mRNA Variants of a Neuronal Sodium Channel in Rat Cardiac Myocytes

Abstract

Death due to ischemic heart disease (IHD) is the result of cardiac arrhythmias and loss of cardiac pump function. One of the putative underlying mechanisms is impaired inactivation of voltage-gated sodium channels (VGSC), resulting in small persistent sodium currents. Nine VGSC (NaV1.1-NaV1.9) have been cloned and functionally expressed. Electrophysiological/pharmacological evidence suggests that “neuronal” isoform(s) exist in cardiomyocytes along with the cardiac-dominant NaV1.5. Given that persistent currents have been shown to be fundamental to the function of neuronal isoforms and that pharmacological evidence suggests that neuronal VGSC underlie increases in persistent currents during ischemic events, we hypothesized that neuronal VGSC are present in cardiomyocytes. Specifically, the purpose of this study was to demonstrate that the neuronal VGSC NaV1.1 exists in rat right ventricular myocytes.

The full-length NaV1.1 coding sequence was cloned in overlapping segments. Through sequencing, we identified one amino acid difference from a published brain sequence (c.2935A>G) and four deletion variants (c.[del266_473], c.[del2012_2044], c.[del4004_4258], and c.[del4003_4284]). The deletion variants were not present in all sequenced amplicons. Deletions at the first and the third deletion sites were of particular interest as these involved regions of conserved sequence, likely essential to channel function. To explore this further, PCR primers were designed to amplify a partial transcript spanning both of these regions. Three RT-PCR bands were produced: 1) an ~4500 bp band that is likely a composite of two amplicons, one with a deletion at the first deletion site (c.[del266_473]) and one with a deletion at the third deletion site (either c.[del4004_4258] or c.[del4003_4284]); 2) an ~4200 bp band confirmed to contain an amplicon with a deletion at both the first and the third deletion sites (either c.[del266_473] and c.[del4004_4258], or c.[del266_473] and c.[del4003_4284]); and 3) an ~4075 bp band suggesting further unidentified deletion variants. We were unable to identify any amplicons that did not contain at least one deletion involving conserved sequence.

Overall, these results demonstrate that multiple sequence variances of NaV1.1 exist in adult rat cardiomyocytes. Moreover it appears that under normal cellular conditions, at least one deletion that is likely to be devastating to channel function is always present.