Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification
- University of British Columbia, Canada
Figures
Figure 1
Calcium Sparks.
(A) Representative line-scan images of Ca2+ sparks recorded from permeabilized rat ventricular cardiomyocytes under the indicated conditions. (B) Box and whisker plots of the aggregated Ca2+ spark …
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Figure 1—source data 1
Statistical analysis of calcium spark data.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig1-data1-v2.pdf
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Figure 1—source data 2
Number of sparks, cells and rats examined.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig1-data2-v2.docx
Figure 2 with 1 supplement
Tomogram of a Rat Ventricular Myocyte Dyad.
Orthogonal views, (A) XY and (B) YZ, 1nm-thick, through a dyad, with the intersecting planes (red line – XY plane, blue line - XZ plane, green line - YZ plane) positioned over a single RyR2. Single …
Figure 2—figure supplement 1
Localization and Distribution of 3 RyR2 Tetramers Identified in Figure 2 (a,b and c) on the Surface of the jSR.
(a) (i) The RyR2 shown in Figure 2D, (D12); the red arrows point at the identified RyR throughout the panel. (ii) Tilt rotation in X (blue lines), centred on the identified tetramer. (iii) The …
Figure 3
Localization, Distribution and NND Measurements of RyR2 on the Surface of the jSR in a Permeabilized Rat Ventricular Myocyte Exposed to the Phosphorylation Cocktail.
Orthogonal planes within the tomogram are outlined in different colors; XY in red, YZ in green and XZ in blue. (A) A single XY plane extracted from the dual-tilt tomogram. Single arrow points to the …
Figure 4
Permeabilized Rat Cardiomyocytes Treated with 10 µM Exogenous, Purified, FKBP12 or FKBP12.6 Either Without (A and C) or With (B and D) the Phosphorylation Cocktail.
(i) A single XY plane (red) through the dyad with the YZ (green) and XZ (blue) planes intersecting a single tetramer. M – mitochondria, t- t tubule, scale bars 30 nm. (ii) Position, orientation and …
Figure 5
Cumulative Distribution Functions of the C-to-C NND.
Each treatment group is associated with a different color and all are significantly different from each other (p<0.01). Left shifted lines have significantly smaller NND than control while those …
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Figure 5—source data 1
Statistical Analysis of the Cumulative Distribution Functions.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig5-data1-v2.pdf
Figure 6 with 1 supplement
Superresolution Images of the Distribnution of RyR2 on the Myocyte Surface.
(A) Superresolution images of the surface of isolated cardiomyocytes labeled for the ryanodine receptor under the indicated conditions. Scale bars are 500 nm. (B) Alpha shapes identify individual …
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Figure 6—source data 1
Statistical Analysis of the Cumulative Distribution Functions for the Alpha Shape Areas.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig6-data1-v2.pdf
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Figure 6—source data 2
Statistical Analysis of the Cumulative Distribution Functions for the Tetramers per Cluster.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig6-data2-v2.pdf
Figure 6—figure supplement 1
Screen Shot of the Output from the Fourier Ring Correlation Program: Resolution = 18.6 nm.
The image is from the Control experimental group. The original image (white) was split into two (red) which were correlated with each other. For further details, see Banterle et al, J. Struct. Vol …
Figure 7
Western Blots.
(A) Western blots probed with anti FKBP12 (two blots displayed, the bottom blot shows a visible band in control lane) and the proteins selected as loading controls, calsequestrin and RyR. (B and C) …
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Figure 7—source data 1
Statistical Analysis of the Western Blots.
- https://cdn.elifesciences.org/articles/51602/elife-51602-fig7-data1-v2.pdf
Figure 8 with 2 supplements
Calcium Spark and TEM Correlation Microscopy Pipeline.
(A) Enzymatically dissociated cardiac cells were plated on laminin coated ibidi u-Dish 35 mm, glass bottom Grid-50 dishes (Laminin, Roche). (B) Permeabilized cardiac myocytes were exposed to …
Figure 8—figure supplement 1
Dual Tilt Electron Tomography; Imaging and Data Processing Pipeline.
(a) A slot grid holding a 200–300 nm thick section of the same cell used for imaging calcium sparks was placed in the rotating, dual tilt stage and observed in the 200 kV Tecnai G2 transmission …
Figure 8—figure supplement 2
2D Electron Micrographs of Treated Isolated Cells.
Scale bars are 1 μm.
Videos
Video 1
Dyad of a control myocyte:tomogram and model.
Video 2
Dyad from a Myocyte Treated with the Phosphorylation Cocktail: Tomogram and Model.
Video 3
Dyad of a Myocyte Treated with FKBP12: Tomogram and Model.
