Fig. 1: AAV-mediated in vivo expression of Kir2.1Δ314-315.
a, AAV-based vector encoding human KCNJ2 (Kir2.1 channel), WT or Δ314-315 mutant, driven by the cTnT proximal promoter, followed by enhanced tdTomato after an IRES. b, Imaging of luciferase transgene expression under cTnT cardiospecific control comparing AAV-infected and non-infected mice. Upper, live C57BL/6J mouse injected (femoral vein) with AAV–Luciferase control vector in packaging serotype 9 (dose 3.5 × 1010 vg in 50 µl of saline solution). Lower, three isolated hearts from Luciferase-injected animals are shown next to three hearts from non-infected mice (saline solution). Images taken 4 weeks after inoculation. c,d, Representative fluorescence images of two non-injected (left) and AAV-Kir2.1∆314-315 transduced (right) mice, the latter showing expression of tdTomato throughout the heart (n = 5 per group). e, Left, representative fluorescence microscopy images of short-axis cross-sections of AAV-transduced hearts illustrate expression of tdTomato throughout the heart. Middle, a cropped image used for quantification. Scale bar, 100 μm. Right, fluorescence intensity staining and quantification (n = 3 mice, n = 1,465 cells) of transduced protein expression, used to assign the number of integrated viral genomes per cardiomyocyte. f, Representative western blots and quantification of total and membrane protein extracts from control and AAV-transduced isolated cardiomyocytes. Levels of expression were corrected by GAPDH expression and normalized by non-injected levels. Statistical analyses were conducted using two-tailed Mann–Whitney t-test. g, Representative immunostaining pattern of Kir2.1 at the plasma membrane in isolated cardiomyocytes from Kir2.1WT-expressing and Kir2.1∆314-315-expressing mice. Graph shows the percentage of cell surface with positive staining. Different colors in the same group identify cells coming from one animal. Statistical analyses were conducted using two-level hierarchical t-test analysis followed by Bonferroni’s post test. Each value is represented as mean ± s.e.m. ****P < 0.0001.
