Partial restoration of mitochondrial dysfunction by AAV-Ant1 protects from dilated cardiomyopathy in Ant1^-/- plus mtDNA mutant mice

Domain: biology
Published: 2025-12-15
Editorial outcome: minor_revision

Abstract
Primary mitochondrial disease (PMD) patients manifesting cardiomyopathy are twice as likely to die as other PMD patients. One PMD with cardiomyopathy is caused by null mutations in the heart-muscle isoform of the adenine nucleotide translocator (SLC25A4, ANT1) gene, with the severity of cardiomyopathy mediated by mitochondrial DNA. To optimize strategies for addressing mitochondrial cardiomyopathy, we generated an Ant1 null mouse and combined it with the ND6P25L mitochondrial DNA mutation to mimic the hypertrophic versus dilated cardiomyopathies observed in patients. Here, we transduce the neonatal Ant1-/- and Ant1-/-+ND6P25L mouse hearts with an AAV2/9-pDes-Gfp-mAnt1 cDNA vector. We show that restoration of just 10% of Ant1 gene expression was sufficient to ameliorate the cardiomyopathies in these mice. Proteomics and single-nucleus RNA sequencing reveal the reversal of dysregulated mitochondrial metabolic genes, including PGC1α, as well as cardiac contractile and extracellular matrix proteins. Hence, a modest increase in cardiac mitochondrial energetics can have profound benefits on cardiac function and is effective in treating mitochondrial cardiomyopathy.

Introduction
Primary mitochondrial disease (PMD) is one of the most common inherited metabolic disorders affecting both children and adults, with an incidence of ~1 in 5000. PMD can result from mutations encoded in over 350 nuclear DNA (nDNA) genes1 or in the 37 mitochondrial DNA (mtDNA) genes2. Pediatric mitochondrial diseases are more often the result of nDNA mutations, while adult mitochondrial diseases are more likely the result of mtDNA mutations1,3,4. 20–40% of PMD patients develop cardiomyopathy between birth and 27 years of age4–8, with the ten-year mortality being 67-71% for PMD children with cardiomyopathy versus 18–26% for PMD children without cardiomyopathy6. Therefore, it is imperative that we develop an approach to treat mitochondrial cardiomyopathy if we are to save the lives of the most vulnerable PMD patients.

To address this critical clinical need, we have focused on a PMD resulting from an nDNA gene mutation whose severity is modulated by the mtDNA genetic background, the combination of the nDNA and mtDNA variant resulting in life-threatening pediatric dilated cardiomyopathy. These patients harbor homozygous null mutations in the nuclear-coded adenine nucleotide translocator-1 (ANT1) gene (SLC25A4), and ANT1-/- patients present with cardiomyopathy and myopathy9–11.

Starting with a thirteen-generation consanguineous pedigree harboring a SLC25A4 frameshift mutation (c.523delC, p.Q175RfsX38), the homozygous null patients paired with the common European mtDNA haplogroup H (mtDNAH) manifest progressive hypertrophic cardiomyopathy associated with myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Echocardiographic studies revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. Their end-stage heart disease is characterized by symmetric, concentric cardiac hypertrophy, widespread cardiomyocyte degeneration, and extensive subendocardial interstitial fibrosis, which is compatible with life into middle age. However, family members with the same ANT1-/- nDNA mutation but who inherit the rarer haplogroup “U2” mtDNA (mtDNAU2) from their mothers develop pediatric dilated cardiomyopathy. Haplogroup “U2” differs from haplogroup “H” in harboring an MT-RNR2 (16S rRNA) m.1811A>G and an MT-TL2 (tRNALeu(CUN)) m.12308A>G mutation, which may diminish the translation of the seven mtDNA-coded oxidative phosphorylation (OXPHOS) complex I polypeptides9.

To model this pedigree, we inactivated the mouse heart-muscle ANT1 gene (Ant1), resulting in autosomal recessive myopathy and hypertrophic cardiomyopathy12,13. We then combined this nDNA mitochondrial gene mutation with our mtDNA complex I missense mutation MT-ND6 m.13997G>A (P25L, ND6P25L)14, the same amino acid change that results in maternally inherited PMD in humans [ND6 m.14600G>A (P25L, ND6 P25)]15. The Ant1-/- plus ND6P25L combination results in early onset dilated cardiomyopathy, affecting echocardiogram measurements and leading to an 80% reduction in cardiac contractility and dramatic ventricular dilation16, thus modeling the severe dilated cardiomyopathy manifest by the ANT1-/- + mtDNAU2 children16.

There are four ANT isoforms in humans17–27 and three isoforms in mouse12,28–31. ANT1 is the predominant ANT in muscle, while ANT1 and ANT2 are present in the heart, raising the possibility that ANT2 may partially compensate for the ATP export deficiency in the ANT1 null heart29.

ANT1/Ant1 has been found to have four functions: ATP/ADP exchange, modulation of the permeability transition pore (mtPTP)30,32, generation of a lipid-modulated proton channel33, and regulation of mitophagy34. Ant1 deficiency in muscle is associated with a massive accumulation of mitochondria with abnormal morphology34,35.

There is no FDA-approved cure for PMD-associated cardiomyopathy. Current treatment is primarily palliative36–38, prompting us to develop a gene therapy. Using the Ant1-/- and Ant1-/- + mtDNA ND6P25L mice as a model system, we show that AAV-Ant1 mediated gene therapy can mitigate the mouse dilated cardiomyopathy. This could save the lives of the ANT1-/- + mtDNAU2 children who typically require a heart transplant at a young age. To treat the nDNA Ant1-/- defect, we use an adeno-associated virus (AAV), which permits the transduction of the Ant1 cDNA into the Ant1-/- and Ant1-/- + mtDNA ND6P25L postnatal mouse hearts. Our results show that even a partial rescue of Ant1 expression significantly ameliorates both the Ant1-/- hypertrophic and the Ant1-/- + mtDNA ND6P25L dilated cardiomyopathies.

Results
AAV-mediated transduction of the mitochondrial Ant1 gene
To transduce the hearts of Ant1-/- mice, we designed a heart-muscle-directed vector using the mouse Desmin promoter (pDes), Gfp reporter gene, and mouse Ant1 cDNA, the cDNAs separated by a 2A translation interruption sequence39,40, encapsulated within the AAV2/9 coat (AAV2/9-pDes-Gfp-mAnt1) and referred to as AAV-Ant1 hereafter (Fig. 1A). As one control, we generated a second AAV2/9 construct without the Ant1 gene (AAV2/9-pDes-Gfp).

We injected the AAV-Ant1 vector into the pericardium of male and female Ant1+/+ (wild-type, WT); Ant1-/- (Ant1-null); and Ant1-/- + ND6P25L (Ant1-null plus partial mtDNA complex I defect) mice at three days of age. Littermates were injected with PBS as a control for the procedure or with AAV2/9-pDes-Gfp to confirm that the cardiac therapeutic benefit was not due to the vector or GFP expression. AAV-Ant1 and PBS-injected animals were continuously monitored, and their cardiac function was evaluated quarterly for 12–14 months post-injection (Fig. 1B).

At one month post-pericardial injection, Gfp mRNA was detected in the hearts of AAV-Ant1 transduced mice, as well as in the diaphragm and liver. Hence, the heart was transduced but some vectors diffused from the pericardium to other tissues (Fig. 1C, top). To further investigate the distribution of the vector following pericardial delivery, we evaluated the presence of the vector genome by performing digital PCR (dPCR) in the heart, liver, lung, gastrocnemius, and diaphragm tissues isolated from Ant1+/+ AAV-Ant1 transduced mice (Supplementary Fig. 1). This revealed that the AAV vector is present in the liver at similar level to the heart and present at the level ~3 fold less in the lung, diaphragm and gastrocnemius than the heart. The expression of the GFP protein was confirmed by immunohistochemical staining in coronal, anterior, and posterior cardiac sections, with GFP protein being observed in cells dispersed throughout the myocardium (Fig. 1C, bottom).

To confirm the expression of the Ant1 cDNA, mitochondria were isolated from the heart of the AAV-Ant1 transduced mice and tested for ANT1 protein by western blot. ANT1 protein was found in transduced Ant1-/- and Ant1-/- + ND6P25L mice but at a much lower level than found in Ant1+/+ mice, about 10% (Fig. 1D, and Supplementary Fig. 2). While the ANT2 protein was present in the Ant1-/- and Ant1-/- + ND6P25L transduced mice, ANT2 levels were not increased relative to WT indicating that the Ant2 gene was not induced to compensate for the cardiac Ant1 defect (Fig. 1D).

To determine if the ~10% production of Ant1 was due to low expression in all the cells or high expression in a small percentage of cells, we determined the percentage of GFP-positive cardiomyocytes. On average, 20% of the cardiomyocytes were positive for GFP (Fig. 2A, B). Hence, a small percentage of cardiomyocytes were transduced but they expressed high levels of transgene products.

Immunohistochemical staining for GFP in cardiac cells was performed on hearts at 3, 6, 9, and 12–14 months post-transduction. This revealed cardiac GFP at all time points, confirming that the AAV-Ant1 transgenes were stably expressed for over one year, though some variability in expression was noted by 12–14 months (Fig. 2C, D, Fig. 3A, and Supplementary Fig. 3). We found no significant tissue damage generated by the injection procedure, as determined by H&E and Trichrome staining, nor any increase in cell death, as shown via TUNEL staining (Fig. 3B and Supplementary Fig. 4) and cleaved caspase 3 (Supplementary Fig. 5), nor inflammation, as shown via interleukin-18 (IL-18) staining (Supplementary Fig. 6). Pericardial transduction with AAV2/9-pDes-Gfp confirmed GFP histochemical expression without altered cardiac function or toxicity (Supplementary Fig. 3B and Supplementary Table 1).

Amelioration of Ant1-deficient cardiomyopathy by AAV-Ant1 transduction
To determine the effect of AAV-Ant1 transduction on cardiac function, we performed echocardiographic analysis on male and female Ant1+/+; Ant1-/-; and Ant1-/- + ND6P25L mice without and with AAV-Ant1 viral transduction at 3, 6, 9, and 12 months post-pericardial injection. Using the M-Mode echocardiographic measurements of the left ventricle in short and long views, we observed marked alterations in the Ant1-/- and Ant1-/- + ND6P25L hearts relative to Ant1+/+ hearts in the stroke volume, ejection fraction, fractional shortening, cardiac output, left ventricle mass, and the thickness of the anterior and posterior walls. The heart rate of the Ant1-/- and Ant1-/- + ND6P25L mice was slightly higher than that of the Ant1+/+ mice but not statistically significant (Figs. 4A, D and Supplementary Table 1). Differences were found between sexes, with the cardiomyopathy in females being less severe than in males (Fig. 4).

Postnatal periventricular AAV-Ant1 transduction markedly improved the echocardiogram parameters of the age-matched Ant1-/- and Ant1-/- + ND6P25L mouse hearts. The transduced Ant1-/- and Ant1-/- + ND6P25L hearts had increased stroke volume, ejection fraction, fractional shortening, and cardiac output, and decreased left ventricular mass (Fig. 4A), and these beneficial effects were maintained for 12 months (Supplementary Table 1). By 12 months of age, the left ventricular mass of the untransduced Ant1-/- + ND6P25L hearts had increased dramatically (Supplementary Table 1) which was reflected in the heart/body weight ratio which increased linearly with age (Fig. 4B). AAV-Ant1 transduction of the Ant1-/- + ND6P25L hearts returned the 12-month heart mass to that of the transduced Ant1-/- hearts in male mice (Fig. 4B). AAV-Ant1 stabilization of the Ant1-/- + ND6P25L heart/body weight was attributed to changes in heart size since the body weight of all mice in the study increased comparably (Fig. 4C).