Video 4
Dyad of a Myocyte treeated with FKBP12.6: Tomogram and Model.
Video 5
Dyad of a Myocyte treated with FKBP12 and the Phosphorylation Cocktail: Tomogram and Model.
Video 6
Dyad of a Myocyte treated with FKBP12.6 and the Phosphorylation Cocktail: Tomogram and Model.
Tables
Table 1
Tetramer Arrangements.
| Checkerboard | Side-by-Side | Isolated | Both | Tomograms | Tetramers | Rats | |
|---|---|---|---|---|---|---|---|
| FKBP12 | 8.4% | 57.1% | 18.5% | 16.0% | 5 | 119 | 3 |
| FKBP12+Phos | 60.5% | 13.2% | 19.4% | 7.0% | 7 | 129 | 3 |
| FKBP12.6 | 8.2% | 82.8% | 7.4% | 1.6% | 7 | 122 | 3 |
| FKBP12.6+Phos | 71.3% | 5.2% | 22.6% | 0.9% | 8 | 115 | 3 |
| Control | 50.0% | 27.6% | 14.3% | 8.2% | 5 | 98 | 3 |
| Phosphorylation | 75.0% | 6.3% | 14.1% | 4.7% | 4 | 64 | 3 |
Table 2
dSTORM Images: Cluster Areas.
| Median Cluster Area (nm2 x 103) | Mean Cluster Area (nm2 x 103) | Number of Clusters Examined | |
|---|---|---|---|
| Control (10) | 12.2 | 27.3 | 5582 |
| Phosphorylated (12) | 11.1 | 32.2 | 4997 |
| FKBP12 (13) | 5.9 | 12.4 | 3615 |
| FKBP12.6 (7) | 6.1 | 13.2 | 2444 |
| FKBP12 + Phos (10) | 16.0 | 33.3 | 7761 |
| FKBP12.6 + Phos (9) | 13.1 | 32.3 | 4248 |
Table 3
Tetramer Density per Cluster.
| Tomograms | dSTORM Images | |||
|---|---|---|---|---|
| Median Tetramer Coverage/Cluster | No. of Tomograms Examined | Median No. of Tetramers/Cluster | Mean No. of Tetramers/Cluster | |
| Control | 49.5% | 13 | 8 | 18.5 |
| Phosphorylated | 52.8% | 9 | 8 | 23.3 |
| FKBP12 | 57.1% | 12 | 5 | 9.7 |
| FKBP12.6 | 62.5% | 9 | 5 | 11.3 |
| FKBP12 + Phos | 44.9% | 7 | 10 | 20.5 |
| FKBP12.6 + Phos | 46.2% | 8 | 8 | 20.5 |
Table 4
Nearest Neighbour Centre-to-Centre Distances.
| Checkerboard | Side-by-Side | ||||
|---|---|---|---|---|---|
| Mean ± SD | Median | Mean ± SD | Median | ||
| FKBP12 | 34.8 ± 2.0 | 35.0 | 28.4 ± 0.9 | 28.5 | |
| FKBP12 + Phos | 34.3 ± 1.9 | 34.6 | 28.6 ± 0.9 | 28.8 | |
| FKBP12.6 | 33.3 ± 2.8 | 33.6 | 28.0 ± 0.8 | 28.0 | |
| FKBP12.6 + Phos | 35.1 ± 2.1 | 34.9 | 28.4 ± 0.2 | 28.4 | |
| Control | 34.2 ± 1.6 | 34.6 | 28.3 ± 0.8 | 28.1 | |
| Phosphorylation | 34.2 ± 1.9 | 34.3 | 27.7 ± 0.3 | 27.7 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Rat) | Primary left ventricular myocytes | Charles River Canada | RRID:RGD_11040548 | Wistar Male rat (150–200 gr) |
| Antibody | Anti FKBP12 (Rabbit polyclonal) | Abcam | RRID:AB_303413 | WB (1:2500) |
| Antibody | Anti Calsequestrin (Rabbit polyclonal) | Abcam | RRID:AB_303865 | WB (1:75000) |
| Antibody | RYR2 Phospho Serine 2814 Anti-Serum antibody (Rabbit polyclonal) | Badrilla | RRID:AB_2617055 | WB (1:2000) |
| Antibody | Ryanodine Receptor Monoclonal Antibody (C3-33) | Thermo Fisher Scientific | RRID:AB_2183054 | Super-res IF(1:100), WB (1:1000) |
| Antibody | Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Thermo Fisher Scientific | RRID:AB_2535805 | Super-res IF (1:50) |
| Antibody | Anti-Rabbit IgG (H+L) Polyclonal Antibody, Horseradish Peroxidase Conjugated | Innovative Research (Invitrogen/Zymed) | RRID:AB_88349 | WB (1:10000) |
| Antibody | Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP | Thermo Fisher Scientific | RRID:AB_2533947 | WB (1:10000) |
| Other | Immobilon-P PVDF membrane | Sigma Millipore | Cat# IPVH00010 | WB (0.45 µm pore size) |
| Other | FluoSpheres Carboxylate-Modified Microspheres, 0.1 µm, infrared fluorescent | Thermo Fisher Scientific | Cat# F8799 | Microspheres used in Super-res IF (Image stabilization) |
