AKAP6 and phospholamban colocalize and interact in HEK‐293T cells and primary murine cardiomyocytes

Abstract Phospholamban (PLN) is an important Ca2+ modulator at the sarcoplasmic reticulum (SR) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca2+ ATPase (SERCA2) function, whereas a protein kinase A (PKA)‐dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post‐translational modification, however, remains not fully understood. Using publicly available databases, we identified A‐kinase anchoring protein 6 (AKAP6) as a candidate that might play some roles in PLN phosphorylation. Immunofluorescence showed colocalization between GFP‐AKAP6 and PLN in transfected HEK‐293T cells and cultured mouse neonatal cardiomyocytes (CMNCs). Co‐immunoprecipitation confirmed the functional interaction between AKAP6 and PLN in HEK‐293T and isolated adult rat cardiomyocytes in response to isoproterenol stimulation. Functionally, AKAP6 promoted Ca2+ uptake activity of SERCA1 in cotransfected HEK‐293T cells despite the presence of PLN. These results were further confirmed in adult rat cardiomyocytes. Immunofluorescence showed colocalization of both proteins around the perinuclear region, while protein–protein interaction was corroborated by immunoprecipitation of the nucleus‐enriched fraction of rat hearts. Our findings suggest AKAP6 as a novel interacting partner to PLN in HEK‐293T and murine cardiomyocytes.


Immunofluorescence of HEK293-T
Cells were in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde on ice and permeabilized with ice cold 90% methanol at -20°C for 1 min. After permeabilization, the cells were blocked with blocking solution (5% FBS in PBS) at room temperature for 1 hr. The cells were incubated overnight at 4°C with the primary antibodies diluted in blocking solution. The next day, the cells were washed in PBS. The fluorophore-conjugated secondary antibodies were diluted in blocking solution, and the staining of the cells was performed at room temperature for 1 hr in the dark. The samples were counterstained with 4', 6-Diamidino-2-Phenylindole (DAPI, Sigma-Aldrich) or Hoechst 33342 (nuclear stain, Sigma) for 30 min in the dark. The slides were mounted in the Fluoromount medium (Sigma). The slides were visualized and Zstacked with a Zeiss spinning disk confocal microscopy (Zeiss Observer.Z1). A three-dimensional interpretation of the multichannel Z-stack images was performed using Imaris software.

Immunofluorescence of Neonatal and Adult Ventricular Myocytes
Isolated cells were washed gently with PBS at 4°C for 20 min. The cells were then fixed with 4% PFA for 30 min at 4°C. Then, they were incubated at 4°C with fresh permeabilization solution (0.2% Tween-20, 0.5% Triton X-100 in 1x PBS). The cells were then washed gently with PBS at 4°C three times for 5 min each. The cells were then incubated in blocking solution (5% FBS, 0.2% Tween-20, 0.5% Triton X-100 in PBS) for 30 min at room temperature. They were then incubated overnight at 4°C with primary antibody which was diluted in blocking solution with gentle shaking. The primary antibodies used for these experiments were mouse monoclonal 2D12 anti-PLN antibody (Abcam, 1:500) and mouse anti-AKAP6 antibody (OR017.720 -Biolegend, 1:500). The next day, the cells were gently washed with the permeabilization solution twice (15 min each) with PBS. The fluorescent conjugated secondary antibodies (Alexa Fluor 488, 1:500 or 633, 1:500, Life Technologies) diluted in blocking solution were applied to the cells and then incubated in the dark for 1 hr at room temperature. Afterwards, the secondary antibodies were removed, and the cells were washed twice (15-min each) with PBS in the dark at room temperature. The cells were then counterstained with 4', 6-Diamidino-2-Phenylindole (DAPI, Sigma-Aldrich) or Hoechst 33342 (nuclear stain, Sigma) for 30 min in the dark. Subsequently, the cells were washed twice (15 min each) with PBS in the dark. The cells were then visualized and Z-stacked using. A three-dimensional interpretation of the multichannel Z-stack images was performed using Imaris software (Bitplane).

