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. 2024 Feb 5;156(2):e202313436.
doi: 10.1085/jgp.202313436. Epub 2024 Jan 16.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes

Tytus Bernas  1 , John Seo  2 , Zachary T Wilson  2 , Bi-Hua Tan  3 , Isabelle Deschenes  3 , Christiane Carter  4 , Jinze Liu  4 , Gea-Ny Tseng  2
Affiliations

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes

Tytus Bernas et al. J Gen Physiol. .

Abstract

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

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Conflict of interest statement

Disclosures: The authors declare no competing interests exist. J Seo and Z.T. Wilson were premed students working in their labs as technicians while waiting for med school admission. They are now in med school and are unreachable.

Figures

Figure 1.
Figure 1.
Persistent PKA activation increased Na current amplitudes without increasing the total NaV1.5 protein level in ventricular myocytes. PKA myocytes had been incubated with 8CPT-cAMP (100 µM)/okadaic acid (100 nM) for 6–15 h before experiments. CON myocytes had been cultured for the same duration under the control conditions. (A) PKA activation is confirmed by the appearance of cAMP response element binding protein 1 with serine at position 133 phosphorylated (CREB1-S133P) and its nuclear entry. Left upper: Immunoblot (IB) images of WCLs from CON and PKA myocytes probed with antibodies specific for CREB1-S133P (top) and CREB1 (bottom). Left lower: PKA to CON ratio of CREB1-S133P band intensities (5.46 + 1.04, dotted line denotes value of 1). Right: CREB1-S133P immunofluorescence (red, nuclei stained blue) in CON and PKA myocytes. Images are presented in orthogonal view (XY, YZ, and XZ planes) to show that CREB1-S133P was within, instead of around, nuclei of the PKA myocyte. *: CREB1-S133P specific band in IB and CREB1-S133P signals within nuclei. (B) Top: Na current (INa) traces recorded using a cell-attached patch clamp with a pipette tip positioned on the top surface of the cell center or close to the cell end (within three sarcomeres) of CON and PKA myocytes. These exceptional traces were obtained on the same day (CON myocytes 5–6 pm, PKA myocytes 11 PM–midnight). During patch clamp recording, myocytes were superfused with nominally Ca-free (supplemented with 2 mM Mg) Tyrode’s without 8CPT-cAMP/okadaic acid. The pipette was filled with Ca-containing (2 mM) Tyrode’s solution. The tip resistance was (in MΩ): CON cell end 1.44 + 0.06, CON cell center 1.41 + 0.06, PKA cell end 1.33 + 0.05, and PKA cell center 1.33 + 0.06 (one-way ANOVA, P = 0.267). (C) Top: Bar graphs (mean + SE) and individual data points of maximal peak INa amplitudes. Bottom: Time constants (τ) of inactivation of maximal INa. Data were pooled from five independent experiments. The numbers of myocytes studied are shown in parentheses. (D) Airyscan images of NaV1.5 immunofluorescence from CON and PKA myocytes in XY and YZ planes. (E) Top: NaV1.5 immunoblot images of SDS extracts of CON and PKA myocytes in two independent experiments. Middle: Coomassie blue (CB) stain of the same gels to confirm even loading. Bottom: Average PKA:CON ratio of NaV1.5 band intensities (0.95 + 0.09), not different from 1 (dotted line). Information on the antibodies used in experiments shown in this and the following figures is listed in Table 1. The listed P values are from t tests against null hypothesis (A and E), or CON versus PKA myocytes (C). Source data are available for this figure: SourceData F1.
Figure 2.
Figure 2.
Persistent PKA activation for 15 h increased the size and density of NaV1.5 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.
Figure S1.
Figure S1.
Using wheat germ agglutinin (WGA) to verify differential distribution patterns of NaV1.5 inside myocytes and on myocyte surface. CON and PKA myocytes (incubation time 15 h) were stained for native NaV1.5 (green) and WGA (red). Z-stack images at 50-nm z steps were collected. Shown are images of NaV1.5, WGA, and their merge at z planes deep inside myocytes (purely inside), and at the interface between surface and subsarcolemmal space (mixed). In the latter, the surface areas are demarcated by dashed white lines. In the “purely inside” views, both NaV1.5 and WGA are in striations of ∼2 µm spacing, and along the lateral cell edge. There are WGA-positive vesicles. In the “mixed” views, NaV1.5 and WGA are in faint striations in the subsarcolemmal space. However, WGA manifests a vague hexagonal pattern while NaV1.5 is in random clusters on the surface. NaV1.5 clusters are much denser on the surface of PKA than CON myocytes.