The injection process proved safe since the buffer-injected hearts did not exhibit cardiac damage or body weight changes, and the efficacious benefit was due to Ant1 expression since AAV2/9-pDes-Gfp transduction had no apparent benefit (Supplementary Table 1).

Amelioration of the metabolic effects of Ant1 deficiency by AAV-Ant1 transduction
To determine the level of Ant1 gene expression following AAV-Ant1 transduction of the Ant1-/- and Ant1-/- + ND6P25L mouse hearts, we performed bulk RNA-seq analysis on the hearts of Ant1+/+ and non-transduced and AAV-Ant1 transduced Ant1-/- and Ant1-/- + ND6P25L littermates at 8–9 months post-transduction. This revealed that AAV-Ant1 transduction of both Ant1-/- and Ant1-/- + ND6P25L mice produced approximately 10% of the cardiac Ant1+/+ Slc25A4 mRNA (Fig. 5A). Further transcriptome Gene Ontology (GO) pathway analysis revealed that the non-transduced Ant1-/- mouse hearts manifested altered cardiac muscle differentiation, development, and contraction pathways (Fig. 5B) and that the Ant1-/- and Ant1-/- + ND6P25L hearts experienced the downregulation of extracellular matrix (ECM) and upregulation of sarcomere gene mRNAs, indicating changes in all components of the contractile machinery (Fig. 5C, D). These deficiencies were partially mitigated by the restoration of 10% Ant1+/+ of the cardiac Ant1 mRNA levels, showing that relatively little OXPHOS gene expression can have a major beneficial functional effect on cardiac function.

These RNA-seq observations were complemented by mass spectrometry proteomic analysis performed on the hearts of 6-month-old Ant1+/+ and Ant1-/- mice and AAV-Ant1 transduced littermates. The inactivation of Ant1 had profound consequences on the cardiac proteome which were partially mitigated by AAV-Ant1 transduction (Supplementary Fig. 7). Tryptic peptides from ANT1 protein were absent in the Ant1-/- mice but present in Ant1-/- transduced hearts (Fig. 5E). ANT2 (Slc25a5) tryptic peptides were detected in all samples but were slightly downregulated in the Ant1-/- hearts (Fig. 5F). Hence, ANT2 is not induced to compensate for ANT1 deficiency in the heart. Volcano plot quantification of heart proteomes confirmed the absence of ANT1 protein (Slc25a4) in the Ant1-/- hearts (Fig. 5G) and the restoration of ANT1 protein following AAV-Ant1 heart transduction, but at low levels (Fig. 5I).

Relative to Ant1+/+ hearts, Ant1-/- hearts (Fig. 5G) exhibited decreased levels of many mitochondrial complex I proteins (NDUFA6, NDUFA10, NDUFA2, NDUFB8, NDUFA9, NDUFB1, MT-ND5, MT-ND1, and MT-ND4), consistent with ANT1 deficiency being associated with the downregulation of complex I proteins16. Additional downregulated proteins in the Ant1-/- hearts included developmental and myogenic proteins (PPP1CA/B, NYX, FHL2, and SERPINA1D) and immunoglobulin proteins (IGHG1 and IGKC). Upregulated proteins in Ant1-/- hearts included proteins of fatty acid metabolism (ECHDC2 and ACSF2) and some mitochondria-related proteins (DGUOK, ANKRD29, AIFM, ACO2, AK2, and ATAD3), as well as collagen VI proteins (COL6A1, COL6A2, and COL6A3), though most of the ECM genes were downregulated (Fig. 5C and G).

AAV-Ant1 transduction of Ant1-/- hearts resulted in a striking contraction in the variation of protein concentrations relative to Ant1+/+ hearts (compare the Log2 fold change in Fig. 5G versus Fig. 5H and I). AAV-Ant1 partially restored the levels of a variety of mitochondria and functionally related proteins (ATP5F1D, ANKRD29, CAND2, UQCRQ, H1.4, MT-ND5, HNRNPU, DOCK10, ATP5MG, ECHDC2, PSMD8, NYX, GLUD1, HSP90B1, GRIN2B, TMEM143, MYOZ2, ATAD3, AIFM1, LRPPRC, and others), including some complex I protein subunits such as MT-ND5. However, the induction of mitochondrial gene expression by AAV-Ant1 transduction was insufficient to fully reverse the Ant1-/- associated complex I deficiency16. Moreover, upregulation of the collagen VI (COL6A1, COL6A2, and COL6A3) proteins was reversed in the AAV-Ant1 transduced hearts (Fig. 5G versus H). We didn’t observe full rescue of age-related accumulation of somatic mtDNA deletion mutations in AAV-Ant1 transduced Ant1-/- heart either16.

Finally, AAV-Ant1 transduced hearts exhibited no induction of innate immune genes or proteins, even in hearts already expressing normal levels of ANT1 (Supplementary Fig. 7). Hence, the current level of transgene expression is not associated with an adverse immune reaction to the therapy.

Mitigation of ventricular cardiomyocyte defects by AAV-Ant1 transduction
To further define mechanistically how different Ant1-/- cardiac cell types respond following AAV-Ant1 transduction, the Ant1+/+, Ant1-/-, and AAV-Ant1 transduced Ant1-/- (Ant1-/- + AAV-Ant1) hearts were subjected to single-nucleus RNA sequencing (snRNA-seq). This was performed using hearts 1-month post AAV transduction to assess the early effects of Ant1 transduction on mitochondrial OXPHOS and cardiac structure and function, and to avoid secondary outcomes. snRNA-seq clearly annotated the various cardiac cell types by their distinct gene expression profiles, with all three groups of mouse hearts showing concordant cell-type identities and composition (Fig. 6A, Supplementary Fig. 8A, B). Because of its low expression, snRNA-seq could not capture the Ant1 gene expression in the cell nuclei of AAV-Ant1 transduced hearts. Rather, qRT-PCR analysis of the same heart samples confirmed the cardiac Ant1 transgene expression in these mice (Supplementary Fig. 8C).

The impact of the Ant1-/- mutation on different cardiac cell types was determined by comparing the cell-type-specific gene expression between Ant1-/- and Ant1+/+ hearts and between Ant1-/- + AAV-Ant1 and Ant1-/- hearts. Ant1 was highly expressed in all cardiac cell types, as was Ant2, though at a lower level than Ant1 (Supplementary Fig. 9A). Overall, Ant2 expression was comparable between Ant1+/+ and Ant1-/- hearts, confirming that its expression was not increased to compensate for the Ant1 deficiency (Supplementary Fig. 9B).

Changes in gene expression for the eight cardiac cell types revealed the biggest alterations in gene expression between Ant1-/- and Ant1+/+ hearts in ventricular cardiomyocytes (vCM), followed by fibroblasts (Fb), endothelial cells (Endo), pericytes (PC), and macrophages (Mac). Transduction of Ant1-/- cells with AAV-Ant1 had a significant effect on the gene expression of these same cell types (Supplementary Fig. 10). Focusing on vCM, the expression of 395 genes were significantly downregulated, and 33 genes were upregulated in Ant1-/- vCMs compared to Ant1+/+ vCMs (Fig. 6B). Pathway analysis revealed that downregulated genes in Ant1-/- vCMs are all related to mitochondrial bioenergetics and cardiac muscle function (Fig. 6C). These included mitochondrial OXPHOS genes such as ATP synthetase (complex V) Atp5b and Atp5o, and cardiac contractile genes Tpm1 and Myl3. Upregulated genes in Ant1-/- vCMs included Nppa and Nppb, markers of cardiomyopathy (Fig. 6B).

The AAV-Ant1 transgene had a substantial impact on Ant1-/- hearts, particularly in vCMs. Ninety-two genes were significantly upregulated in AAV-Ant1 transduced Ant1-/- vCMs compared to Ant1-/- vCMs (Fig. 6D, Supplementary Fig. 10). Strikingly, pathway analysis revealed that the upregulated genes in transduced Ant1-/- vCMs are virtually all related to mitochondrial bioenergetics and cardiac muscle function (Fig. 6E), demonstrating that AAV-Ant1 was able to reverse the mitochondrial bioenergetic and cardiac contractile defects in Ant1-/- vCMs at the transcriptome level. Importantly, the downregulation of 22 genes in the Ant1-/- vCMs was reversed by AAV-Ant1 transgene (Supplementary Fig. 11). These include the master OXPHOS transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (Pgargc1a)41–43, the complex I N-module structural subunit Ndufa644, as well as the muscle function genes calcium/calmodulin-dependent protein kinase ID (Camk1d), cardiac Z-disc protein (Nexn), and α1-adrenergic receptor (Adra1a) in vCMs (Fig. 6F, Supplementary Fig. 11).

Significant gene expression changes in Ant1-/- mice that were reversed by AAV-Ant1 transduction were also observed in other cardiac cell types, though to a lesser extent (Supplementary Fig. 10 and 12–15). For example, 141 genes were downregulated in Ant1-/- fibroblasts and 17 downregulated genes were reversed by AAV-Ant1 transduction (Supplementary Fig. 12). Comparable degrees of change were seen for endothelial cells (Supplementary Fig. 13), pericytes (Supplementary Fig. 14), and macrophages (Supplementary Fig. 15). All cells lacking ANT1 showed induction of Nppa, and all, but macrophages, showed induction of skeletal muscle α-actin (Acta1). Since the Desmin promoter is expected to limit AAV9-Ant1 transduction to cardiomyocytes only, the fact that other cardiac cell types showed significant gene expression changes suggests cell-cell crosstalk that impacts non-cardiomyocytes. Such cell-cell crosstalk could be mediated by secreted signals or cell surface ligand-receptor signaling.

Methods
Reagents and resource sharing
Further information and requests for resources and reagents should be directed to Douglas C. Wallace (wallaced1@chop.edu)

Experimental model-mouse genetics
All mouse studies are approved by CHOP IACUC. Mice were maintained on the C57Bl/6JEiJ background, which was derived from the JAX C56Bl/6J line into which a functional nicotinamide nucleotide transhydrogenase (Nnt) gene was reintroduced53. Mice without specific nDNA or mtDNA mutations were designated as WT (Ant1+/+). These mice were compared to mice harboring the nDNA Ant1 (Slc25a4)-null mutation gene (Ant1-/- mice). The phenotype of these mice was previously reported on the C56Bl/6J Nnt-/- strain12,13. We transferred the Ant1-/- mutation into the C57Bl/6JEiJ strain (Nnt+/+)54,55 and the C57Bl/6JEiJ Ant1-/- males were crossed with female C57Bl/6JEiJ ND6P25L mice to generate Ant1-/- + ND6P25L mice14. Our Ant1+/+ and Ant1-/- mouse mtDNAs differ from the published C57BL/6J mtDNA56 by the addition of a mtDNA MT-ND5 m.12352C>T (ND5 S204F) missense mutation. The phenotype of these mice has been described16. The Ant1+/+ and Ant1-/- mice were maintained by brother-sister mating. The Ant1-/- + ND6P25L mutant mice were maintained by crossing the Ant1-/- + ND6P25L females with Ant1-/- males. All mouse experimental procedures were approved by the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee (IACUC, IAC 25-000925). Mice were pooled at weaning to attain 3 to 5, genotype-, sex-, and age-matched mice per cage with ad libitum access to food (5LOD diet from PicoLab) and water, on a 12:12 h light-dark cycle. The room temperature range is 68–74 degrees Fahrenheit with a relative humidity of 30–70%. A minimum of 3 subjects per strain was used for all experiments. Additional animals were included, as noted, based on the availability of age- and strain-matched mice and the nature of the assay13. Subjects were randomly numbered during sample preparation to blind the experimenter for subsequent tests and analysis. Besides cage changes and daily health checks, these mice were left undisturbed. When mice were determined moribund by the veterinary staff, they were humanely euthanized, and the date of death was recorded.

