Contribution of sodium channel neuronal isoform Nav1.1 to late sodium current in ventricular myocytes from failing hearts.

Late Na+ current (INaL) contributes to action potential (AP) duration and Ca2+ handling in cardiac cells. Augmented INaL was implicated in delayed repolarization and impaired Ca2+ handling in heart failure (HF). We tested if Na+ channel (Nav) neuronal isoforms contribute to INaL and Ca2+ cycling defects in HF in 17 dogs in which HF was achieved via sequential coronary artery embolizations. Six normal dogs served as control. Transient Na+ current (INaT) and INaL in left ventricular cardiomyocytes (VCMs) were recorded by patch clamp while Ca2+ dynamics was monitored using Fluo-4. Virally delivered short interfering RNA (siRNA) ensured Nav1.1 and Nav1.5 post-transcriptional silencing. The expression of six Navs was observed in failing VCMs as follows: Nav1.5 (57.3%) > Nav1.2 (15.3%) > Nav1.1 (11.6%) > Nav2.1 (10.7%) > Nav1.3 (4.6%) > Nav1.6 (0.5%). Failing VCMs showed up-regulation of Nav1.1 expression, but reduction of Nav1.6 mRNA. A similar Nav expression pattern was found in samples from human hearts with ischaemic HF. VCMs with silenced Nav1.5 exhibited residual INaT and INaL (∼30% of control) with rightwardly shifted steady-state activation and inactivation. These currents were tetrodotoxin sensitive but resistant to MTSEA, a specific Nav1.5 blocker. The amplitude of the tetrodotoxin-sensitive INaL was 0.1709 ± 0.0299 pA pF–1 (n = 7 cells) and the decay time constant was τ = 790 ± 76 ms (n = 5). This INaL component was lacking in VCMs with a silenced Nav1.1 gene, indicating that, among neuronal isoforms, Nav1.1 provides the largest contribution to INaL. At –10 mV this contribution is ∼60% of total INaL. Our further experimental and in silico examinations showed that this new Nav1.1 INaL component contributes to Ca2+ accumulation in failing VCMs and modulates AP shape and duration. In conclusion, we have discovered an Nav1.1-originated INaL component in dog heart ventricular cells. This component is physiologically relevant to controlling AP shape and duration, as well as to cell Ca2+ dynamics. Key points Late Na+ current (INaL) contributes to action potential remodelling and Ca2+/Na+ changes in heart failure. The molecular identity of INaL remains unclear. The contributions of different Na+ channel isoforms, apart from the cardiac isoform, remain unknown. We discovered and characterized a substantial contribution of neuronal isoform Nav1.1 to INaL. This new component is physiologically relevant to the control of action potential shape and duration, as well as to cell Ca2+ dynamics, especially in heart failure. Introduction A physiological role of neuronal isoforms of voltage-sensitive Na+ channels (Navs) in heart has been suggested many years ago by Corabeouf (Coraboeuf et al. 1979) based on their result that low concentrations (33 nm) of tetrodotoxin (TTX) significantly shortened action potential (AP) duration recorded in cardiac Purkinje fibres. At this low concentration TTX blocks only so-called ‘TTX-sensitive’ Navs which are neuronal isoforms (Haufe et al. 2005a2005a). Indeed, during the past decade, besides the dominating cardiac isoform main α-subunit, Nav1.5, different transcripts of highly TTX-sensitive neuronal Nav isoforms have been found in mouse (Nav1.1, 1.3, 1.6) and dog (Nav1.1, 1.2, 1.3) heart (Maier et al. 2004; Haufe et al. 2005a2005a,b). These neuronal Navs are responsible for 10% and 20% of the peak transient Na+ current, INaT, in dog myocardial and Purkinje cells, respectively (Haufe et al. 2005b2005b). The contribution of neuronal Navs to the late Na+ current, INaL, has not been studied in cardiac cells, therefore the molecular identity of INaL needs further careful consideration. This problem is especially important in HF where the gene expression profile changes dramatically. Thus an intriguing possibility could be that the differential expression of neuronal Navs can contribute, at least in part, to INaL alterations (augmentation) (Valdivia et al. 2005; Maltsev et al. 2007) and to INaL-related electrophysiological abnormalities observed in HF (for review see Maltsev & Undrovinas, 2008). In the present study we used a combination of molecular and pharmacological approaches, as well as numerical modelling, to test a hypothesis that neuronal Nav isoforms contribute to INaL in a physiologically significant manner in ventricular myocytes (VCMs) isolated from failing adult dog hearts. We performed post-transcriptional silencing of Nav1.1 and Nav1.5 genes by sequence-directed RNA using virally delivered short interfering RNA (siRNA). We found the expression of six Navs in both human heart and adult dog VCMs. Analysis of the expression revealed up-regulation of Nav1.1 and down-regulation of Nav1.6 in failing heart compared to normal heart. We took advantage of the method of culturing adult dog VCMs, established by our group, that preserves INaT and INaL over 5 days, i.e. sufficient time for gene silencing and membrane protein turnover (Maltsev et al. 2008a2008a; Mishra et al. 2011). In VCMs with silenced Nav1.5 we found a substantial residual INaT and INaL. Our biophysical and pharmacological examinations confirmed its neuronal isoform origin. Diastolic Ca2+ accumulation was reduced via blockade of the neuronal INaL component, indicating its physiological importance. Our numerical model simulations also demonstrated the physiological importance of the novel INaL component: the Nav1.1- associated INaL effectively modulates AP shape and duration, as well as intracellular Ca2+ homeostasis.