Ryanodine receptor dispersion disrupts Ca2+ release in failing cardiac myocytes.

Key resources table

Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background ( Rattus norvegicus , M) Male Wistar- Hannover rats Janvier-labs RjHan: WI; RGD: 13792727 Antibody Mouse-anti-RyR2 primary antibody ThermoFischer Scientific Cat# MA3-916; RRID: AB_2183054 IHC, 1:100; WB, 1:1000 Antibody Alexa Fluo 647 conjugated goat-anti-mouse secondary Ab Molecular Probes/Invitrogen Cat# A-21237; RRID: AB_2535806 IHC, 1:200 Antibody Anti-goat IgG-HRP linked whole antibody R and D Systems Cat# HAF109; RRID: AB_357236 WB, 1:3000 Antibody Mouse IgG HRP linked Whole Ab GE Healthcare Cat# NA931V; RRID: AB_772210 WB, 1:3000 Antibody Rabbit IgG HRP linked Whole Ab GE Healthcare Cat# NA934V; RRID: AB_772206 WB, 1:3000 Antibody Goat Anti-GAPDH Polyclonal antibody Santa Cruz Biotechnology Cat# sc-20357; RRID: AB_641107 WB, 1:500 Antibody Goat Anti- Junctophilin-2 Polyclonal Antibody Santa Cruz Biotechnology Cat# sc-51313; RRID: AB_2296391 WB, 1:1000 Antibody BIN1 (Amphiphysin II (2F11) Antibody) Santa Cruz Biotechnology Cat# sc-23918; RRID: AB_667901 WB, 1:500 Software, algorithm Dense Stochastic Sampling Imaging (DSSI) algorithm SoftWoRx, GE Healthcare Software, algorithm dSTORM image post-processing algorithm PMID: 26490742 Described in the Github repository ( Kolstad, 2018 ; copy archived at https://github.com/elifesciences-publications/Ryanodine_Receptor_Dispersion_during_Heart_Failure ) Software, algorithm Mathematical Model PMID: 22495592 ; PMID: 23708355 ; this paper Described in the Github repository ( Kolstad, 2018 ; copy archived at https://github.com/elifesciences-publications/Ryanodine_Receptor_Dispersion_during_Heart_Failure ) Software, algorithm SigmaPlot SigmaPlot RRID: SCR_003210 Rat model of post-myocardial infarction congestive HF All experiments were approved by the Norwegian National Animal Research Authority (project license no. FOTS 5982, 7786), and were performed in accordance with the National Institute of Health guidelines (NIH publication No. 85 – 23, revised 2011) and European Directive 2010/63/EU. Large anterolateral myocardial infarctions were induced in ~300 g male Wistar-Hannover rats, by ligation of the left coronary artery as previously described ( Lunde et al., 2012 ). Development of HF was verified six weeks later using a Vevo 2100 echocardiography imaging system (VisualSonics, Toronto, Canada). Inclusion of failing animals was based on established criteria ( Sjaastad et al., 2000 ), including dilation of the left atrium (diameter >5 mm) and ventricle, and increased lung weight (>2.5 g). Sham-operated rats served as controls. Experiments were performed over a two year period, using animals from 10 rounds of animal surgery. Sample sizes were determined by power analysis, assuming that only 50% of post-infarction animals would be included in the final data set, and based on a pilot project of variability in CRU morphology in healthy controls.

Cell isolation

Cardiac myocytes from failing and Sham-operated rats were isolated using a standard enzymatic dispersion technique ( Louch et al., 2011 ). Excised hearts were mounted on a Langendorff setup, and retrogradely perfused through the aorta with Ca 2+ -free solution containing (in mmol/L): 130 NaCl, 25 Hepes, 5.4 KCl, 0.5 MgCl 2 , 0.4 NaH 2 PO 4 , 5.5 D-glucose, pH 7.4. Once cleared of blood, hearts were then perfused with the above solution including collagenase (2 mg/mL, Worthington Biochemical Corp., Lakewood, NJ, USA) and low [Ca 2+ ] (0.05 mmol/L). After 10 min of digestion, hearts were cut down, minced, and filtered, and isolated cardiomyocytes were allowed to sediment. Immunostaining Isolated cardiomyocytes were transferred to cell culture medium (DMEM 1X, Life Technologies with 10% FBS, Biowest Nuaillé, France and 1% Penicillin-Streptomycin, Sigma), and plated on laminin-coated, glass bottom culture dishes (MatTek corporation, Ashland MA). Staining was performed according to a described protocol ( Swift et al., 2007 ), with consecutive steps for chemical fixation (4% Formaldehyde in 1 mol/L HEPES buffer, 10 min), quenching (PBS + 100 mmol/L Glycine, 10 min), permeabilization (PBS + 0,03% Triton X-100, 10 min), and blocking (NaCl 150 mmol/L, Na 3 citrate 17.5 mmol/L, 5% goat serum, 3% BSA, 0.02% NaN 3 , 2 hr). PBS washing was performed in between each step. The cells were then incubated overnight with 1/100 diluted mouse-anti-RyR2 primary antibody (ThermoFischer Scientific, MA3–916) in low blocking buffer, containing 150 mmol/L NaCl, 17.5 mmol/L Na 3 citrate, 2% goat serum, 1% BSA, and 0.02% NaN 3 at 4°C. This protocol has previously been reported to result in the binding of multiple primary antibodies to each RyR tetramer ( Baddeley et al., 2009 ). The following day, cells were washed with PBS and incubated with 1/200 diluted secondary antibody (Alexa Fluo 647 conjugated goat-anti-mouse secondary Ab, Molecular Probes/Invitrogen) in low blocking buffer for 2 hr. Cells were then washed and stored in PBS until image acquisition. Of note, the fab-fragment secondary antibody employed places the fluorescent label far closer to the epitope than traditional antibodies. Thus, under our experimental conditions, the steric error is generally half the area of a 30 nm square was above threshold, and RyR clusters were defined by occupied, neighbouring grid positions. Based on previous ( Macquaide et al., 2015 ) and present ( Figure 4—figure supplement 1 ) calculations clusters with edge-to-edge distance