| Other | SuperSignal West Femto Chemiluminescent Substrate kit | Thermo Fisher Scientific | Cat# 34095 | Horseradish peroxidase (HRP) substrates- WB |
| Other (Calcium Indicator) | Fluo-4, Pentapotassium Salt | Thermo Fisher Scientific | Cat# F14200 | Concentration: 30 µmol/L |
| Chemical compound, drug | Protease Inhibitor Cocktail | Sigma | Cat#P-8340 | WB- lysis buffer |
| Chemical compound, drug | IBMX (3-isobutyl-1-methylxanthine/) | Sigma | Cat# 15879 | In phosphorylation cocktail as a phosphodiesterase (PDE) inhibitor (10 µmol/L) |
| Chemical compound, drug | Calyculin A from Discodermia calyx | Sigma | Cat# C5552 | In phosphorylation cocktail as an inhibitor of serine-threonine protein phosphatase 2A |
| Chemical compound, drug | Okadaic acid ammonium salt from Prorocentrum concavum | Sigma | Cat#O8010 | In phosphorylation cocktail- Inhibitor of type 1 and type 2A protein phosphatases |
| Chemical compound, drug | 2-mercaptoethanol | Sigma | Cat#M3148 | Super-res IF (140 mmol/L) |
| Chemical compound, drug | Glucose Oxidase from Aspergillus niger | Sigma | Cat#G0543 | Super-res IF (0.5 mg/ml) |
| Chemical compound, drug | Catalase from bovine liver | Sigma | Cat#C3155 | Super-res IF (40 µg/ml) |
| Software, algorithm | Hierarchical statistical data analysis | Sikkel et al., 2017 | PMID: 29016722 | Used for statistical analysis of calcium spark parameters |
| Software, algorithm | Amira for Life Sciences | Thermo Fisher Scientific (FEI) | RRID:SCR_007353 | Tomography 3D visualization and analysis software |
| Software, algorithm | ImageJ | NIH Image | RRID:SCR_003070 | Used in WB analysis |
| Software, algorithm | Fiji | NIH Image | RRID:SCR_002285 | Spark Master plugin, used in calcium spark analysis |
| Transfected construct (include species here) | E. coli RosettaTM (DE3) cells | Novagen | Cat#70954 | Expression and purification of human FKBP12 and FKBP12.6 |
| Other | Superdex 200 16/60 column | GE Healthcare | Cat#GE28-9893-35 | Expression and purification of human FKBP12 and FKBP12 |
| Other | PorosMC column | Thermofisher Scientific | Cat#1542226 | Expression and purification of human FKBP12 and FKBP12 |
| Chemical compound, drug | lysozyme | Thermofisher Scientific | Cat#89833 | 25 μg/ml (Expression and purification of human FKBP12 and FKBP12) |
| Chemical compound, drug | TEV protease | NEB | Cat#P8112S | Expression and purification of human FKBP12 and FKBP12 |
| Chemical compound, drug | imidazole | Sigma | Cat#792527 | 300 mmol/L (Expression and purification of human FKBP12 and FKBP12) |
| Chemical compound, drug | isopropyl-β-D-thiogalactoside (IPTG) | Thermofisher Scientific | Cat#BP-1755 | 0.4 mmol/L (Expression and purification of human FKBP12 and FKBP12) |
| Chemical compound, drug | PMSF | Sigma Millipore | Cat#7110 | 100 μM (Expression and purification of human FKBP12 and FKBP12) |
| Chemical compound, drug | DNase I | Sigma | Cat#11284932001 | 25 μg/ml (Expression and purification of human FKBP12 and FKBP12) |
Additional files
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Source code 1
Program to fit RyR2 tetramers to tomographic images.
- https://cdn.elifesciences.org/articles/51602/elife-51602-code1-v2.pdf
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Source code 2
Program to analyse the significance between cumultative distribution functions.
- https://cdn.elifesciences.org/articles/51602/elife-51602-code2-v2.pdf
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51602/elife-51602-transrepform-v2.docx
