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Abstract: Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types. [Copyright &y& Elsevier]

Abstract: Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary β subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and β subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where β1 and β3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with β2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to β3 and β4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and β subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation–contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”. [Copyright &y& Elsevier]

Abstract: Voltage-gated sodium channels are composed of pore-forming α- and auxiliary β-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel α-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Nav1.5 sodium channel α-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel α- and β-subunits. α-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel α-subunit isoforms Nav1.1, Nav1.2, Nav1.3, Nav1.4 and Nav1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac α-subunit isoform, Nav1.5. Each of the β-subunit isoforms (β1–β4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the α-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel α-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Nav1.5 α-subunit and they contribute to the total sodium current. [Copyright &y& Elsevier]