Figure S2.
Figure S2.
The EB1 transcript was upregulated, while NaV1.5 transcript was downregulated in PKA versus CON myocytes. Total RNAs were extracted from CON and PKA myocytes (incubation time 15 h, five independent experiments) with TRIZOL reagents. After confirming high sample quality (RNA integrity number >7), the RNA concentrations were adjusted to 100 ng/µl and used for two groups of experiments. Group 1 was RNA-seq experiment: mRNA library construction followed by next-generation sequencing (VCU Genomics Core). Raw FASTQ sequences of individual samples were inspected for quality using the tool FastQC and then merged with multiQC. PE 100 bp reads were aligned to the rat primary genome assembly (mRatBN7.2, version 110). Raw counts were normalized between and within samples, using EdgeR’s calcNormFactors scaling factor of trimmed mean of M-values (TMM), that took into account of variations in library size, sequencing depth, and gene lengths. Group 2 was quantitative RT-PCR experiment: mRNA was reverse transcribed followed by PCR reactions using the following primer pairs that cross exon-intron boundaries: (a) EB1: forward 5′-TGT​CGC​TCC​AGC​TTT​GAG​TA-3′, reverse 5′-AGC​AGC​TTC​GTC​ATC​TCC​AT-3′, (b) NaV1.5: forward 5′-TCA​ATG​ACC​CAG​CCA​ATT​ACC​T-3′, reverse 5′-CCC​GGC​ATC​AGA​GCT​GTT-3'. House-keep transcript GAPDH was included in the qPCR reactions. Data of TMM (RNA-seq) or GAPDH (qPCR) normalized transcript levels are presented as individual data points of CON-PKA pairs. Each pair is connected by a dashed line for visual effect clarification. The P values are from paired t tests between CON and PKA data points.
Figure 3.
Figure 3.
Time-dependent increase in EB1 during 8CPT-cAMP/okadaic acid incubation in ventricular myocytes. (A) EB1 and NaV1.5 immunofluorescence signals in CON and PKA myocytes. The incubation times with 8CPT-cAMP/okadaic acid are listed on left. (B) Pixel contents of EB1 and NaV1.5 immunofluorescence in CON and PKA myocytes at different incubation times listed along the abscissa. Data were normalized by the mean value of CON myocytes at the 0.5 h time point (dashed lines). Listed P values are from the t test of PKA versus CON. All scale bars are 10 μm.
Figure 4.
Figure 4.
Persistent PKA activation for 15 h induced microtubule reorganization in ventricular myocytes. (A) Left: Immunoblot images of cytosolic fraction from CON and PKA myocytes in two independent experiments. Antibodies targeted EB1, total α-tubulin (α-Tub), and detyrosinated α-tubulin (deY α-Tub). CB confirms even loading. In the immunoblot image of α-Tub, and immunoblot images in the following figures, the dotted vertical line indicates lane(s) in between removed for presentation (corresponding uncropped images are shown in source data). Right: PKA:CON ratios of band intensities pooled from the number of immunoblots shown in parentheses. (B) XY and YZ plane images of CON and PKA myocytes immunostained for EB1, β-tubulin (β-Tub), and deY α-Tub. “Tubulin merge” is combined β-Tub and deY α-Tub signals. (C) Reduction of interfibrillar microtubules in PKA versus CON myocytes. Top: XY and YZ plane images of β-Tub immunofluorescence in CON and PKA myocytes. In the PKA myocyte’s YZ-plane view, the β-Tub dense region was part of an intercalated disc (ICD, based on nCadherin immunostaining, not shown). Bottom: Microtubule density quantified as “% myocyte central area occupied by β-Tub puncta,” where “myocyte central area” was defined as the cross-sectional area within 1 μm from cell contour. Numbers in parentheses are those of the myocytes studied. (D) Immunofluorescence signals of β-Tub, deY α-Tub, and their merge at a z plane close to the surface of CON and PKA myocytes. Listed P values are from t tests against null hypothesis (A), or PKA versus CON (C). Scale bars are 10 μm for B, 2 μm for C, and 5 μm for D. Source data are available for this figure: SourceData F4.
