Abstract Body (Do not enter title and authors here): Introduction: Na_v1.5, the predominant Na⁺ channel in cardiac myocytes, is encoded by SCN5A and is essential for depolarization and impulse propagation in the heart. Loss of function mutations of Na_v1.5 can lead to tachyarrhythmias, conduction disease and dilated cardiomyopathy by altering channel expression, kinetic properties, and/or membrane trafficking. We previously reported that amino acid K1479 of Na_v1.5 is acetylated, that K1479 acetylation or mutation to the acetylation mimic glutamine (K1479Q) decreases inward Na⁺ current (I_Na) in heterologous expression systems by decreasing membrane trafficking, that cardiac myocytes from mice lacking the deacetylase Sirt1 (Sirt1^-/-) are hyperacetylated and have less I_Na, and that Sirt1^-/- mice have conduction disease and arrhythmias. Here, we studied the effect of the K1479Q mutation that mimics acetylation in-vivo.

Methods: K1479Q knock-in mice were engineered via CRISPR/Cas9 on a C57BL6 background and backcrossed 4 times with C57BL6 wild type (WT) mice. Na_v1.5^WT/Q heterozygous (het) mice were mated, and male (M) and female (F) gene-targeted offspring and littermate controls were studied by electrocardiograms (EKGs) and echocardiograms (Echos) at 3 and 6 months of age. Data is shown as mean±SEM.

Results: The K1479Q mutation was homozygous (hom) embryonic lethal (Na_v1.5^WT/Q x Na_v1.5^WT/Q crosses: WT, n=23; Het, n=49; Hom, n=0). On EKG (WT: n = 5M, 3F; Het: n = 6M, 5F), Na_v1.5^WT/Q mice had a prolonged PR interval at 3 months (42±1 vs. 36±1 ms, p=0.0003) and at 6 months (46±1 vs. 39±1 ms, p=<0.0001) compared to WT (Figure). Na_v1.5^WT/Q mice also showed a prolonged QTc interval (72±1 vs. 65±2 ms, p=0.007) compared to WT controls at 6 months. No significant differences were identified in cardiac size or function between Na_v1.5^WT/Q and WT mice at 6 months.

Conclusions: The K1479Q Na_v1.5 mutation, which mimics the acetylation post-translational modification at K1479, leads to homozygous embryonic lethality and a heterozygous electrophysiological phenotype as severe as targeted deletion of the channel. These findings suggest that Na_v1.5-K1479Q and acetylated Na_v1.5-K1479 do not produce I_Na in-vivo in cardiac myocytes, in part due to failure to traffic to the surface membrane.