The voltage-gated sodium channel NaV1.5 controls cardiac excitability and is an established therapeutic target.
Mutations in the SCN5A gene, which encodes NaV1.5, are associated with inherited arrhythmia syndromes (long
QT syndrome, Brugada syndrome, congenital heart block) and dilated cardiomyopathy. While gain of function
mutations that disrupt NaV1.5 inactivation explain action potential duration (APD) and QTc prolongation, the
mechanisms by which loss of function NaV1.5 mutations cause the other diverse pathogenic outcomes are
unresolved. The physiological significance of other Na+ channel genes expressed in the heart are also uncertain.
Rodent models with gene-targeted Scn5a mutations can recapitulate some clinical features of disease, but their
use is complicated by compensatory mechanisms that may occur early in development. In addition, the available
pharmacological blockers of NaV1.5 block brain Na+ channels and other potential cardiac Na+ channels with
equal or greater potency, limiting their utility. In order to advance our understanding of NaV1.5-related biology,
we have developed a chemical-genetic model to achieve acute and reversible silencing of NaV1.5 in situ. We
engineered a NaV1.5 channel that contains a high-affinity, isoform-specific binding site for acylsulfonamide (GX)
drugs, enabling chemical strategies to pharmacologically drive nonconducting channel conformations. The
NaV1.5-GX channel has WT voltage-dependent gating and, unlike WT NaV1.5 and most other putative cardiac
Na+ channels, is blocked by nanomolar concentrations of GX compounds. We have used CRISPR gene-editing
to replace the endogenous Scn5a locus with the GX binding site in mice, creating a novel NaV1.5GX strain.
Homozygous NaV1.5GX/GX mice have normal cardiac phenotypes, yet the acute application of nanomolar GX
compounds to NaV1.5GX/GX isolated cardiac myocytes ablates Na+ current (INa). Systemic drug application in vivo
results in conduction slowing in NaV1.5GX/WT mice, and conduction block and sudden death in NaV1.5GX/GX mice,
thus providing a facile means to study NaV1.5 function and SCN5A-mediated disease. We propose first to
examine the effects of acute Nav1.5 blockade by GX compounds on gene expression, Ca2+ handling, ROS
production, fibrosis, cardiac function and arrythmias will be studied using NaV1.5GX/WT and NaV1.5GX/GX cardiac
myocytes and mice, and compared to chronic Nav1.5 blockade using Scn5a+/- heterozygous knockout mice. We
will then identify the effects of Na+ channel blockade on structural and electrophysiological remodeling, and on
arrhythmia susceptibility following Transverse Aortic Constriction (TAC). Lastly, we will develop in vivo and ex
vivo platforms to study SCN5A mutations identified in patients. The Scn5aGX mouse presents a unique
opportunity to examine the phenotypes of human SCN5A mutations in a cardiac environment. In total, we
anticipate these efforts will reveal novel molecular mechanisms of genotype-phenotype coupling stemming from
SCN5A's role in controlling cardiac excitability.