Figure S3.
Figure S3.
Colocalization of α-tubulin and β-tubulin (α- and β-Tub, respectively) immunofluorescence in rat ventricular myocytes. Shown are Airyscan images α-Tub and β-Tub immunofluorescence (detected by α-Tub rat Ab and β-Tub mouse Ab) and their merge in control and PKA myocytes (PKA activation 12 h). Images were obtained in a z-plane close to the myocyte’s surface.
Figure 5.
Figure 5.
Quantification of immunofluorescence (IF) intensity and distribution along the lateral surface and at ICD of ventricular myocytes immunostained for NaV1.5, EB1, β-Tub, and nCadherin (nCad). Top: Representative Airyscan images. The central z-plane image with a clearly defined cellular contour was used for quantification. Pixel contents = background-subtracted mean pixel value times cellular area. Bottom left: IF signal within an area 1 μm from cellular contour normalized by total pixel content gave “% pixel contents on the lateral surface.” This value was divided by % cellular area within the 1 μm rim and defined as “degree of enrichment on lateral surface.” Bottom right: IF signals within all areas demarcated by nCad IF signals were summed and normalized by total pixel content to give “% pixel contents at ICD.” This value was divided by “% cellular area demarcated by nCad signals,” and defined as “degree of enrichment at ICD.”
Figure 6.
Figure 6.
Persistent PKA activation for 12 h caused an enrichment of NaV1.5, EB1, and β-Tub immunofluorescence along the lateral surface and at intercalated discs of ventricular myocytes. (A) Airyscan images of NaV1.5, EB1, β-Tub, and nCad immunofluorescence in CON and PKA myocytes. (B) Box plots of whole myocyte pixel content (normalized to the mean values of CON myocytes), enrichment of immunofluorescence along the lateral surface, and enrichment of immunofluorescence at intercalated disc regions. Data were pooled from five independent experiments, with 40–70 myocytes per group. Dotted line indicates CON mean (top) or no enrichment (middle and bottom). Listed P values are from t tests of PKA versus CON myocytes.
Figure 7.
Figure 7.
Persistent PKA activation for 15 h increased the size and density of NaV1.5, EB1, and β-Tub clusters at ICDs. (A) Procedures of analyzing clusters at ICDs. Top: Orthogonal views of cell ends of CON and PKA myocytes, showing merged immunofluorescence signals of nCadherin (nCad, blue), NaV1.5 (green), EB1 (white), and β-tub (red). Middle: YZ-plane views of individual immunofluorescence, with ROI demarcated by nCad (white dotted lines). Bottom: Signals outside ROIs were cleared (black background), and signals inside ROIs were segmented to define clusters (black clusters on white background). This was followed by quantification of cluster density (% ICD area occupied by clusters), average size (µm2), and mean immunofluorescence intensity in clusters. (B) Box plots of cluster parameters for NaV1.5, EB1, and β-Tub at ICDs of CON and PKA myocytes. Data are pooled from four independent experiments. For each myocyte, 10–12 YZ-plane images at 0.25–0.5 µm intervals, advancing from the cell end toward the cell center (before nCad signals disappeared), were exported to ImageJ for analysis. Numbers shown in B are those of ROIs quantified. Listed P values are from t tests between CON and PKA myocytes. In A, scale bars are 2 μm for the top row and 1 μm for the middle and bottom rows.
Figure 8.
Figure 8.
