Figure 1. Cardiac-specific, FLAG-tagged TTX-sensitive Na_V1.5-expressing transgenic mice.

(A) Diagram showing Na_V1.5. The pore-forming α-subunit is a pseudotetramer of transmembrane domains (I–IV) linked by intracellular loops. The channel’s inactivation gate is in the III–IV linker. The best-established CaM binding site is on the C-terminal domain, where the FHF binding site also resides. (B) Schematic of binary transgene system. The expression of reverse tetracycline-controlled transactivator (rtTA) is driven by the cardiac-specific α-myosin heavy chain promoter. The cDNAs for FLAG-F1759A-Na_V1.5 or FLAG-tagged TTX-sensitive Na_V1.5 were ligated behind 7 tandem tetO sequences. (C) Anti-FLAG antibody immunoblots of cleared lysates of hearts from pWT, IQ/AA, and nontransgenic mice. Representative images of 3 independent experiments. (D) Immunostaining of nontransgenic, pWT, and IQ/AA mice cardiomyocytes. Nontransgenic cardiomyocytes: primary antibody, anti-Na_V1.5 antibody. pWT and IQ/AA cardiomyocytes: primary antibody, anti-FLAG antibody. FITC-conjugated secondary antibody was used for all experiments. Scale bar: 5 μm. Representative of 20 cardiomyocytes from at least 3 independent cardiomyocyte isolations for all groups. (E–G) Exemplar whole cell Na⁺ current trace of ventricular cardiomyocyte from nontransgenic, pWT, and IQ/AA transgenic mice in the absence (black) and presence (red) of 20 nM TTX. Representative of n = 13, 21, and 44 cells, from left to right. Vertical scale bars: 10 pA/pF; horizontal scale bars: 5 ms. (H) Graph showing effect of 20 nM TTX on peak Na⁺ current. ****P < 0.0001 by paired t test. For nontransgenic, P = 0.61. n = 13, 21, and 44 cells from left to right. (I) Graph of fraction transgenic Na⁺ current for pWT and IQ/AA. Mean ± SEM. n = 21 and 44 cells from left to right. P = 0.73 by t test.