Molecular identity of the late sodium current in adult dog cardiomyocytes identified by Nav1.5 antisense inhibition

Abstract

Late Na+current ( INaL) is a major component of the action potential plateau in human and canine myocardium. Since INaLis increased in heart failure and ischemia, it represents a novel potential target for cardioprotection. However, the molecular identity of INaLremains unclear. We tested the hypothesis that the cardiac Na+channel isoform (Nav1.5) is a major contributor to INaLin adult dog ventricular cardiomyocytes (VCs). Cultured VCs were exposed to an antisense morpholino-based oligonucleotide (Nav1.5 asOligo) targeting the region around the start codon of Nav1.5 mRNA or a control nonsense oligonucleotide (nsOligo). Densities of both transient Na+current ( INaT) and INaL(both in pA/pF) were monitored by whole cell patch clamp. In HEK293 cells expressing Nav1.5 or Nav1.2, Nav1.5 asOligo specifically silenced functional expression of Nav1.5 (up to 60% of the initial INaT) but not Nav1.2. In both nsOligo-treated controls and untreated VCs, INaTand INaLremained unchanged for up to 5 days. However, both INaTand INaLdecreased exponentially with similar time courses (τ = 46 and 56 h, respectively) after VCs were treated with Nav1.5 asOligo without changes in 1) decay kinetics, 2) steady-state activation and inactivation, and 3) the ratio of INaLto INaT. Four days after exposure to Nav1.5 asOligo, INaTand INaLamounted to 68 ± 6% (mean ± SE; n = 20, P < 0.01) and 60 ± 7% ( n = 11, P < 0.018) of those in VCs treated by nsOligo, respectively. We conclude that in adult dog heart Nav1.5 sodium channels have a “functional half-life” of ∼35 h (0.69τ) and make a major contribution to INaL.