Study Design and Pericardial Injections
All measurements were taken from distinct biological samples. The number of biological samples is labeled in the figure legends. Three-day-old female and male mice were pericardially injected with AAV2/9-pDes-Gfp-mAnt1 or AAV2/9-pDes-Gfp vectors at a dose of 2.5×1011 vg per mouse, diluted in PBS to a final volume of 20 μl. Control littermates were injected with PBS. Pericardial injection was performed without ultrasound guidance57,58. Briefly, three-day-old pups were anesthetized by cooling on paper on top of ice, then carefully injected with the virus or PBS using a Hamilton Gastight 1710 syringe with the needle entering right beneath the edge of the sternum. The needle was inserted in the trans-diaphragm direction towards the heart at a ~30° angle to the handling table surface, and through the chest wall on the left border of the sternum at the axillary level57. A plastic tube was put on the needle such that only ~2–3 mm of the needle enters the chest. The pups were allowed to recover from anesthesia and monitored at 1-, 3-, 6-, 9-, and 12-month post-injection.

Production of adeno-associated virus (AAV)
The Gfp reporter cDNA and mouse Ant1 cDNA were cloned into a bicistronic vector separated by the 2A translational interruption sequence39,40, transcribed from the mouse Desmin promoter. The plasmid was produced by VectorBuilder Inc., USA (https://en.vectorbuilder.com/) and sequenced to confirm the correct insertion of Ant1. The AAV (serotype 2/9) plasmids were packaged at the Children’s Hospital Vector Core with titers from 1.5×1013 vg/ml to 4.9×1013 vg/ml.

Western blotting
Mice were sacrificed by cervical dislocation, and heart tissues for western blot analysis were isolated, rinsed, and homogenized in ice-cold medium (isolation buffer) containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, and 0.1% bovine serum albumin (BSA) (pH adjusted to 7.2 with Trizma® base) using a glass homogenizer with six slow strokes of a Teflon pestle rotating at 600 rotations per minute. The homogenate was centrifuged at 700×g for 5 to 10 min to pellet nuclei and unbroken cells. The resulting supernatant was centrifuged at 8500×g for 10 min to isolate mitochondria. The final pellet was resuspended in the isolation buffer devoid of BSA, and protein concentrations were quantified using the Bradford method. Lysates containing 20 µg of protein were subjected to electrophoresis on 4–12% Bis-Tris SDS-polyacrylamide NuPAGE™ gels (Invitrogen). The separated proteins were transferred onto a nitrocellulose membrane using the iBlot Gel Transfer System (Invitrogen). Following this, the membranes were blocked for one hour in a 5% nonfat milk solution in TBST buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 0.1% Tween® 20). The membranes were incubated overnight at 4°C with shaking in primary isoform-specific antibodies (anti-Ant1 or Ant2)12, diluted to 1:1000 in TBS. Following three washes with TBST, the membranes were exposed to secondary peroxidase-labeled antibodies for 2h at room temperature. Finally, immune-reactive bands were visualized using an Enhanced Chemiluminescence reagent and detected via autoradiography. To estimate the relative level of ANT1 protein generated by transduction (Supplementary Fig. 2), the membranes were incubated overnight at 4°C, shaking in primary isoform-specific antibodies (anti-Ant1)12, diluted to 1:500 in TBS. For porin detection, anti-VDAC1/Porin primary antibody (Abcam, ab14734; 1:5000) was used. Following three washes with TBST, the membranes were exposed to secondary antibody for 2h at room temperature. The relative level of protein fluorescence was quantified using the Licor system.

RNA isolation, PCR, RNA library, and sequencing
For RNA isolation and gene expression analyses58,59, mice were sacrificed by cervical dislocation, and tissues for RNA sequencing analysis were isolated and flash-frozen in liquid nitrogen. Then, total RNA was extracted in TRIzol™ Reagent (Thermo Fisher Scientific, 15596026). Purified RNA was assessed by NanoDrop spectrophotometer (Thermo Fisher Scientific) for a 260/280 ratio and quantified by Qubit Flex (Thermo Fisher Scientific) using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Q32855).

PCR Protocol: cDNA synthesis was generated from total RNA using the SuperScript IV Reverse Transcription kit (Thermo Fisher Scientific, 18090010). PCR amplification was conducted on a PCR Thermocycler (Eppendorf Mastercycler) with specific primers: EGFP-N-Reverse (5′-CGTCGCCGTCCAGCTCGACCA) with an annealing temperature of 66°C and EGFP-C-Forward (5′–CATGGTCCTGCTGGAGTTCGTG) with an annealing temperature of 61°C. The PCR cycling conditions comprised initial activation at 50°C for 2 min, polymerase activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 61°C for 30 s. The resulting cDNA was separated and visualized via DNA gel electrophoresis.

RNA sequencing: The RNA was diluted to 1.43 ng/μl and reverse transcribed into cDNA using the Ion Torrent™ NGS Reverse Transcription Kit (Cat. No. A45003). The RNA library was prepared using the Ion Torrent S5 Chef and Ion AmpliSeq Kit for Chef DL8 (Thermo Fisher Scientific, A29024) and sequenced using the Ion Torrent S5 Sequencer and Ion 540 Kit (Thermo Fisher Scientific, A30011). The raw data was normalized in R (version 4.2.2) using the “DESeq2” (version 1.38.3) package. Volcano plots were generated in R using the “EnhancedVolcano” (version 1.16.0) package. GO data was generated using “clusterProfiler” (version 4.6.2), “AnnotationDbi” (version 1.60.1), and “org.Mm.eg.db” (version 3.16.0) packages in R. GO plots were generated in GraphPad Prism 9.

Total DNA Isolation and cellular transgene levels determined by digital PCR
Total DNA was isolated from tissue samples collected from various organs using the Qiagen DNeasy Blood & Tissue Kit. Tissues were homogenized in lysis buffer and digested with Proteinase K, followed by DNA purification using silica-based spin columns. In addition to the organ-derived samples, C2C12, a non-transduced cell line, served as the negative control. Genomic DNA from the C2C12 cell line was extracted using the DNeasy Blood & Tissue Kit. DNA concentration and purity for all samples were measured using a Qubit fluorometer (Thermo Fisher Scientific), and samples were diluted to a uniform concentration prior to downstream analysis. To quantify the viral genomes in the transduced mouse tissues, Gfp in the viral vector genome and Gapdh in the cell nuclear genome were quantified by digital PCR (dPCR) performed using the QuantStudio™ AbsoluteQ™ dPCR System. Each 10 μL reaction mixture consisted of 2 μL of 5× Absolute Q Digital PCR Master Mix, 0.5 μL of FAM- or VIC-labeled Applied Biosystems TaqMan probe for GFP and GAPDH, respectively. 1 μL of 0.05 and 0.5 ng of genomic DNA template for eGFP and GAPDH, respectively, with nuclease-free water added to reach the final volume. Reactions were loaded onto Absolute Q dPCR plates and placed into the Absolute Q instrument. Following amplification, the Absolute Q Analysis Software was used to obtain absolute quantification results for each target. Copy number per microliter (copies/μL) for GAPDH and eGFP was directly exported from the software output and analyzed in Microsoft Excel. Vector genomes per diploid genome equivalent were calculated as GFP/GAPDH x 2.

Mass spectrometry
Mice were sacrificed by cervical dislocation, and tissues for RNA sequencing analysis were isolated and flash-frozen in liquid nitrogen. Flash-frozen mouse hearts were resuspended in 100 mM ammonium bicarbonate and 8 M urea at a ratio of 1:5 g:mL. Hearts were homogenized using an Omni TH, centrifuged at 20,000×g for 5 min at 4°C, and then a protein assay using BC. Fifty µg of protein were diluted to 50 µL in 100 mM ammonium bicarbonate, and dithiothreitol (DTT) was added to a concentration of 10 mM DTT. Proteins were incubated in DTT at 37°C for 45 min, and then cysteines were alkylated by adding 5.5 µL of 0.5 M iodoacetamide in the dark and incubating at RT. Ammonium bicarbonate (50 mM) was used to dilute samples to 150 µL, and proteins were digested by adding 2 µg of Sequencing Grade Modified Trypsin (Promega) and incubating overnight at 37°C. Samples were acidified to 0.1% trifluoroacetic acid (TFA) and desalted using C18 stage tips: tips were conditioned using acetonitrile (ACN) and then 0.1% formic acid (FA). Samples were then loaded and washed with 0.1% FA and eluted in 0.1% FA in 60% ACN. Peptides were dried in a Savant SpeedVac and then resuspended in 20 µL of 0.1% TFA. A NanoDrop (Thermo Fisher Scientific) was used to measure absorbance at 280 nm to normalize injection volumes. Proteomics was performed using a Dionex UltiMate 3000 nanoLC and a Q Exactive HF (Thermo Fisher Scientific). The solvent system was comprised of buffer A (0.1% FA) and buffer B (0.1% FA in 80% ACN). A C18 trap column (Thermo Fisher Scientific) and an in-house packed C18 (Dr. Maisch, GMBH) analytical column were used. The analytical gradient was run at 300 nL/min with 5–25% buffer B over 90 min and then 25–45% buffer B over 30 min. Data independent acquisition (DIA) was used with 24 m/z MS2 windows. Data were searched with DIA-NN using a complete UniProt FASTA digest library-free search without heuristic protein inference and processed using MSstats. Mass spec data and additional experimental details are deposited in PRIDE.

Heart/body weight ratio and histology
Mice were sacrificed by cervical dislocation, and heart tissues were collected for histological analysis and rinsed, and fixed in 10% formalin-neutral buffer for 48 h and processed by Histowiz Inc. (https://home.histowiz.com/). Before proceeding with the fixation process, each mouse and the excised mouse heart were weighed, and the heart/body weight ratio was determined.

Evaluation of the number of transduced cells. To assess the number of GFP-positive cells, we analyzed heart sections from 3 Ant1+/+ mice 1-month post AAV-Ant1 injection. Three sections from each heart representing different anatomical planes (coronal, anterior, and posterior) were analyzed, with 5 random fields analyzed per section for each of 3 hearts, a total of 5×3×3=45 random fields. We then performed Wheat Germ Agglutinin (WGA, conjugated with red fluorescence) staining to label cell plasma membranes to locate each cell, and GFP-positive cells were visible on the same section/field. We set the minimal area (size) threshold to exclude non-cardiomyocytes. We then counted the total number of cardiomyocytes and GFP-positive cardiomyocytes in the field and calculated the % of GFP-positive cells manually. A representative picture and quantification of % GFP-positive cells are represented in Fig. 2A, B. We found that, on average, 20% of the cardiomyocytes express GFP.

