of AAV gene therapy.

A The relative expression of hNdufs6 mRNA in mice liver of Ndufs6^gt/gt, AAV-Neonatal and AAV-Adult groups at 3 months post gene therapy (n = 4). B Relative protein expression of NDUFS6 in mice liver was detected in Ndufs6^gt/gt, AAV-Neonatal and AAV-Adult groups by Western blot. The quantification results were calculated following two steps by Image J: 1) gray scale for each sample was normalized to GAPDH (WT-1 = WT gray scale/GAPDH gray scale; Ndufs6^gt/gt-1 = Ndufs6^gt/gt gray scale/GAPDH gray scale; AAV-Neonatal-1 = AAV-Neonatal gray scale/GAPDH gray scale; AAV-Adult-1 = AAV-Adult gray scale/GAPDH gray scale); 2) then Ndufts6^gt/gt-1, AAV-Neonatal-1 and AAV-Adult-1 are expressed as percentage of WT levels. (n = 4). C Immunofluorescence analysis of macrophages displaying DAPI, mF4/80, and Latex beads. The DAPI staining (blue) marks the cell nuclei, mF4/80 staining (red) identifies the macrophage surface receptor, and green fluorescence highlights the phagocytosed Latex beads within the cells. The merged image shows the co-localization of mF4/80 with the Latex beads, confirming the phagocytic activity of mF4/80-positive cells. Scale bar = 20 μm. D Quantification of fluorescence intensity and representative photos of phagocytic activity of KCs in neonatal (PDN1) mice and Adult (3-month-old) mice at 2 h, respectively. Scale bar = 20 μm.

Fig. 7. The higher LamR expression in neonatal mice enhances the efficacy of AAV gene therapy in heart.

A The relative expression of hNdufs6 mRNA in cardiac tissue of Ndufs6^gt/gt, AAV-Neonatal and AAV-Adult groups at 3 months post AAV gene therapy (n = 4). B Relative protein expression of NDUFS6 in cardiac tissue was detected in Ndufs6^gt/gt, AAV-Neonatal and AAV-Adult groups by Western blot. The quantification results were calculated following two steps by Image J: 1) gray scale for each sample was normalized to GAPDH (WT-1 = WT gray scale/GAPDH gray scale; Ndufs6^gt/gt-1 = Ndufs6^gt/gt gray scale/GAPDH gray scale; AAV-Neonatal-1 = AAV-Neonatal gray scale/GAPDH gray scale; AAV-Adult-1 = AAV-Adult gray scale/GAPDH gray scale); 2) then Ndufts6^gt/gt-1, AAV-Neonatal-1 and AAV-Adult-1 are expressed as percentage of WT levels. C Representative images and quantification IHC showed Ndufs6 protein expression in the cytoplasm of heart tissues in Ndufs6^gt/gt, AAV-Neonatal and AAV-Adult mice (n = 4). Scale bar=50μm. ***p = 0.0004. D LamR mRNA expression in AC16 cell line after siRNA treatment at different concentration. (n = 3). E LamR protein expression in AC16 cell line after siRNA treatment at different concentration. (n = 3). F Representative images of AC16 cells and AC16-LamR-KD cells 48 h post AAV9-CAG-eGFP infection. Scale bar = 20 um. (n = 3). G LamR mRNA relative expression level in PND0 and 3-month-old mice. (n = 4) H LamR protein expression and quantitative analysis result on PND0 and 3-Month-old mice (n = 4). ***p = 0.0001.