Ma3 916

Heart lysates were prepared as previously described [22 (link)]. Briefly, the whole hearts were homogenized and sonicated in lysis buffer (in mmol/L: Tris-HCl 50 [pH 7.4], NaCl 150, NaF 10, Na₃VO₄ 1, EGTA 5, EDTA 5, 0.5% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, and protease inhibitors (#P8340, Sigma-Aldrich)). After standing on ice for 1 h allowing lysis, the homogenates were centrifuged at 13,000g 4 °C for 15 min. The supernatants were kept as whole cell proteins. Protein concentration was measured by BCA assay.
Proteins were separated on 4–12% Bis-Tris gels and transferred to PVDF membrane. The membrane was probed with primary antibodies against MG53 (provided by Dr. Jianjie Ma), NCX1 (1:1000, #R3F1, Swant, Switzerland), RyR2 (#MA3-916, Thermo Scientific), SERCA2a (#MA3-919, Thermo Scientific), Junctophilin-2 (JP2, #SC51313, Santa Cruz Biotechnology), Amphiphysin 2 (Bin1, #SC23918, Santa Cruz Biotechnology) and GAPDH (1:10,000, #G8975, Sigma-Aldrich) overnight at 4 °C, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000–1:10,000 dilution in PBS solution). The immunoreactions were visualized using an enhanced-chemiluminescent detection kit and the protein bands were quantified with Quantity One 1-D Analysis Software (Bio-Rad, USA).

Samples were snap-frozen in optimal cutting temperature (OCT) compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands) and stored at −80°C until usage. Tissue sections of 100μm thickness were acquired with a cryotome (CM 1950, Leica AG, Wetzlar, Germany), immediately immersed in 1% paraformaldehyde for 15min and subsequently washed in PBS. RyRs were immunolabeled (MA3-916 and A-21121, ThermoFisher Scientific, Waltham, MA, USA), the sarcolemma and extracellular matrix stained with wheat germ agglutinin (WGA, ThermoFisher), and nuclei with DAPI as described previously.^{15 (link)} To stain L-type Ca channels, we used the same protocol on non-fixed tissue slices and applied rabbit anti-CACNA1C (1:200, ab58552, abcam) as primary and goat anti-rabbit conjugated to AF488 (1:400, A27034, ThermoFisher) as secondary antibodies. Tissue slices were mounted on a glass slide, embedded in Fluoromount-G (#17984-25, Electron Microscopy Science, Hatfield, PA, USA) and then dried for at least 24h at room temperature and <40% relative humidity.

Protein extraction and Western blotting were performed as previously described.^{2 (link)} Briefly, mouse or human tissue lysates or SR fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyl difluoride (PVDF) membranes. The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na⁺/Ca²⁺-exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca²⁺/calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made),^{16 (link)} and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA). Membranes were then washed in tris-buffered saline and incubated with secondary anti-mouse and anti-rabbit antibodies (both 1:10,000) conjugated respectively to Alexa-Fluor 680 (Life Technologies, Carlsbad, CA) and IR800Dye (Rockland Immunochemicals, Gilbertsville, PA). The fluorescence was visualized on an Odyssey infrared scanner (Li-Cor, Lincoln, NE) and the bands were quantified using ImageJ (National Institute of Health, Bethesda, MD).