Evaluation of the time course of cardiac Gfp expression. To establish the time course of expression of the GFP transgene, we used immunofluorescence of GFP (antibody: Abcam #183734) to determine the extent of GFP-positive area relative to the total area of heart sections. Whole-slide images were captured using identical scanner settings. We used FIJI to quantify the images. Background brightness was stable at 240–244 (8-bit), and GFP-negative tissue appeared at 214–230 across all slides. Ventricular myocardium regions of interest were manually outlined (Fig. 2C, D), and pixels were classified as DAB-positive if saturation was ≥5 and brightness was ≤201. Multiple representative slides were analyzed to distinguish valid DAB signals from GFP-negative areas, thus minimizing false positives in controls. Automatic thresholding was avoided because it recalculates the cutoff based on each image’s histogram, causing the thresholds to shift with variations in stain amount and background, leading to inconsistent performance across sample types in our tests. Fixed thresholds were applied uniformly to all slides in batch mode, and the percentage of DAB-positive area was calculated for each region of interest. This allowed us to evaluate samples injected with AAV2/9-pDes-Gfp-mAnt1 for each genotype (WT=Ant1+/+, Ant1-/-, and Ant1-/- + ND6P25L) at each time point (1, 3, 6, 9, and 12 months). The analysis confirmed the stability of the transduction until 12 months after injection, at which point appreciable downregulation was observed.

Evaluation of cardiac pathology and inflammation. To detect and quantify any AAV-Ant1 transduction-induced pathology of the hearts, coronal longitudinal sections of paraffin-embedded tissues were sliced (samples were trisected to have three coronal views per sample) and stained for H&E, Trichrome, and TUNEL. At least two animals per genotype (Ant1+/+, Ant1-/-, and Ant1-/- + ND6P25L) were histologically evaluated at each time point (3, 6, 9, and 12 months) for each of the three genotypes. There was no sign of tissue damage or increased cell death (Fig. 3B and Supplementary Figs. 3–6). Approximately 30 mice were studied. IHC for GFP was performed using the Abcam #183734 antibody. Heart sections from 3-month-old PBS-injected or AAV-Ant1 transduced mice (n=12 for all treatments and genotypes) were analyzed and stained for cleaved caspase 3 (antibody: Cell Signaling #9661S) and IL-18 (antibody: Abcam #71495).

Echocardiography
Echocardiography was performed using a VEVO 3100 and an MS550D transducer (VisualSonics)60. Mice were anesthetized with 2% isoflurane in 80% O2, which was lowered to 1.5% during the procedure. The heart rate was targeted to 400-450 bpm for the imaging. The average anesthesia length was ~10 min per mouse. First, B-mode images of the parasternal long-axis view were taken, followed by M-mode images, and by B- and M-mode of the parasternal short-axis view. For analysis, the LV trace function of VEVOLAB 3.2.6 (VisualSonics) was used on the M-mode images of the parasternal short and long-axis views. Analysis of short and long views provided comparable results, and only short-view data have been included in the manuscript.

snRNA-seq and data analysis
snRNA-seq and data analysis61,62 involved Isolated nuclei processed using the Chromium Single Cell 3ʹ Reagent V3.1 kit from 10X Genomics. A total of 8000 nuclei per sample were loaded into a Chip G. Barcoding, cDNA amplification, and library construction were performed according to the Chromium 3ʹ V3.1 protocol. Sequencing was performed on a DNBSEQ-T7 platform targeting 20,000 reads per nucleus. Raw data was aligned to the reference GRCm38 genome using the Cell Ranger V7.2.0 software (10X Genomics) according to the 10X Genomics’ instructions. The snRNA-seq data of each sample were processed using Seurat (v5). Nuclei were filtered for mitochondrial reads <10%, nFeature_RNA>800, and nCount_RNA<10,000, and three datasets were integrated using Harmony63. The cluster UMAP, violin plots, feature plots, and volcano plots were generated61,62. We used clusterProfiler64 to perform GO pathway analysis.

