Ryanodine

Nanoscale imaging was performed 1 d after pAcGFP-actinin plasmid transfection. In experiments under electric field stimulation, 2 mM Ca²⁺-HEPES-Tyrode’s solution was used as the bathing solution, and myocytes were electrically stimulated (Serizawa et al., 2011 (link) and references therein). For experiments on cell-SPOC, myocytes were treated with 4 µM ionomycin (Sigma-Aldrich) to increase the Ca²⁺ permeability of the membranes. Then, they were bathed in Ca-SPOC solution (pCa 5.75 buffered by Ca²⁺/10 mM EGTA; see the Methods section of the online supplemental material) containing 4 µM thapsigargin (Sigma-Aldrich) and 200 µM ryanodine (Sigma-Aldrich). OM (Selleck) was initially dissolved in ethanol and diluted with 0.6 µM Ca-SPOC solution. The final concentration of ethanol was 0.1%, having no effect on cell-SPOC.
Except for the experiment on spontaneous beating (performed at 27 ± 0.5°C), all the mechanical experiments were performed at 36.0 ± 0.5°C in this study. When changes in [Ca²⁺]_i were measured simultaneously with sarcomeric motion, the myocytes were bathed in the 2 mM Ca²⁺-HEPES-Tyrode’s solution containing 2 µM Fluo-4-AM for 20 min at 25 ± 0.5°C. Thereafter, the imaging experiments were performed at 36.0 ± 0.5°C.

Eugenol (purity=99%), ryanodine, BDM, procaine, MgATP, disodium phosphocreatine,
EGTA, imidazole, methanesulfonic acid, calcium chloride, and creatine kinase were
purchased from Sigma Chemical Co. (USA). All other reagents were analytical grade and
were purchased from Merck (Germany).