Persistent PKA activation promoted the correlative distribution of NaV1.5, β-Tub, and EB1 on myocytes’ surface and ICDs. Top: Cartoon depicting the planes of view. (A) Left: 2-D views of NaV1.5, β-Tub, and EB1 immunofluorescence on myocytes’ surface. Right: 3-D views of NaV1.5, β-Tub, and EB1 immunofluorescence at myocytes’ ICD. For each myocyte, six panels are shown: NaV1.5, β-Tub, and EB1 individual immunofluorescence (upper row), and merged (lower row). In the PKA myocyte’s surface images (lower left), cyan dotted circles denote looped microtubules (β-Tub panel) overlapped with EB1 puncta (EB1 panel). In the ICD images (right), semitransparent blue surfaces denote nCadherin-demarcated ICD volumes. (B) Box plots of thresholded Pearson correlation coefficients between the pairs of immunofluorescence signals listed along the abscissa in CON and PKA myocytes. Data were pooled from 29 to 41 myocytes per group from two independent experiments. Listed P values are from t tests between CON and PKA myocytes. In A, scale bars are 5 μm for the myocyte surface views (left) and 2 μm for the myocyte ICD 3-D views (right).
Figure 9.
Figure 9.
NaV1.5 and EB1 could be coimmunoprecipitated reciprocally when expressed in HEK293 cells but not in ventricular myocytes, while FRAP experiments suggested NaV1.5/EB1 interactions at the ICD region of myocytes. (A) GFP-NaV1.5 expressed in HEK293 cells was coimmunoprecipitated with native EB1 reciprocally. WCL was prepared from HEK293 cells incubated with 8CPT-cAMP/okadaic acid for 4 h or time control with 1% Triton lysis buffer. (−) IP lanes were loaded with eluates from protein A/G beads incubated with WCL without antibody. (+) IP lanes were loaded with eluates from protein A/G beads incubated with WCL and immunoprecipitating (IP) antibody: GFP rabbit Ab (left) or EB1 rat Ab (right). The immunoblot (IB) Abs are listed on the left. Double and single blue circles denote EB1 dimer and monomer bands. (B) GFP-NaV1.5 and EB1-mRFP coexpressed in cardiac myocytes did not coimmunoprecipitate. Experiments validating GFP-NaV1.5 and EB1-mRFP as surrogates of native NaV1.5 and EB1 in myocytes are presented in Fig. S4. Shown are protein(s) expressed in myocytes and conditions (CON or PKA; top), proteins loaded in lanes: WCL (prepared with 1% Triton lysis buffer), IP with mCherry “mChr” or GFP rabbit “rab” Ab, and supernatant (WCL after immunoprecipitation; middle), and immunoblot images probed with GFP goat Ab (upper row) or EB1 rat Ab (lower row). Left: Specificity of immunoprecipitation. EB1-mRFP expressed alone could be immunoprecipitated with mCherry Ab but not by GFP Ab, and GFP-NaV1.5 expressed alone could be immunoprecipitated with GFP Ab but not by mCherry Ab. Right: In WCLs prepared from myocytes coexpressing GFP-NaV1.5 and EB1-mRFP cultured under CON or PKA conditions for 15 h, mCherry rabbit Ab immunoprecipitated EB1-mRFP but not GFP-NaV1.5, and GFP rabbit Ab immunoprecipitated GFP-NaV1.5 but not EB1-mRFP or native EB1. Red star and blue circle denote the band positions of EB1-mRFP and native EB1, respectively. (C) GFP-NaV1.5 in HEK293 cells was present in Triton-soluble fraction (detected in 1% Triton WCL), while NaV1.5 in ventricular myocytes was not present in Triton-soluble fraction (undetectable in 1% Triton lane) but could be extracted with 2% SDS RIPA buffer (detected in 2% SDS lane). (D) Contrasting the subcellular environment of NaV1.5 and EB1 in ventricular myocytes. Shown are immunoblot images of cytosolic and SDS extracted fractions of CON and PKA myocytes (incubation 15 h) probed for NaV1.5 and EB1. CB stain shows loading levels. The CB stain of the cytosolic fraction is modified from the one shown in Fig. 4 A. (E) Using FRAP to monitor mobilities of GFP-NaV1.5 and EB1-mRFP expressed in ventricular myocytes. Top left: Representative images of a live myocyte with four ROIs marked: red—cell center, green—cell end, blue—reference in cell area not photobleached, yellow—background in cell-free area. The corresponding time courses of FRAP are plotted below. Background bleach was corrected based on fluorescence decline in ROI 3, and the fluorescence intensity was normalized to between 1 (right before photobleaching) and 0 (the first scan after photobleaching). Bottom: Average time courses of FRAP of GFP-NaV1.5 and EB1-mRFP. Shown are the mean (colored bright and dark green for GFP-NaV1.5 or bright and dark red for EB1-mRFP) and standard error (gray) values superimposed on double-exponential fit (black curve). Left most panel illustrates the calculation of “% of fluorescence recovered 2 min after photobleaching.” Top right: Bar graphs (mean and SE) and individual data points of percentage of fluorescence recovered 2 min after photobleaching for GFP-NaV1.5 and EB1-mRFP measured from cell center and cell end. Listed P values are from t tests between specified groups. Source data are available for this figure: SourceData F9.