Transduction of CMNC
Polybrene was diluted to the final concentration of 10 µg/mL in Prewarmed DMEM/F12. The amount of this mixture was the same as the amount of virus required (2 mL per each well of a 6-well plate). The mixture was then gently administered on the CMNCs and incubated at 37°C for 90 min. During this time, the required amount of viral stock was thawed at room temperature. After the incubation time, the mixture was removed, and the virus was added to the culture. CMNCs were then incubated for 21 hr with lentiviral solution at 37°C, 5% CO2. The next day the media was changed to DMEM/F12 supplemented with 2% FBS and 1% P/S for an additional 24 hr prior to any downstream application. For the cells were kept for 96 hr post-transduction while the media was changed every other day.

SERCA1 activity measurements
Cells were collected and homogenized in phenylmethylsulfonyl fluoride (PMSF) solution (250mM sucrose, 5nM HEPES, 0.2mM phenylmethylsulfonyl fluoride, 0.2% sodium azide, and 1%triton X-100), sonicated (2secs on: 5 secs off for 20secs total). They were then centrifuged at 10,000g for 30 min at 4 o C. The supernatant (75ul) was then diluted into 5ml of ATPase assay solution (100 mM KCl, 20 mM HEPES, 10 mM MgCl2, 10 mM NaN3, 10 mM phosphoenolpyruvate, 5 mM ATP, 1 mM EGTA, pH 7.0), 18U/ml lactate dehydrogenase, 18 U/ml pyruvate kinase, and 4uM ionophore A23187 (Sigma C7522). Each reaction was measured in duplicate with a reaction volume of 100 μl. The reaction was started by adding 0.3 mM NADH. Total ATPase activity was measured using a spectrophotometric plate reader (SPECTRAMAX plus; Molecular Devices) across Ca 2+ concentrations ranging from pCa 7.0 to 4.5. Basal activity was determined in the presence of 40 μM of the Ca 2+ -ATPase inhibitor cyclopiazonic acid in dimethyl sulfoxide. The free Ca 2+ concentration corresponding to each CaCl2 addition was assessed separately using dual-emission spectrofluorometry and the fluorescent Ca 2+ -binding dye indo 1. The data were analyzed by nonlinear regression with computer software (Graph Pad Software), and the apparent Ca 2+ affinity (KCa) values were calculated by using an equation for a general cooperative model for substrate activation. The values for maximal SERCA2a activity that occurred at pCa 6.423 to 5.407 were taken directly from the experimental data and normalized to protein content measured by the BCA assay.

Adenoviral transduction of AVC
The adenoviruses used in this experiment were kindly contributed by Kapiloff laboratory. The cardiomyocytes were transfected with a titer of 1000 MOI for 72 hr with Ad-U6 AKAP6 7210D mp2A adenovirus containing rat specific AKAP6 knock down shRNA and rAKAP6 7210D mut.C adenovirus containing control scrambled shRNA. The media was collected after 72 hr and the cells were fixed for IF.

Imaris analysis
The 3D reconstructions and colocalization analysis were performed using imaris software and automatic thresholding developed by Costes and Lockett at the National Institute of Health, NCI/SAIC was used (Costes et al., 2004).

Statistical analysis
Using GraphPad Prism 5, one-way ANOVA analysis was utilized to test the differences and the significance among more than two groups. To compared two groups unpaired t-test was performed. The data are expressed as mean ± SEM. A P-value of < 0.05 was considered significant.    Table S2. Normalized Mean and SEM of RNA-seq data for AKAPs in human hearts. HPM (n=85, individuals, unit: Fragments Per Kilobase Mil-lion (FPKM)), HPA (n=4, heart tissues, unit: Transcripts Per Kilobase Million (TPM)), GTEx (n=412, heart tissues, unit: Reads Per Kilobase Million(RPKM)), FANTOM5 (n=4, individuals, unit: Tags Per Million) and GEO (n=4, individuals, unit: Reads Per Kilobase Million(RPKM)) database. An 'X' is incorporated where RNA-seq data is not found. The data shows the Mean± SEM expressions normalized to the maximum mean signal all across within each dataset, except for the HPM results that only provided Mean expression values. Table S3. Normalized Mean and SEM of RNA-seq data of human AKAP candidates in non-hypertrophic and hypertrophic adult human. RNA seq data were extracted from GEO data and was normalized relative to the highest expression across all samples. These results are shown as mean ± SEM; n=4 for normal and n=5 for hypertrophic samples. Two-tailed unpaired t-test. *p<0.05 comparing normal versus hypertrophic patients' RNA expression in the heart for each AKAP.