Abstract-Mutations in SCN5A encoding the principal Na ϩ channel ␣-subunit expressed in human heart (Na V 1.5) have recently been linked to an inherited form of dilated cardiomyopathy with atrial and ventricular arrhythmia. We compared the biophysical properties of 2 novel Na V 1.5 mutations associated with this syndrome (D2/S4 -R814W; D4/S3 -D1595H) with the wild-type (WT) channel using heterologous expression in cultured tsA201 cells and whole-cell patch-clamp recording. Expression levels were similar among WT and mutant channels, and neither mutation affected persistent sodium current. R814W channels exhibited prominent and novel defects in the kinetics and voltage dependence of activation characterized by slower rise times and a hyperpolarized conductance-voltage relationship resulting in an increased "window current." This mutant also displayed enhanced slow inactivation and greater use-dependent reduction in peak current at fast pulsing frequencies. By contrast, D1595H channels exhibited impaired fast inactivation characterized by slower entry into the inactivated state and a hyperpolarized steady-state inactivation curve. Our findings illustrate the divergent biophysical defects caused by 2 different SCN5A mutations associated with familial dilated cardiomyopathy. Retrospective review of the published clinical data suggested that cardiomyopathy was not common in the family with D1595H, but rather sinus bradycardia was the predominant clinical finding. However, for R814W, we speculate that an increased window current coupled with enhanced slow inactivation and rate-dependent loss of channel availability provided a unique substrate predisposing myocytes to disordered Na ϩ and Ca 2ϩ homeostasis leading to myocardial dysfunction. (Circ Res. 2008;102:364-371.) Key Words: arrhythmia Ⅲ ion channels Ⅲ cardiomyopathy Ⅲ genetics M utations in SCN5A, the gene encoding the principle ␣-subunit of the human cardiac voltage-gated sodium channel Na V 1.5, cause inherited susceptibility to ventricular tachyarrhythmia (congenital long-QT syndrome [LQTS], Brugada syndrome [BrS]), 1-3 familial heart block with or without sinus node dysfunction, 4 -6 and other more complex phenotypes with features of both arrhythmia susceptibility and impaired atrioventricular conduction. 7,8 Subclinical expression of SCN5A mutations may also manifest as druginduced arrhythmias, 9,10 sudden infant death syndrome, 11,12 and increased arrhythmia risk in specific populations. 13 Conventional wisdom in the era of molecular cardiology predicts that mutations affecting plasma membrane ion channels will primarily predispose to rhythm disorders, but will not directly affect contractile function. A possible exception to this paradigm was provided by the discovery of SCN5A mutations associated with familial dilated cardiomyopathy associated with atrial and ventricular arrhythmias (DCM-arrhythmia syndrome). In 2004, McNair et al 14 reported that a missense mutation, SCN5A-D1275N, segregated with a phenotype including DCM of variable expression, abnormal ...