Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry

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.

Keywords: Localization; Sodium channels; Ventricular myocytes.

Fig. 1

Laser Stability and Analysis of Laser Intensities. (A) The stability of the Leica SL confocal microscope Arg laser (green line) or GrHeNe (red line) was measured over a period of more than three hours by collecting whole field images from a fluorescent slide. Wide fluctuations in laser stability dissipated by about 2.5 hr (verticle line) after startup of the lasers demonstrating the need to allow adequate warming up time of lasers before quantitative image collection. Mouse ventricular myocytes were stained with anti-Na_V1.1 B and D) or anti-Na_V1.5 (C and E) antibodies. Single confocal plane images were collected using two different laser intensities using the acousto optical tunable filter (AOTF) settings of 80% (B and C) or laser settings of 20% (D and E) demonstrating an over saturated image for anti-Na_V1.5 with the laser setting of AOTF=80% while anti-Na_V1.1 using this setting is in the linear range. In contrast, the image of the myocyte stained with anti-Na_V1.1 (AOTF of 20%) is under saturated and undetectable while Na_V1.5 is in the linear range for image collection. (F) Comparison of average pixel intensity for various AOTF settings collected from either a fluorescent slide (green line) or from ventricular myocytes (blue line) stained with anti-Na_V1.1 antibodies illustrating a similar pattern of linearity for laser intensity settings using AOTF=20% to AOTF=80% using these two sample types. Scale bar = 35um

Fig. 2

Antibody Dilution Series. TsA201 cells transfected with A.) Na_V1.1 cDNA, B.) Na_V1.2 cDNA, C.) Na_V1.3 cDNA, D.) Na_V1.4 cDNA, E.) Na_V1.5 cDNA or F.) Na_V1.6 cDNA were labeled with anti-Na_V1.1-Nav1.6 antibodies, respectively. Average pixel intensity for each group of cells stained with different concentrations of a given antibody were analyzed in Igor and plotted to illustrate that when each antibody was used at high concentrations (red vertical line) it was saturating.

Fig. 3

Representative Staining Patterns of Ventricular Myocytes using Sodium Channel Antibodies. Images are z-stacks of ventricular myocyte images stained with (A) anti-Na_V1.1 (B) anti-Na_V1.2 (C) anti-Na_V1.3 (D) anti-Na_V1.4 (E) anti-Na_V1.5 or (F) anti-Na_V1.6 at the indicated AOTF setting illustrating typical myocytes used for the analysis of the relative sodium channel density. Note staining of z-lines with anti-Na_V1.1, anti-Na_V1.3, anti-Na_V1.4 (G) Quantitative analysis of staining intensities of sodium channel antibodies in ventricular myocytes. The graph indicates the normalized pixel intensity for each subtype-specific sodium channel antibody used. Scale bar=25 um.

Fig. 4

Representative staining densities of Na_V1.1-Na_V1.6 antibodies at various levels throughout ventricular myocytes. Images (1 µm z- steps) of the staining in ventricular mouse myocytes were collected, analyzed using lines scans and the results were plotted as a function of their position within the myocyte. A) Schematic diagram of the image planes used to construct the plots in B–H. The dashed line represents the orientation of the line scan used for analysis. (BH) Staining densities at the surface of the myocyte is represented by the black line for each antibody. The red, green, light blue, dark blue and purple lines represent analysis from distances of 2, 4, 6, 8, 10, or 12 um, respectively below the surface image shown in black.

Fig. 5

Quantification of surface and internal staining patterns of Nav channels in mouse ventricular myocytes. A–B) Optical sections from the middle of mouse ventricular myocytes were analyzed using a line scan to compare the density of staining on the surface (edge) of the myocyte and internally (middle region) for each antibody investigated. Red represents surface staining averaged over 3 pixels. Blue is internal staining density levels averaged over 50 pixels.

Fig. 6

Comparison of Na_V1.5 staining at intercalated disc and surface membrane. (A–C) A representative myocyte is shown in the upper panels that was double labeled with anti-Na_V1.5 antibodies (A) and anti connexin 43 antibodies (B) illustrating colocalization of these proteins in intercalated disc regions as shown in the merged image (C). The lower panels of A–C are the representative line scans taken from the regions between the dotted lines of their respective panels illustrating colocalization of Na_V1.5 and connexin 43. D) Another representatative image of ventricular myocytes labeled with anti-Na_V1.5 antibodies demonstrating staining at both intercalated disc and on surface membrane. E) Computer generated image of myocytes shown in (D) illustrating staining of hot spots at disc (red and yellow peaks) compared to less dense staining (blue) at the membrane. Image is tilted to align with the graph in panel F. F) Cross sectional line scan analysis of staining intensity through the region of the myocyte indicated by the white, dashed lines shown in (D). (G) Quantification of mean pixel intensity of fluorescence in disc compared to cell surface (edge) of myocytes. Scale bars: A–C = 10 um; D = 20 um.