Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: Figure 3. Time-dependent increase in EB1 during 8CPT-cAMP/okadaic acid incubation in ventricular myocytes. (A) EB1 and NaV1.5 immunofluores- cence 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 im- munofluorescence 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Incubation

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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 inter- calated 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Immunostaining

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: Figure 5. Quantification of immunofluorescence (IF) intensity and distribution along the lateral surface and at ICD of ventricular myocytes im- munostained 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.”

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Immunofluorescence

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, Immunofluorescence

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, Immunofluorescence

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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).

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, Immunofluorescence

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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 envi- ronment 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Incubation, Control, Lysis, Western Blot, Immunoprecipitation, Cell Culture, Staining, Modification, Fluorescence

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, In Situ, Proximity Ligation Assay, Immunofluorescence, Transduction, Cell Culture, Expressing, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: Figure 11. EB1 and β-Tub coimmunoprecipitation was reduced, while NaV1.5 and β-Tub coimmunoprecipitation (co-IP) trended higher with per- sistent 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Co-Immunoprecipitation Assay, Activation Assay, Expressing, Lysis, Immunoprecipitation, Negative Control, Incubation, Western Blot, Modification

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202313436

Figure Lengend Snippet: 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.

Article Snippet: Human EB1 with monomeric red fluorescent protein fused to the C-terminus through a peptide (RDP) linker (EB1-mRFP) was a gift from TimMitchison and Jennifer Tirnauer (plasmid #39323; Addgene; Harvard Medical School, Boston, MA).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Expressing, Western Blot, Incubation, Control, Immunofluorescence, Fluorescence, Marker