Fig 2: Myocyte-Specific β-Catenin GOF and Acute GSK-3 Inhibition by SB2 Reduces Conduction Velocity in Murine Hearts(A) Representative activation maps showing slowed electrical activation in β-catenin GOF hearts (right) compared to noninduced controls (left). (B) Activation map showing ventricular tachycardia induced from electrical stimulation of β-catenin GOF ventricles. The white arrows show irregular propagation of 2 different wave fronts, which results in a functional block. Five of 6 β-catenin GOF and 0 of 6 control hearts had spontaneous or stimulation-induced ventricular tachycardia. (C) Conduction velocities of RVs and LVs are significantly reduced in β-catenin GOF hearts compared to controls. (D) Western blot shows expression of stabilized β-catenin (lower-molecule-weight band associated with loss of exon 3) in β-catenin GOF mice and decreased NaV1.5 protein in β-catenin GOF compared to control hearts. (E) Transcription of Wnt/β-catenin target gene Axin2 is increased, and Scn5a is decreased, in β-catenin GOF compared to control hearts. Statistics were performed using Student’s t-test: ∗P < 0.05; ∗∗P < 0.01. β-cat = β-catenin; GOF = gain of function; RV = right ventricle; other abbreviations as in Figure 2.

Fig 3: Structural analysis and mapping of the Nav1.5 mutations. (A) At the top, structural mapping of the Nav1.5 mutations reported in this study based on their cryo-EM structure (PDB id: 7DTC); on the bottom, a left extracellular view of Nav1.5 electrostatic potentials calculated with the APBS method and close-up of Eα1 on the bottom right. Interactions between p.A344 (B), p.N347, (C) and p.D349 (D) in WT Nav1.5. The Cα atoms of these residues are shown in magenta. The SF-supporting helices P1 and P2 are depicted in blue and yellow, respectively. The DEKA residues are shown as orange spheres. (E) Multiple sequence alignment of SCN5A genes in Eukaryota and (F) multiple sequence alignment of human SCN genes with their isoforms. Black stars indicate the mutation sites presented in this study. The selectivity filter (SF)-supporting pore helices P1 and P2 regions are lined above the sequence in black, and the residue p.D372, belonging to the DEKA motif (SF), is marked with a black dot.

Fig 4: Wnt/β‐catenin signaling reduces SCN5A mRNA and Nav1.5 protein in female iPSC‐derived cardiomyocytes. (a) qRT‐PCR showing reduced SCN5A mRNA in female iPSC‐CMs after treatment with CHIR for 48 h (5 μM, n = 8 samples from two cell differentiations). Control group was maintained in culture medium containing equal amount of DMSO (n = 9 samples from two cell differentiations). Data were analyzed by two‐tailed t‐test. (b) Representative western blot showing reduced Nav1.5 (using a polyclonal antibody) in female iPSC‐CMs after treatment with CHIR or DMSO (control) for 48 h. Two Nav1.5 bands were observed with one near 260 kDa and another with a higher molecular weight (arrowhead). (c) Quantification of Nav1.5 band densities in panel B (normalized to calnexin), showing reduced Nav1.5 higher band in CHIR group (n = 5 samples from two cell differentiations) as compared to control, DMSO‐treated group (n = 6 samples from two cell differentiations). Data were analyzed by two‐tailed t‐test. (d) Left panel, diagram showing the binding sites of the two anti‐Nav1.5 antibodies. The polyclonal antibody binds to the intracellular loop between domains I and II and the monoclonal antibody binds to the C‐terminus of Nav1.5. Right panel, western blot showing time‐dependent reductions in Nav1.5 in female iPSC‐CMs after treatment with CHIR (5 μM) for 24, 48, or 72 h. The same samples were probed sequentially with two different anti‐Nav1.5 antibodies shown in left panel. The lower band (near 260 kDa) was not detected by the monoclonal antibody. (e) qRT‐PCR showing CHIR‐induced upregulation of TBX3 mRNA, a known repressor of Scn5a/Nav1.5 expression, in female iPSC‐CMs (n = 8 samples from three cell differentiations) after CHIR treatment for 48 h. Control group was maintained in culture medium containing equal amount of DMSO. *p < 0.0001 by two‐tailed t‐test. (f) Western blot showing increased Tbx3 protein in female iPSC‐CMs after treatment with CHIR for 48 h.

Fig 5: Localization of Nav1.5 WT and mutants transiently transfected in HEK293 cells. Representative immunofluorescence confocal images showing the plasma membrane and cytosolic localization of WT Nav1.5 protein (A), along with its p.A344S (B), p.N347K (C), and p.D349D (D) mutants. Nav1.5 is labeled in green. Scale bar: 10 µm.