br Methods Ventricular cardiomyocytes from adult male Wistar
Methods Ventricular cardiomyocytes from adult male Wistar rats were isolated using a standard enzymatic digestion . Cells were incubated at 37°C for 4 to 6h with Tyrode solution (in mM: 140 NaCl, 4 KCl, 1.1 MgCl2, 10 HEPES, 10 glucose, 1.8 CaCl2; pH7.4, with NaOH) supplemented or not with 10μM 8-(4-chlorophenylthio)-2-O-methyladenosine-3,5-cyclic monophosphate (8-pCPT). In some experiments, myocytes were incubated during 4 to 6h in the presence of selective EPAC inhibitors: 10μM 4-Methylphenyl-2,4,6-trimethylphenylsulfone, an EPAC 2 inhibitor (ESI-05) or 10μM 6-Fluoro-5,7-dibromo-2-methyl-1-formyl-1,2,3,4-tetrahydroquinoline, an EPAC 1 inhibitor (CE3F4) . The non-specific TRPC channel blocker SKF-96365 (30μM) and the selective TRPC3 antagonist Pyr3 (10μM) were directly applied during the recordings. To record variations in [Ca2+]i, myocytes were loaded with Fluo-3AM , . Images were obtained with confocal microscopy Leica TCS SP5, objective w.i. 63×, n.a. 1.2 by scanning the cell with a white light laser fitted at 500nm every 1.43ms; emissions were collected at >510nm. Image analyses were performed by homemade routines using IDL software. Images were corrected for the background fluorescence. The whole-cell patch-clamp currents were recorded using an Axopatch 1D, filtered at 2kHz and digitized at 10kHz using pClamp 8 software. 1–1.5MΩ pipettes were filled with solution containing (in mM): CsCl 130, MgCl2 2, EGTA 11, CaCl2 4.87, HEPES 20, Na2ATP 5, Na2GTP 0.4 and Na2 creatine phosphate 5; pH7.2 with CsOH and protein kinase g were perfused with a solution containing (in mM): NaCl 117, MgCl2 1.8, CsCl 20, CaCl2 2, glucose 10, and HEPES 10; pH7.4 with CsOH. Series resistance was electronically compensated up to 50% and was continually monitored during the experiment. All experiments were performed at 22±1°C. For Western Blot, cardiomyocytes were lysed in a buffer containing (in mM): 50 Tris, 500 NaCl, 20 MgCl2, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% Sodium Dodecyl Sulfate (SDS), and protease inhibitors (1mM phenylmethylsulphonylfluoride, 10μg/L aprotinine, 10μg/L leupeptin, pH7.5). After 20min centrifugation (15,000g), 40μg proteins were denaturated in Laemmli's buffer, separated on 10% SDS-polyacrylamide gel, and transferred onto a polyvinylidene difluoride membrane. Membranes were hybridized overnight (4°C) with the primary antibody against TRPC3 or C4 (1:200, Alomone Labs). For Immunocytochemistry, cells were fixed (15min, paraformaldehyde 2%). After washing and neutralization (0.5M NH4Cl), cells were permeabilized (0.5% Triton X-100 in PBS, 5min). After washing (1% BSA in PBS), myocytes were labeled with the TRPC antibodies in PBS containing 10% goat serum and 0.25% Triton X-100. After overnight incubation (4°C) and washing (1% BSA in PBS), cells were incubated with AlexaFluor® 488 conjugated anti-rabbit IgG and then resuspended in Moviol medium (15μL). Images were acquired using a Leica TCS SP5 confocal microscope. For qPCR, total RNA was isolated using the TRIzol procedure (Life technologies) and 1μg was reverse transcribed using iScript cDNA synthesis kit (Biorad). The cDNA was used as a template for real-time PCR with SYBR Green Supermix (Biorad). Quantitative determination of the different mRNA expression levels was performed in duplicate with a CFX96 Touch™ Real-Time PCR Detection System (Biorad) using 0.5μM specific primers (Eurofins MWG Operon, France): for TRPC3 fwd; 5′-GCATTCTCAATCAGCCAACA-3′, rev: 5′-TTCACCTTCGTTCACCTCATC-3′, TRPC4 fwd: 5′-AGGCTGGAGGAGAAGACACT-3′, rev: 5′-CAGGTAGCACACGGAGAAGA-3′; YWaz 14-3-3 Protein zeta/delta fwd: 5′-AGACGGAAGGTGCTGAGAAA-3′, rev: 5′-GAAGCATTGGGGATCAAGAA-3′, TATA box Binding Protein fwd: 5′-AAAGACCATTGCACTTCGTG-3′, rev: 5′-GCTCCTGTGCACACCATTTT-3, Ribosomal Protein L 32 fwd: 5′-GCTGCTGATGTGCAACAAA-3′, rev: 5′-GGGATTGGTGACTCTGATGG-3. mRNA levels were normalized to housekeeping genes and were expressed as fold change of that determined in untreated condition.