Figure S4.
Figure S4.
Validating GFP-NaV1.5 and EB1-mRFP as surrogates for native NaV1.5 and EB1 in ventricular myocytes. Validating GFP-NaV1.5 and EB1-mRFP as surrogates for native NaV1.5 and EB1 in ventricular myocytes. (A) Myocytes were incubated without (no Adv) or with Adv-GFP-NaV1.5 for 12 h. The medium was exchanged for virus-free fresh medium and culture continued for 0, 6, 20, 36, or 48 h (total culture times: 12, 18, 32, 48, and 60 h). At specified times, myocytes were fixed for experiments. Top: Airyscan images of GFP-NaV1.5 immunofluorescence detected with GFP goat Ab (culture time: 18–48 h), and GFP fluorescence (FP) of a myocyte cultured for 60 h. Right: Immunofluorescence of native NaV1.5 in a freshly isolated myocyte. At 18 h, GFP-NaV1.5 clustered around nuclear envelope representing protein translation in the NE/rER. At 24 h, GFP-NaV1.5 spread out in striations along z-lines, similar to the nuclear envelope to SR along t-tubules or NEST pathway (He et al., 2020). By 32 h, GFP-NaV1.5 reached the lateral surface and ICD. The distribution pattern was stable at 48 and 60 h culture times. These data showed that the steady-state distribution pattern of GFP-NaV1.5 was similar to that of native NaV1.5, except active GFP-NaV1.5 translation at NE/rER. Bottom: Degree of GFP-NaV1.5 expression versus native NaV1.5 based on pixel contents. Myocytes without or with Adv-GFP-NaV1.5 for the total culture times shown along the abscissa were immunostained with Alomone asc005 rabbit Ab, which detected both GFP-NaV1.5 and native NaV1.5. The total pixel contents were quantified and normalized to the mean pixel contents of “No Adv” myocytes of the same culture time. Shown is a bar graph of mean + SE superimposed with individual data points. “Adv-GFP-NaV1.5” myocytes did not have more immunofluorescence than “no Adv” myocytes until the 32 h time point. By the 48 h time point, “Adv-GFP-NaV1.5” myocytes had 30% more immunofluorescence than “no Adv” myocytes. These data showed a modest expression of GFP-NaV1.5 over native NaV1.5. The native NaV1.5 immunofluorescence image is a duplicate from Fig. 3 A, NaV1.5 immunofluorescence image from a control myocyte after culture for 1 h. (B) (a) Confirming immunoblot banding pattern of EB1-mRFP and native EB1 in HEK293 cells. EB1 rat Ab detected only the native EB1 band (30 kD, blue dot) in untransfected cells, while it detected both native EB1 and EB1-mRFP (expected 55.7 kD, two bands at and above 50 kD, red star) in transfected cells. The identity of the two EB1-mRFP bands was confirmed by reprobing the membrane with mCherry rabbit Ab. (b) EB1-mRFP expressed in ventricular myocytes. Left: IP lane shows EB1-mRFP coimmunoprecipitation with native EB1 (red star and blue circle), indicating dimerization between the two to form active EB1 (Chen et al., 2014). The WCL lane shows that Adv-mediated EB1-mRFP expression was ∼150% over native EB1 (band intensity ratio 2.5:1). This immunoblot image is modified from the same experiment shown in Fig. 9 B, bottom right. Right: In live myocytes (images obtained during FRAP experiments, Fig. 9 E), EB1-mRFP had a wavy strand morphology along the long axis of CON myocyte, suggesting binding along the microtubule lattice. In PKA myocyte, EB1-mRFP clustered to ICD and manifested striations at the z-plane adjacent to myocyte surface. The difference in EB1-mRFP patterns between CON and PKA myocytes is similar that of native EB1 between CON and PKA myocytes (Figs. 4 and 8). Scale bars are 10 mm in A and 5 mm in B. Source data are available for this figure: SourceData FS4.
