Persistent PKA activation for 15 h increased the size and density of Na V 1.5 clusters on ventricular myocytes’ surface. (A) Procedures of detecting and analyzing NaV1.5 clusters on the myocyte surface. Airyscan images of NaV1.5 immunofluorescence were acquired with X and Y pixel dimensions set at 50 nm. The Z plane was advancing in 50 nm steps from intracellular to extracellular space across the myocyte surface. Top: Two orthogonal views of NaV1.5 in the same area of a myocyte, illustrating how NaV1.5 distribution pattern varied depending on the Z position (noted by the cyan dotted lines in XY and YZ views). The one on the right was at a Z plane 0.3 µm beneath the surface. NaV1.5 was in striations (open triangles) and on lateral surfaces, typical of NaV1.5 distribution in myocyte interior. The one on the left shows random NaV1.5 clusters, representing its distribution on myocyte surface at a Z plane 0 μm. The XY plane image of the latter was exported to ImageJ and analyzed in the following steps: (a) demarcating ROI, (b) clearing signals outside ROI, (c) segmenting signals inside ROI to define NaV1.5 clusters, and (d) calculating cluster parameters: average size (μm2), density (% of ROI area occupied by clusters), and mean immunofluorescence intensity in clusters. (B) Examples of NaV1.5 clusters on the surface of CON and PKA myocytes (Z plane at 0 μm). Top: Orthogonal views. Middle: NaV1.5 clusters outlined. Bottom: Cluster parameters. (C) Bar graphs (mean + SE) and individual data points of cluster parameters from CON and PKA myocytes. Date are pooled from two independent experiments with number of ROIs analyzed and number of clusters detected listed on the left.
