a Representative western blottings (W.B.) of homogenates (top panels) with Flag antibody for AAV9-mitoARCUS samples and GFP antibody for AAV-GFP samples. RFLP “last-cycle hot” PCR analysis (RFLP, bottom panels) of DNA samples from the same injected animals, at 6, 12, and 24 weeks PI. Similar analyses were performed for each animal. b Quantification of heteroplasmy shift shown as percent change in heteroplasmy across all tissues at 6, 12, and 24 weeks PI normalized to brain tissue. Heteroplasmy of the heart (H), tibialis anterior (TA), quadriceps (Q), gastrocnemius (G), kidney (K), liver (L), and spleen (Sp) were compared to that of the brain (negative for expression of mitoARCUS). c Quantification by qPCR of total mtDNA levels were measured in the heart, skeletal muscle, liver, and brain (B) at 6 and 24 weeks PI using ND1 and ND5 mitochondrial primer/probes normalized to 18S (nuclear DNA). Data are mean ± SEM of n = 4. Statistical analysis was performed using two-tailed Student’s t-test.