Figure 10.
Figure 10.
Persistent PKA activation promoted NaV1.5/EB1 interactions in myocytes. (A) Detecting GFP-NaV1.5 and EB1-mRFP interaction in ventricular myocytes by in situ proximity ligation assay (PLA). Shown are PLA signals and immunofluorescence of GFP-NaV1.5 and EB1-mRFP in myocytes transduced with Adv-GFP-NaV1.5 and Adv-EB1-mRFP and cultured for 24 h (low expression) and 48 h (strong expression), the latter under the control conditions or with PKA activation for 15 h. These images are z-stack projections of maxima. (B) Persistent PKA activation increased PLA puncta density. The images of z-projection of maxima were segmented in a systemic manner across CON and PKA myocytes to define PLA puncta, followed by calculating the percentage of cellular area occupied by PLA puncta. To correct for the differences in GFP-NaV1.5 and EB1-mRFP expression level, the PLA puncta density of each of the myocytes was divided by the product of normalized GFP-NaV1.5 and EB1-mRFP pixel content of the same myocyte from the same myocyte. Numbers in parentheses are myocytes studied from two independent experiments, with P value from t test between the two groups. (C) Top: Cartoon depicting plane of view. Bottom: 3-D views of PLA puncta in a CON myocyte, as a merge of PLA puncta (white), GFP-NaV1.5 (green), and EB1-mRFP (salmon) or PLA puncta within cell contour (salmon line). Scale bars in all image panels are 10 μm.
Figure 11.
Figure 11.
EB1 and β-Tub coimmunoprecipitation was reduced, while NaV1.5 and β-Tub coimmunoprecipitation (co-IP) trended higher with persistent PKA activation. Experiments were done in HEK293 cells expressing GFP-NaV1.5 and native EB1 and β-Tub, without or with PKA activation (4 h). Cells were lysed in 1% Triton lysis buffer, and WCLs were subject to immunoprecipitation with GFP rabbit or EB1 rat Ab and protein A/G beads ([+] IP). “(−) IPs” were negative control (WCL incubated with protein A/G beads without immunoprecipitating antibodies). (A) Representative immunoblot images probed with Abs listed on the left. The EB1 rabbit Ab IB image of EB1 rat Ab IP is modified from the same experiment image shown in Fig. 9 A. (B) Top: Degrees of β-Tub co-IP with GFP-NaV1.5 or EB1 from CON and PKA cells quantified by β-Tub band intensity in (+) IP lane divided by that in WCL ([+] IP/WCL). Bottom: PKA:CON ratio of degree of β-Tub co-IP with GFP-NaV1.5 or with EB1. The dotted line denotes PKA:CON of 1. Numbers in parentheses are those of independent experiments. Listed P values are from t tests against null hypothesis. Source data are available for this figure: SourceData F11.
Figure 12.
Figure 12.
Persistent PKA activation promoted trafficking of NaV1.5 from a cytosolic reservoir to the plasma membrane. (A) Biotinylation experiments in HEK293 cells expressing GFP-NaV1.5. Top: Representative immunoblot images of WCL and biotinylated fraction (Biot’) from HEK293 cells expressing GFP-NaV1.5 and native EB1, exposed to PKA for 4–6 h or without PKA for the same duration, without or with pretreatment with chloroquine 100 µM (chloroquine was present during incubation with PKA or CON). Abs used in immunoblotting are listed on the left. NKA = Na/K pump α-subunit as the loading control. The absence of EB1 in Biot’ lanes confirms the lack of contamination from cytosolic proteins. Bottom left: Summary of WCL GFP-NaV1.5 (normalized to CON) and cell surface GFP-NaV1.5 (Biot’/WCL), shown as mean + SE with individual data points. Right: PKA:CON ratio of cell surface GFP-NaV1.5 in “No chloroquine” and “With chloroquine” groups (n =10 and 4, respectively; t test between the two groups: P = 0.046). (B) Immunofluorescence images of native NaV1.5 or GFP-NaV1.5, RyR2, and fluorescence images of wheat germ agglutinin (WGA, marker of plasma membrane and t-tubules) from the types of myocytes listed above. A detailed description is in the Discussion section. LS: lateral surface. The myocyte image second from right is a duplicate from Fig. 3 A, NaV1.5 immunofluorescence image from a control myocyte after culture for 1 h. (C) Cartoon of working hypothesis. Scale bars in B are 10 mm. Source data are available for this figure: SourceData F12.
