Partial restoration of the mitochondrial dysfunction by AAV-Ant1 (Slc25a4) transduction protects against dilated cardiomyopathy in Slc25a4-/- plus mtDNA mutant mice
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, the severity of the cardiomyopathy being mediated by the mitochondrial DNA (mtDNA). To optimize strategies for addressing mitochondrial cardiomyopathy, we generated an Ant1 null mouse and combined it with the ND6P25L mtDNA 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 PGC1a, 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. Overall design: Bulk RNA sequencing and single nucleus RNA Sequencing (snRNA-seq) analysis on C57BL/6JEiJ, C57BL/6JEiJ Ant1-/-, and C57BL/6JEiJ Ant1-/- ND6P25L mice (WT, ANT1, ANT1 ND6), pericardially injected with 2.5x10^11 vg AAV2/9Des-Gfp-mAnt1 or 2.5x10^11 vg AAV2/9Des-Gfp . 1, 3, 6, 9, and 12 months post-infection RNA was collected using TRIzol™ Reagent (Invitrogen, 15596026) from heart samples. The RNA was assessed by NanoDrop spectrophotometer (ThermoFisher Scientific) and quantified by Qubit Flex (Thermofisher Scientific) using the Qubit RNA HS Assay Kit (Thermofisher Scientific, Q32855). The RNA was diluted to 1.43 ng/ul 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 (Thermofisher Scientific, A29024) and sequenced using the Ion Torrent S5 Sequencer and Ion 540 Kit (Thermofisher Scientific, A30011). For snRNA-seq, isolated nuclei were processed using the Chromium Single Cell 3' Reagent V3.1 kit from 10X Genomics. A total of 8,000 nuclei per sample were loaded into a Chip G. Barcoding, cDNA amplification, and library construction were performed according to the Chromium 3' V3.1 protocol. Sequencing was performed on a DNBSEQ-T7 platform targeting 20,000 reads per nucleus.