Xestospongin c

Drugs were dissolved in HBSS, which was used as the experimental vehicle, and applied directly to the media containing NGF. Drugs concentrations are based on published literature: choline (Acros Organics, Geel, Belgium) (10 mM, 3 mM, and 1 mM) [22 (link)]; α-bungarotoxin (Thermofisher) (BGTX) (50 nM) [32 (link)]; calpeptin (Sigma Aldrich, St. Louis MO, USA)(26 μM) [33 ], ryanodine (Santa Cruz) (30 μM) [21 (link)]; Xestospongin C. (Tocris Biosciences, Bristol, UK.) (Xest C.) (1 μM) [20 (link)]; FK506 (Tocris Biosciences) (40 μM) [34 (link)]; Substance P (Tocris Biosciences) (Sub P) (1 μM) [20 (link)]; PNU 282987 (Tocris Biosciences) (10 μM) [35 (link)].

Methylglyoxal, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), N‐acetylcysteine (NAC), 4‐phenyl butyric acid (4‐PBA), MRS1845, YM‐58483, caffeine, dichlorodihydrofluorescein diacetate (H₂DCFDA), dihydroethidium (DHE) and propidium iodide (PI) were obtained from Sigma‐Aldrich Co (St Louis, MO, USA). Xestospongin C was purchased from Tocris Bioscience (Bristol, UK). Z‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐FMK), salubrinal and 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis/acetoxymethyl ester (BAPTA/AM) were purchased from Calbiochem (Darmstadt, Germany). The antibodies specific for caspase‐3, caspase‐9, eIF2α, phospho‐eIF2α and poly(ADP‐ribose) polymerase 1 (PARP1) were purchased from Cell Signalling Technology (Beverly, MA, USA). The antibodies specific for glucose‐regulated protein 78 (GRP78), CHOP, PERK, phospho‐PERK and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for ATF4, ATF6, IRE1β and XBP1 were purchased from Abcam (Cambridge, UK).

The pharmacological and small‐molecule inhibitors and agonists used were the following: 7rh (2.5 µm; Sigma) for DDR1 inhibition, rat anti‐human CD29 (1:100; BD Bioscience) to block integrin β1, blebbistatin (1 µm; Enzo Life Sciences) for myosin II inhibition, GM6001 (10 µm; Calbiochem) as broad‐spectrum MMP inhibitor, L‐mimosine (400 µm; Santa Cruz Biotechnology) to arrest the cell cycle at G1, S‐trityl‐L‐cysteine (STLC; 20 µm; Santa Cruz Biotechnology) to arrest at mitosis, ionomycin (10 µm; MedChemExpress) to increase cell membrane permeability to calcium, gadolinium chloride (10 µm; MedChemExpress) to inhibit mechanosensitive calcium channels, BAPTA (10 µm; MedChemExpress) to chelate extracellular calcium, BAPTA‐AM (10 µm; MedChemExpress) to chelate intracellular calcium, arachidonic acid (70 µm; MedChemExpress) to mimic signaling downstream of nuclear mechanosensing, AACOCF3 (28 µm; Tocris) to inhibit signaling downstream of nuclear mechanosensing, 2‐aminoethoxydiphenylborane (20 µm; MedChemExpress) as IP3 receptor agonist, Xestospongin c (10 µm; Tocris) as IP3 receptor agonist, GSMT×4 (10 µm; MedChemExpress) as Piezo 1 inhibitor, Yoda1 (6 µm; MedChemExpress) as Piezo 1 agonist, GSK205 (10 µm; MedChemExpress) as TRPV4 inhibitor, and GSK1016790A (50 µm; MedChemExpress) as TRPV4 agonist.

The chemical reagents and recombinant proteins were obtained from different companies: “Spectrum Collection” (MicroSource Discovery Systems, Inc., 10 mM stock solutions in dimethylsulfoxid (DMSO), purity > 90%), 6-bromoindirubin-3'-oxime (BIO), osajin, peruvotoxin, efaroxan hydrochloride, xestospongin C (Tocris bioscience), SB203580 (Calbiochem), fibroblast growth factor 1 (FGF1, R&D Systems), convallatoxin (Sigma), valeryl salicylate, bpV(HOpic) (Santa Cruz Biotechnology). Inhibitors were added 1 h before cardiomyocyte stimulation.