Statistical analysis
The ultrasound data and the heart/body weight ratio were analyzed using Prism 6.0 (GraphPad, La Jolla, CA, USA), and samples were compared between two conditions at a time using the two-tailed t-test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This novel study explored the interplay of the mitochondrial genetic systems in PMD-mediated cardiomyopathy (CM). AAV-mediated gene therapy targeting the deficient nuclear gene was investigated in two models of PMD-CM. The study was intriguing in many ways. The interaction between pathogenic nuclear and mitochondrial DNA variants leading to a change in CM phenotype (hypertrophic vs dilated) was noteworthy. Also noteworthy was the very low threshold for phenotypic correction for gene therapy in the context of PMD-mediated CM.\nComments:\nRegarding gene therapy, the extent of in vivo vector transduction is undefined. A 10 % increase in ANT1 transcript is helpful but insufficient in quantifying transduction. A 10% signal increase from 1% of cells is different to 1% signal from 10% of cells. Either of these results merits further consideration about the mechanisms of disease correction at such low levels of transduction. This is important in defining the actual threshold for therapeutic effect and has downstream implications if this strategy is to be pursued as a therapeutic option. I suggest the authors quantify the percentage of GFP positive cells in a reasonable sample size. This could be done as a time course study but would be most relevant at the final time point.\nFurthermore, complimentary data about transduction efficiency ought to be sought using PCR (quantitative or digital) for vector genomes. This ought to be done to determine biodistribution of the vector following pericardial delivery, particularly for unintended liver transduction.\nFurthermore, the very low level of transduction is unanticipated and requires some reflection. A dose of 2.5 x10e11 vg/mouse was given. This amounts to 1.25 x 10e14 vg/kg assuming 3 day old pups weigh 2g. This is a very high dose of AAV2/9 and a high-level cardiac transduction is usually achieved with systemic vector delivery at this dose. It is confusing as to why this has not been achieved and what has been the biodistribution following pericardial delivery. This needs to be explored further in biodistribution studies.\nThe method of vector delivery is atypical for gene replacement approaches. Neonatal pericardial delivery of AAV is not used in any of the currently approved AAV gene therapy clinical trials for cardiac indications. These protocols employ systemic delivery (intravenous) or intracoronary injection of vector. The authors need to provide the method for pericardial delivery (currently not included in the methods section) and a justification for this approach. I note that gene-editing was not the molecular strategy employed, a common reason for neonatal vector injection. Given the translational emphasis of this study, discussion is required as to how pericardial vector delivery might play out for patients receiving this therapy. Furthermore, the question of whether neonatal pericardial delivery is necessary to achieve the observed therapeutic effects also needs to be addressed. To my mind a much higher level of transduction could have been achieved with a less invasive delivery method.\nAbstract:\nWhat is meant by – “To perfect strategies for addressing”?\nIntroduction:\n“Between 20 and 40% of PMD are associated with cardiomyopathy, and 71% of PMD children with cardiomyopathy die while only 26% of PMD children without cardiomyopathy die.” Could these mortality rates be placed in a context? Are these rates within a certain time frame?\n“Electrocardiographic studies revealed abnormal contractile mechanics…” Did the authors intend echocardiographic studies?\n“Currently, there is no approved therapy for PMD-associated cardiomyopathy,” Is this true? Can these patients be treated with conventional heart failure therapies such as anti-failure medications, ICD or CRT therapy and heart transplantation?\nFigure 1 (and supplement)\nAs discussed above, the extent of vector transduction needs to be quantified and shown as group data. The transduction biological variability is important to quantify. Toward this end the authors need to provide more GFP and ANT1 westerns demonstrating transduction.\nFingure 2 (and supplement)\nAlthough no differences in inflammation, fibrosis or cell death can be seen in the images provided, the authors need to quantify and present the group data regarding these variables to conclude that the vector did not affect these variables.\nFigure 3\nThe method for pericardial vector injection needs to be provided. Was there any acute morbidity or mortality during vector injection?\nFigure 4\nWhat is the sample size for the RNAseq and mass spectrometry data?\nFigure 5\n“The fact that all cardiac cell types responded to the AAV-ANT1 transduction suggests that either all cells were transduced by the virus or that vCM transduction is functionally coupled to the gene expression of the other cardiac cell types.” This statement requires more exploration. The extent of transduction needs to be quantified far more accurately to enable conclusions to be made about the phenotypic effects of transduction. If there is low level transduction, as suspected, then the authors need to elaborate on the functional coupling resulting in gene expression changes across most other cardiac cells. This kind of bystander effect on gene expression has not been proven in the current study.\nDiscussion\nCould the authors provide precedents for the “sharp threshold effect” in other studies of mitochondrial bioenergetics of relevance do this study? A more in-depth analysis is required to assist readers in understanding the connection between a low transduction event and the observed phenotypic correction.\nCould the authors provide a more in-depth discussion about the pros and cons of pericardial vector inject for gene therapy of cardiac disease?", "reviewer_2": "The authors present a study where they investigate the impact of restoring partial expression of a heart-muscle isoform of Ant (Ant1) in previously established Ant-/- and Ant-/- + ND6P25L mouse PMD cardiomyopathy models. Using a gene therapy approach (AAV2/9), the authors show that after transduction of ANT1 cDNA at P3 into the pericardium, they observe a mild increase in the expression of Ant1, which is sufficient to mitigate the cardiomyopathy up to 12 months old and they demonstrate the reversal of changes in RNAseq (9 months), proteomics (6 months) and with single nucleus sequencing (1 month) post-transduction. The authors suggest that gene therapy could be used to lower the disease severity of patients with more severe forms of ANT-/- associated disease.\nThere is growing evidence that digenic variants influence disease manifestation or severity of many mitochondrial diseases. And the ability to mitigate these effects is so far largely unexplored. Here, the demonstration that even a mild change in expression of a disease gene can partially reduce the severe disease phenotype indicates not only a treatment option for the disease in question, but that modest molecular changes may be sufficient to change the pathology of PMDs.\nMajor points:\n1. In the author’s previous publication, McManus, M.J. et al 2019, they performed characterisation of the two mouse models used in this study. It was established, that the increased severity of the cardiomyopathy in the ANT-/- + ND6P25L model was also accompanied with, amongst other factors, impaired complex I activity, increased somatic mtDNA mutation levels and a shortened lifespan. The authors show that gene therapy can ameliorate the cardiomyopathy phenotype but it would be nice to know whether previously reported abberrations also are impacted, e.g., complex I activity and mtDNA mutation (deletion) levels.\n2. The authors display data from the AAV-Ant1 or PBS injected groups, but they also state that they performed injections with a second control construct lacking the ANT1 cDNA, why was this not shown alongside the other two conditions?\n3. Line 106 &107 – there is no data for the gastrocnemius, this should be included to support this statement.\n4. In supplementary figure 4 A there appears to be a loss of vCMs in the transduced hearts compared to both WT and untreated mutant (from the UMAPs), while the other cell types seem constant across conditions. What is the cause of this? Does ANT cDNA transduction cause a loss of vCMs?\nMinor points\n1. Further, I think it is interesting that Dguok is elevated in both the KO vs WT and rescue vs WT (figures 4G & I), indicating that perhaps there is a demand for mitochondrial purine nucleotide salvage that the treatment does not mitigate. The authors should explore this aspect of the model rather than just focusing on OMICs changes.\n2. What was the rational for snRNA-seq over scRNA-seq?\n3. To simplify for readers, it would be nice to provide a heatmap of at least some of the protemics data in Fig. 4G-I. Perhaps as a supplementary figure.\n4. Line 45 – 46 – Why is ANT classified as a nDNA OXPHOS gene? This is misleading because it belongs to the transporter family.\n5. Figure 3 - It looks like the colour of the Ant1-/- + ND6 is different between the legend and the data points.\n6. Figure 3B – use black stars to show significance to be consistent.\n7. Figure 3B&C – The standard is to use g for grams.", "reviewer_3": "This manuscript presents a detailed evaluation of AAV2/9-based gene replacement therapy for primary mitochondrial disease manifesting dilated cardiomyopathy caused by a null mutation in the heart muscle isoform of the ANT1 gene. The authors generated an Ant1 null mouse model and combined it with an already generated ND6 P25L mtDNA mutation to increase the severity of the dilated cardiomyopathy. The experimental approach to evaluate the efficacy of the gene-based therapeutic approach is well-designed. They subsequently demonstrated partial restoration of the mitochondrial dysfunction by AAV-ANT1(Slc25a4) transduction, which protects against dilated cardiomyopathy in Slc25a4-/- plus mt DNA mutant.\nThe experimental approach to evaluate the efficacy of the gene-based therapeutic approach is well-designed and sound. However, several areas require improvement before the manuscript can be considered for publication.\n(1) Can the authors please elaborate on the chosen dosage? How was it determined? Could the level of restoration be enhanced with a higher dosage?\n(2) How did they ensure minimal damages when they injected 3 day-old pups pericardially?\n(3) While partial restoration of (10%) ANT1 expression in the Ant1-/- and Ant1-/- + mtDNA ND6P25L mouse models led to significant improvement in cardiac pathology by molecular and functional assessment, it is important to discuss whether this level of restoration is adequate for clinical translation, given the patient's null mutation. Additionally, the potential translational relevance of the findings, including limitations and challenges in moving toward clinical application, should be addressed in the discussion.\n(4) The introduction lacks a clear statement of how combining ND6 P25L mtDNA mutation with the ANT-/- null mouse model could lead to dilated cardiomyopathy.\n(5) The Authors believes relatively small improvement in OXPHOS gene expression can have major beneficial functional effects. It is recommended to conduct additional mitochondrial functional assessments such as mito respiration and complex activity assays to validate these findings.\n(6) Figure 3 needs to be re-done. The symbols chosen were confusing.\nMinor issues:\n“Trichome” was mis-spelled in Supplementary Fig. 1\nLine 78-79: Ant1 deficiency in muscle is associated with a massive accumulation of abnormal mitochondrial : Did they mean abnormal mitochondria or mitochondrial components (please specify).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We want to thank the Reviewers for taking time to review our manuscript and providing their constructive comments and insightful suggestions. We have addressed every comment with additional experiments, analyses, and/or textual changes. We believe that these changes significantly strengthen the manuscript. Below, we address the Reviewers’ comments point-by-point. The original Reviewers’ comments are in black font and our responses are in blue font. Similarly, additions and alterations to the manuscript addressing the reviewer comments and suggestions are tracked in blue fonts, with the specific lines in the text referred to in our responses to the Reviewer comments for ease of assessing the changes made.\nReviewer #1\nThis novel study explored the interplay of the mitochondrial genetic systems in PMD-mediated cardiomyopathy (CM). AAV-mediated gene therapy targeting the deficient nuclear gene was investigated in two models of PMD-CM. The study was intriguing in many ways. The interaction between pathogenic nuclear and mitochondrial DNA variants leading to a change in CM phenotype (hypertrophic vs dilated) was noteworthy. Also noteworthy was the very low threshold for phenotypic correction for gene therapy in the context of PMD-mediated CM.\nResponse: We thank the Reviewer #1 for these supportive comments. Following Reviewer #1’s suggestions below, we have addressed each with new experiments and textual changes.\nComments:\nRegarding gene therapy, the extent of in vivo vector transduction is undefined. A 10 % increase in ANT1 transcript is helpful but insufficient in quantifying transduction. A 10% signal increase from 1% of cells is different to 1% signal from 10% of cells. Either of these results merits further consideration about the mechanisms of disease correction at such low levels of transduction. This is important in defining the actual threshold for therapeutic effect and has downstream implications if this strategy is to be pursued as a therapeutic option. I suggest the authors quantify the percentage of GFP-positive cells in a reasonable sample size. This could be done as a time course study but would be most relevant at the final time point.\nResponse #1: As suggested by the Reviewer, we have quantified the number of GFP-positive cells and assessed the time course of retention of GFP expression. The results are presented in a new Supplementary Figure 3 and in the Methods under “Heart/Body Weight Ratio and Histology” (line 496-529).\nEvaluation of the number of transduced cells: To assess the number of GFP-positive cells, we analyzed heart sections from 3 Ant1+/+ mice 1 month post AAV-Ant1 injection. 