Tram 34

Exogenous K⁺ was added as an isotonic physiological salt solution in which all the NaCl was replaced with an equivalent amount of KCl. Concentrations of K⁺ used are expressed as final bath concentration. Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one), L-NAME (N^G-nitro-L-arginine methyl ester), Nordihydroguaiaretic acid (NDGA), papaverine HCl, PD 146176 (6,11-Dihydro[1]benzothiopyrano[4,3-b]indole), tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), TRAM-34 (1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole) and U46619 (9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F_2α) were all obtained from Sigma (Poole, U.K.). Apamin and iberotoxin, from Latoxan (Valence, France). SLIGRL-NH₂ (serine, leucine, isoleucine, glycine, arginine, leucine) from Auspep (Parkville, Australia). 8-iso PGF_2α from Cayman-Europe (Tallinn, Estonia). tAUCB (trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) was a generous gift from Professor Bruce D. Hammock (University of California, Davis). All stock solutions (100 mM) were prepared in dimethylsulfoxide (DMSO) except L-NAME, apamin, iberiotoxin, papaverine and SLIGRL that were dissolved in 0.9% NaCl and tempol which was dissolved in ultrapure water. Vehicle controls were performed when necessary.

TRAM-34 (Sigma Aldrich, T6700) was dissolved in DMSO (10 mM stock solution, Sigma Aldrich, D2650) and used at final concentrations of 1 µM (patch-clamp) or 5 µM (all other experiments). The K_Ca3.1 channel opener 1-EBIO (1-ethyl-2-benzimidazolinone, 200 µM; Sigma Aldrich, SML0034) was diluted from a 20 mM stock solution in DMSO. Temozolomide (Sigma Aldrich, T2577) was dissolved in DMSO (100 mM stock solution) and used at final concentrations of 30 µM. Drugs were added to the cells 1 h before irradiation. Equivalent volumes of DMSO were used as control conditions. Except electrophysiology, all experiments were conducted in a blinded fashion until statistical analysis.

DiBAC₄(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K⁺ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).

The sample was imaged in phenol red-free complete medium. For calcium experiments, the imaging medium was supplemented with 5 mM sodium pyruvate, 10 µM Trolox (Santa Cruz 53188-07-1) and 0.25 mM CaCl₂. For experiments involving inhibitors, either 100 µg/mL R1 peptide (China Peptides), 1 µg/mL cytochalasin D (Sigma Aldrich C8273), or 10 µM TRAM34 (Sigma Aldrich T6700) were added to the imaging medium. For confocal microscopy, 200 µL of imaging medium and 30 µL of stained erythrocytes per well were loaded to eight-well plate (Ibidi 80826). Then, 5–10 µL of stained schizonts were added to the stained erythrocytes in the first well right before imaging. For LLSM, an acid-washed 5 mm round glass coverslip (Warner Instruments CS-5R) was attached to the bottom of each well before loading 200 µL of phenol red-free RPMI-HEPES and 30 µL of stained erythrocytes to the well. Then, 5–10 µL of stained schizonts were gently added to the top of the coverslip and left to settle on the coverslip for 15 minutes. A tweezer was used to attach the coverslip to the sample stage, then the coverslip was embedded in the microscope bath filled with 8 mL of imaging medium.

8-week-old male C57BL/6 mice (SLAC Laboratory Animals, China) were randomized to subject to permanent ligations of the left anterior descending branch of the coronary artery or to a sham operation without ligation, as described previously 13 (link). In brief, mice were anesthetized by 2% isoflurane inhalation and mechanically ventilated, the thorax was opened via left thoracotomy to expose the heart, and the coronary artery was ligated with a 6-0 silk suture. Successful occlusion of the vessel was confirmed both by the presence of myocardial blanching in the perfusion bed and dynamic changes of electrocardiogram (ECG). Sham-operated animals underwent the same procedure without ligation. Mice died within 24 h after surgery were excluded from the experiment. Immediately after MI induction or sham operation, mice were randomized to receive TRAM34 (120mg/kg, Sigma) or same volume of vehicle (corn oil, Sigma) subcutaneously every day until sacrifice.

TRAM‐34 (Wulff et al., 2000) was synthesized in‐house and dissolved in ethanol (Sigma Aldrich, Taufkirchen, Germany) to a stock solution of 5 mM and then diluted to a final concentration of 0.1, 1, or 10 μM. Apoptosis initiator staurosporine (Cell Signaling/New England Biolabs, Frankfurt, Germany) was dissolved in DMSO to a stock concentration of 1 mM and then diluted to a final concentration of 1 μM in cell culture medium.