Figure S5.
Figure S5.
Acute β-adrenergic stimulation does not induce NaV1.5 clustering to intercalated disc region. Adult rat ventricular myocytes were incubated under the control conditions or with isoproterenol (ISO, 100 nM) for 15 min before the experiment. Myocytes from the same heart were cultured under the control conditions or with 8CPT-cAMP (100 μM)/okadaic acid (100 nM) for 12 h before the experiment. (A) Representative Airyscan images of NaV1.5 immunofluorescence from the four groups of myocytes. (B) Bar graph (mean + SE) and individual data points of NaV1.5 enrichment at intercalated discs. The numbers of myocytes analyzed are in parentheses. Listed P values are from t test between PKA and CON myocytes. Scale bars in A are 10 μm.
Figure 13.
Figure 13.
Differential effects of nocodazole on newly translated versuss existing NaV1.5. (A and B) Disrupting microtubules by nocodazole (10 µM, 12 h) prevented newly translated GFP-NaV1.5 from reaching the lateral surface and intercalated discs (A), but did not alter the distribution pattern of native NaV1.5 in ventricular myocytes (B). Scale bars are 10 μm.
Figure 14.
Figure 14.
Forskolin (adenylate cyclase activator) mimics the effects of PKA activation, while a membrane-permeable PKA peptide inhibitor (PKI-14-22 amide, myristoylated) reduces the effects of PKA without affecting CON myocytes. Five groups of myocytes isolated from the same heart were incubated for 4 h under the control conditions (CON), with 10 µM PKI (CON+PKI), with 10 µM forskolin, with 8CPT-cAMP (100 µM)/okadaic acid (100 nM) (PKA), and further 10 µM PKI (PKK+PKI). Myocytes were immunostained for NaV1.5 and nCadherin, and subject to Airyscan imaging followed by quantification. (A) Representative merged NaV1.5 and nCadherin views of myocytes corresponding to the groups in B. (B) Summary of NaV1.5 enrichment at ICDs (nCad-positive areas) from the five groups listed along the abscissa. The dotted line at the y-axis 1 denotes no enrichment. Numbers in parentheses are those of myocytes quantified. Statistical analysis was one-way ANOVA on ranks, P < 0.05, followed by all-pairwise comparisons using Dunn’s method. Shown are P values <0.05 or >0.05 between specified groups.
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References

    1. Barry, D.M., Trimmer J.S., Merlie J.P., and Nerbonne J.M.. 1995. Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ. Res. 77:361–369. 10.1161/01.RES.77.2.361 - DOI - PubMed
    1. Bartsch, D., Casadio A., Karl K.A., Serodio P., and Kandel E.R.. 1998. CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic modulator that form a regulatory unit critical for long-term facilitation. Cell. 95:211–223. 10.1016/S0092-8674(00)81752-3 - DOI - PubMed
    1. Bers, D.M. 2002. Cardiac excitation-contraction coupling. Nature. 415:198–205. 10.1038/415198a - DOI - PubMed
    1. Bogdanov, V., Soltisz A.M., Moise N., Sakuta G., Orengo B.H., Janssen P.M.L., Weinberg S.H., Davis J.P., Veeraraghavan R., and Györke S.. 2021. Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes. Basic Res. Cardiol. 116:63. 10.1007/s00395-021-00895-3 - DOI - PMC - PubMed
    1. Caporizzo, M.A., Chen C.Y., and Prosser B.L.. 2019. Cardiac microtubules in health and heart disease. Exp. Biol. Med. 244:1255–1272. 10.1177/1535370219868960 - DOI - PMC - PubMed
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