3 sections from each heart representing different anatomical planes (coronal, anterior, and posterior) were analyzed, with 5 random fields analysed per section for each of 3 hearts, a total of 5x3x3=45 random fields. We then performed Wheat Germ Agglutinin (WGA, conjugated with red fluorescence) staining to label cell plasma membranes to locate each cell, and GFP-positive cells were visible as green fluorescence in the same section/field. We set the minimal area (size) threshold to exclude non-cardiomyocytes. We then counted total number of cardiomyocytes and GFP-positive cardiomyocytes in the field and calculated the % of GFP-positive cells manually. A representative picture and quantification of %GFP-positive cells are resented in Supplementary Fig. 3A-B and also shown below. We found that on average 20% of the cardiomyocytes express GFP (line 501-511). Hence, we can conclude that the ~10% expression of Ant1 protein is due to a small proportion of the cardiomyocytes being transduced with each expressing on average ~50% of normal level of ANT1 protein. This is reported in the text in line 125-129.\nEvaluation of the time course of cardiac Gfp expression: To establish the time course of expression of the GFP transgene, we used immunofluorescence of GFP to determine the extent of GFP-positive area relative to the total area of heart sections. Since we don’t have remaining sections for most time points to perform WGA staining to quantify % of GFP-positive cells as done above, we instead quantified GFP-positive area using existing GFP-stained sections. Whole-slide images were captured using identical scanner settings. We used FIJI to quantify the images. Background brightness was stable at 240-244 (8-bit), and GFP-negative tissue appeared at 214-230 across all slides. Ventricular myocardium regions for interest were manually outlined (new Supplementary Fig. 3C), and pixels were classified as DAB-positive if saturation was ≥ 5 and brightness was ≤ 201. Multiple representative slides were analysed to distinguish valid DAB signals from GFP-negative areas, thus minimizing false positives in controls. Automatic thresholding was avoided because it recalculates the cutoff based on each image's histogram, causing the thresholds to shift with variations in stain amount and background, leading to inconsistent performance across sample types in our tests. Fixed thresholds were applied uniformly to all slides in batch mode, and the percentage of DAB-positive area was calculated for each region of interest. This allowed us to evaluate samples injected with AAV2/9-pDes-Gfp-mAnt1 for each genotype (WT = Ant1+/+, Ant1-/-, and Ant1-/-+ND6P25L) at each time point (1, 3, 6, 9, and 12 months). These results are presented in the new Supplementary Fig. 3D and also shown below. The analysis confirmed the stability of the transduction until 12 months after injection, at which point appreciable downregulation was observed (line 512-529).\nWe modified text to describe these results: “Immunohistochemical staining of GFP for cardiac cells was performed for hearts at 3, 6, 9, and 12-14 months post-transduction. This revealed cardiac GFP at all time points, confirming that the AAV-Ant1 transgenes were stably expressed for over one year, though some variability in expression was noted by 12-14 months (Fig. 2A, Supplementary Fig. 3C-D and Supplementary Fig. 4)” (line 130-134).\nFurthermore, complementary data about transduction efficiency ought to be sought using PCR (quantitative or digital) for vector genomes. This ought to be done to determine biodistribution of the vector following pericardial delivery, particularly for unintended liver transduction.\nResponse #2: As suggested, we performed dPCR to quantify vector genomes from samples isolated from three 6-month-old Ant1+/+ mice injected with AAV-Ant1. We assessed the presence of the vector genome by measuring the Gfp DNA copy number in the vector genome normalized to Gapdh copy number in the mouse nuclear genome in the heart, liver, lung, gastrocnemius, and diaphragm. C2C12 cells were used as a negative control. The results showed that the AAV vector is present in the liver at similar level to the heart and present at the level ~3 fold less in the lung, diaphragm and gastrocnemius than the heart (new Supplementary Fig. 1, also shown below). It is well known that AAV9 vectors are not completely heart specific and often results in significant unintended liver transduction. For example, a mouse study showed that tail vein-injected AAV9-CMV vectors are abundant in the liver, heart, muscle, and pancreas, with the liver cell containing 15-100 fold more AAV9 vector genomes per diploid genomic equivalent1. Our results showed a much more favourable distribution in the heart (about equal to liver), likely due to (1) pericardial injection and (2) the use of pDesmin promoter. We have modified the text to include a description of these new results (line 107-117) and provided experimental details in the Methods section under “Total DNA Isolation and cellular transgene levels determined by digital PCR” (line 451-469).\nFurthermore, the very low level of transduction is unanticipated and requires some reflection. A dose of 2.5 x10e11 vg/mouse was given. This amounts to 1.25 x 10e14 vg/kg assuming 3 day old pups weigh 2g. This is a very high dose of AAV2/9 and a high-level cardiac transduction is usually achieved with systemic vector delivery at this dose. It is confusing as to why this has not been achieved and what has been the biodistribution following pericardial delivery. This needs to be explored further in biodistribution studies.\nResponse #3: We thank the reviewer for raising this point. The goal of this research is to demonstrate, as a proof of principle, that we could intervene with gene therapy to correct mitochondrial cardiomyopathy without significant toxicity. That is why we used this relatively high dose of AAV. Of note, it is within the range of doses used for clinical AAV gene therapy trials, which according to a recent meta-analysis is between ~1010 to 1017 vg per patient, or ~108 to 1015 vg/kg assuming body weight of 100kg1. To move this proof of principle study forward closer to clinical applications, we are currently testing varying doses and delivery methods, other AAV vectors such as self-complementary AAV and myoAAV, and broader assessments of potential side effects; however, this is beyond the scope of the current manuscript.\nAlthough this AAV dose achieved only ~10% ANT1 protein compared to levels seen in the WT mouse hearts, it produced significant functional consequences by alleviating cardiomyopathy in Ant1-/- mice. As detailed in our response #1 and #2 above, this AAV dose and pericardial injection transduced ~20% of cardiomyocytes and favoured the transduction of the heart over other tissues.\nWe added details of the viral dosage and administration in the Methods under “Study design and pericardial injection” (line 379-390). We significantly expanded our discussion of the AAV vector and delivery strategy and results in the Discussion (line 275-322).\nThe method of vector delivery is atypical for gene replacement approaches. Neonatal pericardial delivery of AAV is not used in any of the currently approved AAV gene therapy clinical trials for cardiac indications. These protocols employ systemic delivery (intravenous) or intracoronary injection of vector. The authors need to provide the method for pericardial delivery (currently not included in the methods section) and a justification for this approach.\nResponse #4: We thank the reviewer for this important point. The detailed the pericardial injection procedure is in the Methods section “Study Design and Pericardial Injections” (line 379-390). We expanded our discussion of the AAV vector and delivery strategy and results in the Discussion (line 275-322).\nI note that gene-editing was not the molecular strategy employed, a common reason for neonatal vector injection. Given the translational emphasis of this study, discussion is required as to how pericardial vector delivery might play out for patients receiving this therapy. Furthermore, the question of whether neonatal pericardial delivery is necessary to achieve the observed therapeutic effects also needs to be addressed. To my mind a much higher level of transduction could have been achieved with a less invasive delivery method.\nResponse #5: There are two aspects of this concern that we have addressed. First is the relative benefit of gene editing versus gene transduction, and second is the reason we employed pericardial transduction rather than another vector delivery approach. AAV-gene transduction is preferable to gene editing because gene editing changes only one mutation site at a time while AAV-transduction can treat any recessive loss-of-function mutation in the gene. Since recessive mutations can occur at many different nucleotides in a PMD genes, the same AAV-gene vector can treat them all. This is covered in the Discussion (line 275-322).\nThe reason we used pericardial injection in this study was to specifically determine if AAV-Ant1 transduction was both efficacious and safe for treating PMD cardiomyopathy. By injecting the pericardium, we have established that AAV-Ant1 is able to correct the cardiac Ant1-null defect without adverse effects. Since most PMD cardiomyopathy patients also present with myopathy, as is the case for ANT1-null mutation patients2, we will ultimately need to treat both the heart and muscle with the same vector. To achieve this objective, we previously demonstrated that AAV-Ant1 transduction can efficiently and safely cure the myopathy of our Ant1-/- mouse3. This muscle study showed that a modest expression of Ant1 resulted in a marked increase in muscle ATP levels and the rapid removal of the massive accumulation of abnormal mitochondria, the product of Ant1 requirement for successful PINK-Parkin pathway of mitophagy4. With the current paper, we have shown that targeted treatment of both the muscle and the heart with AAV-Ant1 transduction is both safe and effective. By employing the Desmin promoter to drive expression of the Ant1 cDNA in both heart and muscle, we plan to proceed with systemic AAV-Ant1 transduction of the Ant1-/- and Ant1-/-+ND6P25L myopathy and cardiomyopathy. We have summarized the logic of our approach to first muscle-specific transduction and the pericardial heart-specific transduction to our ultimate goal of combined systemic muscle and heart treatment of patients with myopathy and cardiomyopathy using systemic self-complementary AAV or MyoAAV, now presented in the Discussion (line 275-311).\nAbstract:\nWhat does “To perfect strategies for addressing\" mean?\nResponse #6: We meant “To optimize strategies for addressing mitochondrial cardiomyopathy”, and we modified the abstract accordingly (line 24-25).\nIntroduction:\n“Between 20 and 40% of PMD are associated with cardiomyopathy, and 71% of PMD children with cardiomyopathy die while only 26% of PMD children without cardiomyopathy die.” Could these mortality rates be placed in a context? Are these rates within a certain time frame?\nResponse #7: These numbers are based on studies from 1984 to 20195-9, and encompass ~2,000 pediatric patients with PMD and cardiomyopathy, carrying nDNA or mtDNA mutations. The onset of symptomatic mitochondrial disease ranged from birth to 10 years of age, and cardiomyopathy was diagnosed between birth and 27 years of age, occurring in 20-30% of patients with mitochondrial disease. The main symptoms were increased left ventricular posterior wall and septal thickness, left ventricular hypertrophy, decreased fractional shortening, atrioventricular block, and sinus node dysfunction. Most patients with cardiomyopathy developed heart failure, underwent resuscitated cardiac arrest, third-degree atrioventricular block, sinus node dysfunction, heart transplantation, or hospitalization for heart failure management, and most subsequently died. After a follow-up of 3 to 7 years, the overall survival rate was significantly lower in patients with cardiomyopathy than in those without. The 10-year mortality rate according to the Kaplan-Meier scale in children with mitochondrial disease was higher in patients with cardiomyopathy (67-71%) than in those without (18-26%).\nWe have summarized this data in line 41-43 which state: “20 to 40% of PMD patients develop cardiomyopathy between birth and 27 years of age5-9, with the ten year mortality being 67-71% for PMD children with cardiomyopathy versus 18-26% for PMD children without cardiomyopathy7”.\n“Electrocardiographic studies revealed abnormal contractile mechanics…” Did the authors intend echocardiographic studies?\nResponse #8: Thank you. We have changed electrocardiographic to echocardiographic (line 56).\n“Currently, there is no approved therapy for PMD-associated cardiomyopathy,” Is this true? Can these patients be treated with conventional heart failure therapies such as anti-failure medications, ICD or CRT therapy and heart transplantation?\nResponse #9: We have modified this sentence for clarity in the revised text as below: “There is no FDA-approved cure for PMD-associated cardiomyopathy. Current treatment is primarily palliative10-12” (line 83-84). Examples of palliative treatments include ACE inhibitors, beta blockers, and mitochondrial cocktail10-12.\nFigure 1 (and supplement)\nAs discussed above, the extent of vector transduction needs to be quantified and shown as group data. The transduction biological variability is important to quantify. Toward this end the authors need to provide more GFP and ANT1 westerns demonstrating transduction.\nResponse #10: As suggested, we performed additional Western blots and quantified Ant1 levels to strengthen our data (new Supplementary Fig. 2). These Western blots were conducted on new samples of isolated mitochondria from Ant1+/+, Ant1-/-, and Ant1-/-+ND6P25L mice, at 6 months post AAV-Ant1 transduction or PBS injection (3 mice per group). The results confirmed our previous results shown in Fig. 1 that AAV-Ant1 produces ~10% of ANT1 protein compared to the level in WT (Ant1+/+) mouse hearts. Together with those shown in Fig. 1D and Fig. 4A, we have now evaluated Ant1 protein levels in AAV-Ant1 transduced hearts from a total of 16 mice via Western blot or mass spectrometry. We have modified text to include description of these new results and provided experimental details in the Methods “Western Blotting” section (line 419-423). For technical reasons, the GFP antibody used in our IHC didn’t work for Western blot in our hands. However, we have measured the percentage of GFP-positive cells in the transduced hearts (new Supplementary Fig. 3) and the tissue distribution of the vector by dPCR (new Supplementary Fig. 1).\nFigure 2 (and supplement)\nAlthough no differences in inflammation, fibrosis or cell death can be seen in the images provided, the authors need to quantify and present the group data regarding these variables to conclude that the vector did not affect these variables.\nResponse #11: As suggested, we performed IHC to assess caspase-3 cleavage and IL-18 (new Supplementary Fig. 6 and new Supplementary Fig. 7) using heart sections from 3-month-old PBS injected or AAV-Ant1 transduced mice (n=12 for all treatments and genotypes). In addition, at least two animals per genotype (Ant1+/+, Ant1-/-, and Ant1-/-+ND6P25L) at each time point (3, 6, 9, and 12 months) were histologically evaluated for H&E, Trichrome, and TUNEL staining. There was no sign of tissue damage or increased cell death (Fig. 2B and Supplementary Fig. 5). Although only representative images are included in the manuscript, approximately 30 mice were studied. In addition, snRNA-seq studies detected no sign of altered inflammation or cell death pathways in any cell type (Fig. 5 and Supplementary Fig. 12-17). We did observe a slight increase in extracellular matrix/fibrotic gene expression 1 month post AAV-Ant1 injection (Supplementary Fig. 14D). However, trichrome stain at 3, 6, 9 and 12 months (Fig. 2B, Supplementary Fig. 4B and Supplementary Fig. 5) showed no sign of histologically noticeable fibrosis.\nThese new results are described in the text (line 136-138). Description of the method in included in Methods under the section “Heart/Body Weight Ratio and Histology” (line 530-540).\nFigure 3\nThe method for pericardial vector injection needs to be provided. Was there any acute morbidity or mortality during vector injection?\nResponse #12: The method for pericardial vector injection has now been added to the revised Methods section under “Study Design and Pericardial Injections” (line 379-390). We didn’t observe any acute morbidity or significant mortality during the injection or in the days afterward in our hands.\nFigure 4\nWhat is the sample size for the RNAseq and mass spectrometry data?\nResponse #13: The sample size for the both bulk RNA-seq and mass spectrometry data is N = 3 per condition. This information is added to the legend of Fig. 4.\nFigure 5\n“The fact that all cardiac cell types responded to the AAV-ANT1 transduction suggests that either all cells were transduced by the virus or that vCM transduction is functionally coupled to the gene expression of the other cardiac cell types.” This statement requires more exploration. The extent of transduction needs to be quantified far more accurately to enable conclusions to be made about the phenotypic effects of transduction. If there is low level transduction, as suspected, then the authors need to elaborate on the functional coupling resulting in gene expression changes across most other cardiac cells. This kind of bystander effect on gene expression has not been proven in the current study.\nResponse #14: As detailed in response #1, we have performed additional experiments to quantify the AAV transduction efficiency. The results showed ~20% of the cardiomyocytes were transduced but to a high level of transgene expression. This analysis also showed that GFP-positive cells observed are cardiomyocytes in morphology (new Supplementary Fig. 3A) and we din’t observe substantial Ant1 expression in non-cardiomyocyte cell types in AAV-Ant1 transduced Ant1-/- mice (Supplementary Fig. 14-17). Therefore, we modified this sentence in the text as the following: “Since the Desmin promoter is expected to limit AAV9-Ant1 transduction to cardiomyocytes only, the fact that other cardiac cell types showed significant gene expression changes suggests cell-cell crosstalk that impact non-cardiomyocytes. Such cell-cell crosstalk could be mediated by secreted signals or cell surface ligand-receptor signaling” (line 270-274).\nDiscussion\nCould the authors provide precedents for the “sharp threshold effect” in other studies of mitochondrial bioenergetics of relevance do this study? A more in-depth analysis is required to assist readers in understanding the connection between a low transduction event and the observed phenotypic correction.\nResponse #15: The fact that only ~10% of the normal level of Ant1 is sufficient to markedly improve cardiac performance is consistent with observed threshold effects of other mitochondrial inner membrane proteins encoded by the mtDNA. In Leber Hereditary Optic Neuropathy (LHON) the mtDNA MT-ND4 m.11778G>A mutation must be essentially homoplasmic before the patient has a high probability of vision loss13,14 and in MELAS subtle changes in the percentage of the mtDNA MT-TL1 m.3242A>G have dramatic effects on the phenotype15. This effect is even more dramatic in the case of null mutations in nuclear DNA coded mitochondrial genes where the absence of the enzyme blocks a function, such as inhibition of mitophagy by the absence Ant1 in skeletal muscle resulting in the massive accumulation of abnormal mitochondria which is abrogated by expression of only ~20% of Ant1 protein3. These are added to the Discussion (line 305-309).\nCould the authors provide a more in-depth discussion about the pros and cons of pericardial vector inject for gene therapy of cardiac disease?\nResponse #16: As suggested, we significantly expanded our discussion of the AAV vector and delivery strategy and results in the Discussion (line 275-322). This is also covered in Response #5.\nReviewer #2\nThe authors present a study where they investigate the impact of restoring partial expression of a heart-muscle isoform of Ant (Ant1) in previously established Ant-/- and Ant-/- + ND6P25L mouse PMD cardiomyopathy models. Using a gene therapy approach (AAV2/9), the authors show that after transduction of ANT1 cDNA at P3 into the pericardium, they observe a mild increase in the expression of Ant1, which is sufficient to mitigate the cardiomyopathy up to 12 months old and they demonstrate the reversal of changes in RNAseq (9 months), proteomics (6 months) and with single nucleus sequencing (1 month) post-transduction. The authors suggest that gene therapy could be used to lower the disease severity of patients with more severe forms of ANT-/- associated disease.\nThere is growing evidence that digenic variants influence disease manifestation or severity of many mitochondrial diseases. And the ability to mitigate these effects is so far largely unexplored. Here, the demonstration that even a mild change in expression of a disease gene can partially reduce the severe disease phenotype indicates not only a treatment option for the disease in question, but that modest molecular changes may be sufficient to change the pathology of PMDs.\nResponse: We thank the Reviewer for these supportive comments. Following these suggestions, we have performed new experiments with results and textual changes that further strengthen our manuscript.\nMajor points:\n1. In the author’s previous publication, McManus, M.J. et al 2019, they performed characterisation of the two mouse models used in this study. It was established that the increased severity of the cardiomyopathy in the ANT-/- + ND6P25L model was also accompanied by, amongst other factors, impaired complex I activity, increased somatic mtDNA mutation levels, and a shortened lifespan. The authors show that gene therapy can ameliorate the cardiomyopathy phenotype, but it would be nice to know whether previously reported aberrations are also impacted, e.g., complex I activity and mtDNA mutation (deletion) levels.\nResponse #1: We performed suggested experiments, including the evaluation of complex I activity and mtDNA mutation (deletion) levels.\n(1) For complex I activity, we used blue native PAGE to study isolated heart mitochondria from WT, Ant1-/-, and Ant1-/-+ND6P25L mice 6 months post AAV-Ant1 or PBS injection (3 mice per condition). These are the same mice shown in the new Supplementary Fig. 2. The loading was normalized using protein quantification and verified by running a western blot for VDAC/Porin. As shown below, a decrease in complex I activity is evident in Ant1-/- mouse hearts compared to WT (as observed in McManus et al 2019), but no obvious restoration of complex I activity recovery is observed in AAV-Ant1-injected Ant1-/- mice compared to those injected with PBS, even though some complex I subunits seem to change expression based on bulk RNA-seq and mass spectrometry data (Fig. 4).\nSamples from 12-month-old mice showed the same results (below).\nThe 10% increase in Ant1 in AAV9-Ant1 transduced Ant1-/- and Ant1-/- + ND6P25L mouse hearts was insufficient to fully restore complex I activity to the Ant1+/+ level. This is partly because the level of Ant1 is coupled to the level of complex I16. We observed that Ant1-/- hearts (Fig. 4G) exhibited decreased levels of many mitochondrial complex I proteins (NDUFA6, NDUFA10, NDUFA2, NDUFB8, NDUFA9, NDUFB1, MT-ND5, MT-ND1, and MT-ND4), and AAV-Ant1 partially restored the expression of some of the complex I protein levels such as MT-ND5, but this was insufficient to fully restore complex I activity (Fig. 4G versus H&I). This is a topic worth further investigation in the future. For this reason, we feel that it is best to present these results here for the reviewers rather than in the revised manuscript.\nWe reported these results in the text: “AAV-Ant1 partially restored the levels of a variety of mitochondria and functionally related proteins (ATP5F1D, ANKRD29, CAND2, UQCRQ, H1.4, MTND5, HNRNPU, DOCK10, ATP5MG, ECHDC2, PSMD8, NYX, GLUD1, HSP90B1, GRIN2B, TMEM143, MYOZ2, ATAD3, AIFM1, LRPPRC, and others) including some complex I protein subunits such as MT-ND5, though this was insufficient to fully restore complex I activity the Ant1-/--associated complex I deficiency16.” (line 205-210).\n(2) To assess the effect of AAV-Ant1 transduction on Ant1-/-, and Ant1-/-+ND6P25L heart mtDNA somatic mutations, we used long-extension PCR to amplify the mtDNA of 12 months post transduction hearts (n=3 per condition) and assessed the presence of deletions by agarose gel. The full length 16.5kb mtDNA is evident, indicated by the red arrow (Panel B is overexposed version of Panel A). Ant1-/- heart mitochondria showed more deletions (lower molecular weight smears) compared to WT ones, and this is largely restored by AAV-Ant1. However, we didn’t observe such effects in Ant1−/−+ND6P25L mice. The reason is not entirely clear. It is possible that the mtDNA ND6P25L mutation itself prevented ANT1 protein’s effect on mtDNA integrity. This is a topic worth further investigation in the future. For this reason, we feel that it is best to present these results here for the reviewers rather than in the revised manuscript.\nPanel A Panel B\n2. The authors display data from the AAV-Ant1 or PBS injected groups, but they also state that they performed injections with a second control construct lacking the ANT1 cDNA, why was this not shown alongside the other two conditions?\nResponse #2: We apologize for not making this clearer. We in fact included these mice receiving AAV-Gfp in many experiments and analyses, as shown in the echocardiographic data in Table 1 – they are in the 9 and 12-month cohort but not in the 3-month cohort. The results are comparable to PBS injected mice. Financial and mouse breeding limitations prevented us from phenotypically analyze AAV-Gfp transduced mice at every time point studied.\n3. Line 106 &107 – there is no data for the gastrocnemius, this should be included to support this statement.\nResponse #3: We apologize for our oversight on this. We have since used digital (dPCR) to quantify the distribution of AAV vector genomes in different tissues: heart, liver, diaphragm and gastrocnemius. These results are shown in the new Supplementary Fig. 1, described in line 109-115, with the method reported in the Methods under “Total DNA isolation and cellular transgene levels determined by digital PCR” (line 451-469).\n4. In supplementary figure 4 A there appears to be a loss of vCMs in the transduced hearts compared to both WT and untreated mutant (from the UMAPs), while the other cell types seem constant across conditions. What is the cause of this? Does ANT cDNA transduction cause a loss of vCMs?\nResponse #4: This apparent discrepancy is the result of sampling variation in the preparation of nuclei for the snRNA-seq studies. The cell composition for each heart analysed is provided in the table below. Pooled heart samples from each condition were sequenced via snRNA-seq (n = 1 per condition), and we only used a small portion of the heart for snRNA-seq (we typically load 8,000 nuclei to capture 3,000-5,000 nuclei with high quality data, so we need only small portion of the tissue; and the rest of the heart we used for other molecular analyses). It is therefore expected that some variation in cell composition exist. We agree that vCM in AAV-Ant1 transduced Ant1-/- hearts in this specific experiment are less than the other 2 conditions. In our experience having performed >100 human/mouse heart snRNA-seq, this falls into the normal variation range when only a portion of the tissue was sampled for analysis. Therefore, we don’t think that AAV-Ant1 results in loss of vCM. For the same reason the Ant1-/- hearts appear to have ~10% more vCM than WT hearts in this experiment, but an increase in cardiomyocytes has not been observed in previous studies of Ant1-/- hearts using alternative methods17-19. We want to emphasize that this does not affect our differential gene expression analysis for abundant cell types like vCM (577 cells) as our normalization methods take into account of cell number variations.\nAll Freq Percent WT Freq Percent KO Freq Percent ANT1 Freq Percent\nvCM 4523 41.75 vCM 1592 43.54 vCM 2354 54.49 vCM 577 20.19\nFibroblast 1860 17.17 Fibroblast 461 12.61 Fibroblast 558 12.92 Fibroblast 841 29.43\nEndothelial 1445 13.34 Endothelial 515 14.09 Endothelial 405 9.38 Endothelial 525 18.37\nPericyte 1373 12.67 Pericyte 487 13.32 Pericyte 522 12.08 Pericyte 364 12.74\nMacrophage 1211 11.18 Macrophage 501 13.7 Macrophage 286 6.62 Macrophage 424 14.84\naCM 171 1.58 aCM 21 0.57 aCM 125 2.89 aCM 25 0.87\nLEC 140 1.29 LEC 28 0.77 LEC 32 0.74 LEC 80 2.8\nNLC 111 1.02 NLC 51 1.39 NLC 38 0.88 NLC 22 0.77\nTotal 10834 3656 4320 2858\nMinor points:\n1. Further, I think it is interesting that Dguok is elevated in both the KO vs WT and rescue vs WT (figures 4G & I), indicating that perhaps there is a demand for mitochondrial purine nucleotide salvage that the treatment does not mitigate. The authors should explore this aspect of the model rather than just focusing on OMICs changes.\nResponse #5: Inactivation of either of the Dguok isoforms results in mtDNA depletion. Hence, it is likely that the increased levels of Dguok in the Ant1-/- and Ant1-/-+AAV9-Ant1 transduced hearts, relative to Ant1+/+ hearts, represents a compensatory induction of mtDNA biogenesis in the Ant1-/- hearts. However, to fully evaluate the effects nucleotide biogenesis in the Ant1-/- mutant and AAV9-Ant1 transduced hearts is beyond the scope of this manuscript.\n2. What was the rational for snRNA-seq over scRNA-seq?\nResponse #6: Both snRNA-seq (single-nucleus RNA-seq) and scRNA-seq (single-cell RNA-seq) measure gene expression in individual cells, and numerous direct-comparison studies have demonstrated that the overall results are highly correlative in key outcomes such as cell type composition, gene expression and cell trajectory20-23. Although snRNA-seq often detects a lower number of RNAs than scRNA-seq, as it excludes transcripts outside the nucleus, it holds several technical advantages when applied to tissues that are difficult to dissociate the constituent cells, such as mammalian hearts. First, snRNA-seq bypasses preparation of intact single-cell suspension and thus can be used to study frozen and archived primary tissues. Second, snRNA-seq can minimize the bias of recovering only cells that are easily dissociated or cells of a certain size (e.g., cardiomyocytes that are 10-20 times larger than other cardiac cells and optimal for platform-specific cell capture). Furthermore, snRNA-seq reduces the impact of aberrant transcriptional changes induced by enzymatic dissociation. For these reasons we used snRNA-seq for our study.\n3. To simplify for readers, it would be nice to provide a heatmap of at least some of the proteomics data in Fig. 4G-I. Perhaps as a supplementary figure.\nResponse #7: As suggested, we provided a heatmap of the proteomics data to help visualize the AAV-Ant1 effects in the new Supplementary Fig. 9 and updated the text accordingly (line 184-185). As evident from this proteomic analysis, the AAV-Ant1 Ant1-/- transduced heart protein composition is intermediate between the Ant1-/- and Ant1+/+ protein compositions. The partial restoration of the protein composition of the AAV-Ant1 transduced Ant1-/- hearts again demonstrates why the ~10% increase in Ant1 expression following AAV-Ant1 transduction does not fully restore the complex I complex level/activity. It also highlights the fact that a partial restoration of a null activity which abrogates a biochemical block, in this case export of mitochondrial ATP, can have a disproportionally positive effect on the physiological status of the heart.\n4. Line 45 – 46 – Why is ANT classified as a nDNA OXPHOS gene? This is misleading because it belongs to the transporter family.\nResponse #8: The text linking ANT1 to OXPHOS has been deleted.\n5. Figure 3 - It looks like the colour of the Ant1-/- + ND6 is different between the legend and the data points.\n6. Figure 3B – use black stars to show significance to be consistent.\n7. Figure 3B&C – The standard is to use g for grams.\nResponse #9: We have modified the figures as suggested.\nReviewer #3\nThis manuscript presents a detailed evaluation of AAV2/9-based gene replacement therapy for primary mitochondrial disease manifesting dilated cardiomyopathy caused by a null mutation in the heart muscle isoform of the ANT1 gene. The authors generated an Ant1 null mouse model and combined it with an already generated ND6 P25L mtDNA mutation to increase the severity of the dilated cardiomyopathy. The experimental approach to evaluate the efficacy of the gene-based therapeutic approach is well-designed. They subsequently demonstrated partial restoration of the mitochondrial dysfunction by AAV-ANT1(Slc25a4) transduction, which protects against dilated cardiomyopathy in Slc25a4-/- plus mtDNA mutant.\nThe experimental approach to evaluate the efficacy of the gene-based therapeutic approach is well-designed and sound. However, several areas require improvement before the manuscript can be considered for publication.\nResponse: We thank the Reviewer for these suggestions. We have performed new experiments with results and textual changes that further strengthen our manuscript.\n(1) Can the authors please elaborate on the chosen dosage? How was it determined? Could the level of restoration be enhanced with a higher dosage?\nResponse #1: The typical range of doses used for clinical AAV gene therapy trials according to a recent meta-analysis is between 1010 to 1017 vg per patient, or ~108 to 1015 vg/kg assuming body weight of 100kg1. Our chosen dose of 2.5 x1011 vg/mouse (or 1.25x1014 vg/kg assuming 3 day old pups of 2g) is at the higher end of this range. We chose this dose because our goal is to demonstrate, as a proof of principle, that we could intervene with gene therapy to correct mitochondrial cardiomyopathy without significant toxicity. We wanted to ensure we delivered enough AAV into the heart. We also used digital PCR (dPCR) to measure the AAV distribution in different organs, and it is more favourable toward the heart vs. other organs, including the liver (new Supplementary Fig. 1).\nThe rational for employing pericardiac vector injection and the use of high dose AAV-Ant1 is now discussed in the Discussion (line 295-311). This clarifies the logic of the current experiments in light of our successful transduction on Ant1-/- mouse muscle, the results published in 20053.\n(2) How did they ensure minimal damages when they injected 3 day-old pups pericardially?\nResponse #2: First, we expected minimal damage with our tiny Hamilton needles for injection because the neonatal mouse heart in the first 7 days after birth is well known to retain significant regenerative capacity to resection, infarction and cryoinjury24-26. Second, we carefully monitored the mice during and post observation and observed no acute morbidity or mortality. Histological analyses post heart collection also revealed no signs of noticeable tissue damage. We also didn’t see any increase in inflammatory gene expression in our RNA-seq data. The injection design is further elaborated in the Methods under the heading “Study Design and Pericardial Injections” (line 379-390).\n(3) While partial restoration of (10%) ANT1 expression in the Ant1-/- and Ant1-/- + mtDNA ND6P25L mouse models led to significant improvement in cardiac pathology by molecular and functional assessment, it is important to discuss whether this level of restoration is adequate for clinical translation, given the patient's null mutation. Additionally, the potential translational relevance of the findings, including limitations and challenges in moving toward clinical application, should be addressed in the discussion.\nResponse #3: We have significantly expanded our Discussion section to discuss these important questions (line 275-311). The role of threshold effects in mitochondrial bioenergetics demonstrates the striking effects of relatively small changes in OXPHOS function can have on the overall health status of the patient (line 303-311).\n(4) The introduction lacks a clear statement of how combining ND6 P25L mtDNA mutation with the ANT-/- null mouse model could lead to dilated cardiomyopathy.\nResponse #4: We have made such a clear statement in our revised manuscript as the following: “The Ant1-/- plus ND6P25L combination results in early onset dilated cardiomyopathy, affecting echocardiogram measurement and leading to an 80% reduction in cardiac contractility and dramatic ventricular dilation, thus modeling the severe dilated cardiomyopathy manifest by the ANT1-/-+mtDNAU2 children” (line 70-74).\n(5) The Authors believes relatively small improvement in OXPHOS gene expression can have major beneficial functional effects. It is recommended to conduct additional mitochondrial functional assessments such as mito respiration and complex activity assays to validate these findings.\nResponse #5: As suggested, we measured complex I activity with blue native PAGE in isolated heart mitochondria from WT, Ant1-/-, and Ant1-/-+ND6P25L mice 6 months post AAV-Ant1 or PBS injection (3 mice per condition). These are the same mice shown in the new Supplementary Fig. 2. The loading was normalized using protein quantification and verified by running a western blot for VDAC/Porin. As shown below, a decrease in complex I activity is evident in Ant1-/- mouse hearts compared to WT (as observed in McManus et al 2019), but no obvious restoration of complex I activity recovery is observed in AAV-Ant1-injected Ant1-/- mice compared to those injected with PBS, even though some complex I subunits seem to change expression based on bulk RNA-seq and mass spectrometry data (Fig. 4).\nSamples from 12-month-old mice showed the same results (below).\nThe 10% increase in Ant1 in AAV9-Ant1 transduced Ant1-/- and Ant1-/- + ND6P25L mouse hearts was insufficient to fully restore complex I activity to the Ant1+/+ level. This is partly because the level of Ant1 is coupled to the level of complex I16. We observed that Ant1-/- hearts (Fig. 4G) exhibited decreased levels of many mitochondrial complex I proteins (NDUFA6, NDUFA10, NDUFA2, NDUFB8, NDUFA9, NDUFB1, MT-ND5, MT-ND1, and MT-ND4), and AAV-Ant1 partially restored the expression of some of the complex I protein levels such as MT-ND5, but this was insufficient to fully restore complex I activity (Fig. 4G versus H&I). This is a topic worth further investigation in the future. For this reason, we feel that it is best to present these results here for the reviewers rather than in the revised manuscript.\nHowever, 10% restoration of Ant1 did increase the overall expression of many of the complex I nDNA and mtDNA coded subunits (Fig. 4G-I) as well as increase the flux of mitochondrial ATP into the cytosol17. Because of the threshold effect (see Discission, line 305-311), this likely changed the mitochondrial physiology sufficiently to significantly improve cardiac function.\n(6) Figure 3 needs to be re-done. The symbols chosen were confusing.\nResponse #6: We have updated Fig. 3 and Supplementary Fig. 8, which we believe are clearer.\nMinor issues:\n“Trichome” was mis-spelled in Supplementary Fig. 1\nResponse #7: Thank you. We corrected this as suggested (now Supplementary Fig. 4).\nLine 78-79: Ant1 deficiency in muscle is associated with a massive accumulation of abnormal mitochondria: Did they mean abnormal mitochondria or mitochondrial components (please specify).\nResponse #8: We meant abnormal mitochondrial morphology27. This accumulation of abnormal mitochondrial is the result of the requirement of functional ANTs to support PINK-Parkin based mitophagy. Since only Ant1 is the only ANT protein abundantly expressed in the skeletal muscle, Ant1-deficiency block muscle mitophagy and results in the accumulation of damaged mitochondria4. We have modified this sentence for clarity as the following: “Ant1 deficiency in muscle is associated with a massive accumulation of mitochondria with abnormal morphology” (line 81-82).\nComment from Reviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank you for co-review our manuscript and appreciate your comments and time.\nReferences\n1. Au, H.K.E., Isalan, M. & Mielcarek, M. Gene therapy advances: a meta-analysis of AAV usage in clinical settings. Frontiers in Medicine 8, 809118 (2021).\n2. Strauss, K.A. et al. Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup. Proceedings of the National Academy of Sciences of the United States of America 110, 3253-3458 (2013).\n3. Flierl, A., Chen, Y., Coskun, P.E., Samulski, R.J. & Wallace, D.C. Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse. Gene Therapy 12, 570-578 (2005).\n4. Hoshino, A. et al. The ADP/ATP translocase drives mitophagy independent of nucleotide exchange. Nature 575, 375-379 (2019).\n5. Wahbi, K. et al. Long-term cardiac prognosis and risk stratification in 260 adults presenting with mitochondrial diseases. European Heart Journal 36, 2886-2893 (2015).\n6. Honzik, T. et al. Neonatal onset of mitochondrial disorders in 129 patients: clinical and laboratory characteristics and a new approach to diagnosis. Journal of Inherited Metabolic Disease 35, 749-759 (2012).\n7. Holmgren, D. et al. Cardiomyopathy in children with mitochondrial disease; clinical course and cardiological findings. European Heart Journal 24, 280-288 (2003).\n8. Imai-Okazaki, A. et al. Long-term prognosis and genetic background of cardiomyopathy in 223 pediatric mitochondrial disease patients. International Journal of Cardiology S0167-5273, 01080-01089 (2021).\n9. Imai-Okazaki, A. et al. Cardiomyopathy in children with mitochondrial disease: Prognosis and genetic background. International Journal of Cardiology 279, 115-121 (2019).\n10. Meyers, D.E., Basha, H.I. & Koenig, M.K. Mitochondrial cardiomyopathy: pathophysiology, diagnosis, and management. Texas Heart Institute Journal 40, 385-394 (2013).\n11. Yang, J. et al. Mitochondrial cardiomyopathy: molecular epidemiology, diagnosis, models, and therapeutic management. Cells 11(2022).\n12. Bogle, C. et al. Treatment strategies for cardiomyopathy in children: a scientific statement from the American Heart Association. Circulation 148, 174-195 (2023).\n13. Sadun, A.A., La Morgia, C. & Carelli, V. Leber's Hereditary Optic Neuropathy. Current Treatment Options in Neurology 13, 109-117 (2011).\n14. Wallace, D.C. et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242, 1427-1430 (1988).\n15. Picard, M. et al. Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming. Proceedings of the National Academy of Sciences of the United States of America 111, E4033-E4042 (2014).\n16. McManus, M.J. et al. Mitochondrial DNA variation dictates expressivity and progression of nuclear DNA mutations causing cardiomyopathy. Cell Metabolism 29, 78-90 (2019).\n17. Graham, B.H. et al. A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/skeletal muscle isoform of the adenine nucleotide translocator. Nature Genetics 16, 226-234 (1997).\n18. Narula, N. et al. Adenine nucleotide translocase 1 deficiency results in dilated cardiomyopathy with defects in myocardial mechanics, histopathological alterations, and activation of apoptosis. JACC. Cardiovascular imaging 4, 1-10 (2011).\n19. Palmieri, L. et al. Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy. Human Molecular Genetics 14, 3079-3088 (2005).\n20. Bakken, T.E. et al. Single-nucleus and single-cell transcriptomes compared in matched cortical cell types. PLoS One 13, e0209648 (2018).\n21. Ding, J. et al. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nature Biotechnology 38, 737-746 (2020).\n22. Wen, F., Tang, X., Xu, L. & Qu, H. Comparison of single‑nucleus and single‑cell transcriptomes in hepatocellular carcinoma tissue. Molecular Medicine Reports 26, 339 (2022).\n23. Santiago, C.P. et al. Comparative analysis of single-cell and single-nucleus RNA-sequencing in a rabbit model of retinal detachment-related proliferative vitreoretinopathy. Ophthalmology Science 3, 100335 (2023).\n24. Porrello, E.R. et al. Transient regenerative potential of the neonatal mouse heart. Science 331, 1078-1080 (2011).\n25. Jesty, S.A. et al. c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart. Proceedings of the National Academy of Sciences of the United States of America 109, 13380-13385 (2012).\n26. Porrello, E.R. et al. Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proceedings of the National Academy of Sciences of the United States of America 110, 187-192 (2013).\n27. Morrow, R.M. et al. Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity. Proceedings of the National Academy of Sciences of the United States of America 114, 2705-2710 (2017)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "To their credit the authors have undertaken substantial additional experiments to address all reviewers comments. I am satisfied that my concerns have been addressed. The manuscript has been strengthened by these additional data and modifications to the manuscript.", "reviewer_2": "This paper represents a substantial amount of high-quality experimental work in the mouse and provides a nice preclinical framework for treating cardiomyopathy caused by ANT1 mutations. The authors have fully responded to my critique and I now feel this paper is now ready for publication.\nNils-Göran Larsson", "reviewer_3": "The authors have made an arduous effort to address the critiques of the previous review. This reviewer finds the revisions satisfactory and has no more technical concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter