Cells
Primary rat ventricular cardiomyocytes (RVCM) were obtained from 3 to 5 day old Sprague Dawley (Scanbur, Sollentuna, Sweden) and cultured on 18 mm diameter coverslips for 5 days previously described [14] using a modified growth medium. Growth medium was either a 2:1 mixture of DMEM/F-12:PC-1, supplemented with 2.5% FBS and 0.05 pM of AngII or DMEM for primary cell isolation (Gibco), 1:1000 Cardiomyocyte Growth Supplement (Pierce), 10% FBS and 50 pM AngII. Rats were euthanized by rapid decapitation and the heart removed for generation of cardiomyocyte cultures. Quality of culture was determined using a cardiomyocyte characterization kit (Chemicon). Cardiomyocytes were cultured for 5 days prior to experiment and contracting clusters, seen with transmission light, were selected for recording. Expression of cardiomyocyte markers were confirmed using Troponin I (Chemicon) and Desmin (Chemicon) antibody staining according to manufacturer’s protocol. Rat aortic smooth muscle cells (ASMC, catalog number R6110, 3H Biomedical, ScienCell) were cultured according to manufacturer’s instruction. Briefly, cells were thawed and plated on poly-L-lysine coated coverslips in complete smooth muscle cell medium (SMCM, ScienCell) including 2% FBS and supplemented with 0.05 pM of AngII. Cells were cultured for three to 5 days before experiments, cells from initial plating and first passage were used. Cells were confirmed to express anti-α-smooth muscle actin by immunostaining (antibody from Abcam). Human Embryonic Kidney 293a (HEK, catalog number R70507, Thermo Fisher Scientific) cells were plated on 18 mm diameter coverslips 2 days prior to transfection with Exgen500 (Fermentas), experiments were performed 24 h after transfection. Manufacturers protocols were used for transfection using 1 μg DNA per transfection. HEK cells between passage 3 and 20 was used in experiments. RT-PCR was used to confirm RNA expression of CaV1.2 (Fig 2a).
All experiments were performed according to Karolinska Institutet regulations concerning care and use of laboratory animals, and were approved by the Stockholm North ethical evaluation board for animal research.
Reagents and constructs
All chemicals and drugs were obtained from Sigma-Aldrich, if not otherwise stated. Bradykinin was purchased from Abcam. 3,5-bis(trifluoromethyl)pyrazole (BTP2) a TRPC channel blocker was obtained from Santa Cruz. Rat angiotensin II type 1 receptor, AT1R, with Venus fused at C-terminus was used for Ca2+ experiments [15]. The HA-tagged AT1R was made by insertion of PvuI restriction site through mutagenesis in the first extracellular loop between Pro331 and Phe332. Agilent QuikChangeII Site-Directed Mutagenesis kit together with the following primers were used: sense 5′ – GGTGATTGCCGAACGATCGGGGCCAGCGGTAC and antisense 5′ GTACCGCTGGCCCCGATCGTTCGGCAATCACC. The product was digested with PvuI (Thermo Scientific), and RSYPYDVPDYARS (Hemaglutinin flanked by PvuI sites) was inserted through ligation (Phusion Hot Start II, ThermoFisher). Structure of the construct was verified by using BigDye® Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems). pGβ-2A-YFP-Gγ2-IRES-GαqmTq [16] was kindly provided by the lab of Th. W. J. Gadella and used for Gαq-Gβγ FRET (Förster Resonance Energy Transfer) measurements.
Membrane recruitment
Control and cells exposed to AngII were fixed using 2% paraformaldehyde for 10 min and stained using 1:1000 dilution of Alexa-594 fluorescence conjugated hemagglutinin (HA) antibody (Invitrogen). Cells were imaged using a Zeiss LSM780 in two channels for Venus and Alexa-594 fluorescence. Mean membrane and cytosol fluorescence intensities were calculated. Membrane fraction is reported as normalized to control ratio between mean membrane to cytosolic intensities.
PCR
Approximately 107 HEK cells were used for RNA extraction using manufacturer’s protocols (RNeasy Plus, Qiagen). cDNA was obtained using iScript cDNA kit (BioRad) following manufacturers protocols. Primers for CaV1.1, CaV1.2 and CaV1.4 were designed and used for PCR amplification (Phusion High-Fidelity DNA polymerase, Thermo Scientific). PCR amplification was run using the manufacturer’s protocols with the following program changes: 40 cycles with 10s extension time. Annealing temperature was 56 °C for CaV1.4, 58 °C for CaV1.1 and CaV1.2. The product was run on a 2% Agarose gel (1:10,000 GelRed, Biotium) and imaged on a Li-Cor Oddyssey system. The following primer pairs were used at 0.5 μM:
CaV11_4689F CAAGAGGAGTATTATGGCTATCGG,
CaV11_5252R GGTCAGCAGTCCCTTCAGCA,
CaV12_6568F CTGCGACATGACCATAGAGG,
CaV12_7188R CAGCGAGGCTCGTACAGA,
CaV14_5045F CAAAGCCACGATGGTCTCCC and.
CaV14_5545R TTTCGCCCACGATGATAGG (Cybergene).
[Ca2+]i measurements
Intracellular Ca2+ concentration ([Ca2+]i) measurements carried out in HEK cells were performed using the fluorescent Ca2+ indicator Fura Red (Invitrogen). Cells were loaded with Fura Red using a buffer consisting of 5 μM Fura Red and 1 μl of 20% Pluronic F-127 (Invitrogen) diluted in KREBs (110 mM NaCl, 4 mM KCl, 1 mM NaH2PO4xH2O, 25 mM NaHCO3, 1.5 mM CaCl2x2H2O, 1.25 mM MgCl2x6H20, 10 mM Glucose and 20 mM HEPES, pH 7.40, sterile filtered). Cells grown on #1.5 18 mm diameter coverslips (Warner Instruments) were rinsed with KREBs buffer and loaded for 45 min with the loading buffer at 37 °C with 5% CO2. After 45 min cells were washed again with KREBs prior to being mounted in a perfusion chamber (Chamlide). Through the use of heated optical table and in-line perfusion feed heater the cells were kept at 37 °C throughout experiments. Loading protocol was evaluated by testing different loading times, both at room temperature and in incubator. The minimal dye concentration usable was identified through Ca2+ calibration of serial dilution of dye.
CaV1.2 inhibitors, nifedipine and verapamil, were both mixed fresh for each experimental day. Solutions were kept in fridge and protected from light. We chose to use a high concentration of nifedipine, 100 μM, to compensate for loss of active drugs during the longer experiments. Degradation was unavoidable within each experiment with cells and solutions kept at 37 °C. UV and 488 nm laser excitation also contributed to some drug degradation throughout experiments.
[Ca2+] calibration was done by the following protocol, first a 3 min long washout of the KREBs buffer using a 0 [Ca2+] containing buffer (110 mM NaCl, 4 mM KCl, 1 mM NaH2PO4xH2O, 25 mM NaHCO3, 1.25 mM MgCl2x6H20, 250 μM EGTA, 10 mM glucose and 20 mM HEPES, pH 7.40, sterile filtered) followed by 1 μM ionomycin in 0 [Ca2+] buffer for 4 min. Calibration ended with 7 min of 1.5 mM [Ca2+] KREBs buffer perfusion.
The strongest AT1R-expressing cells were excluded from the analysis. Cells not responding to the first stimuli were also excluded. Cells were individually selected and the mean Ca2+ dye intensity was calculated and calibrated using the data from the end of each experiment. The mean intensity ratio, R, between the 488 nm excitation image and the 405 nm excitation image for each cell was calculated for each timepoint. Rmin and Rmax are the minimum and maximum intensity ratios obtained during calibration. Calibrated [Ca2+]i, was calculated with kd provided by Invitrogen as:
$$ {\left[{Ca}^{2+}\right]}_i={k}_d\frac{R-{R}_{min}}{R_{max}- R} $$
Ca2+ measurements of RVCM were performed using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (Invitrogen) with the same protocol as described for Fura Red. Oregon Green BAPTA1 was chosen as its weaker binding affinity compared to Fura Red made it insensitive to background Ca2+ sparklets in RVCM. Calibration was not possible for the RVCM Ca2+ experiments, thus the non-calibrated Ca2+ response for each cell was obtained by the mean intensity within each selected cell.
Nifedipine effects on bradykinin activation of bradykinin B2 receptors were evaluated through comparing signaling strength with or without pretreatment of 100 μM nifedipine on 100 nM bradykinin (Abcam) treatment. Experiments were performed on Oregon Green Bapta 1 loaded HEK cells. Cells were loaded in the same manner as for Fura Red but with 5 μM Oregon Green BAPTA-1. All other parameters were kept identical as for the 1 nM AngII experiments described above. Each experiment was calibrated as per the protocol described above.
Peak amplitudes were in all experiments calculated as peak above baseline. Baseline was defined as the mean intensity of a stable part of the trace prior to the peak. This calculation reflects the cytosolic increase in concentration rather than the absolute concentration.
Gαq dissociation
HEK cells expressing AT1R and Gαq-Gβγ FRET sensor were exposed to 1 nM AngII with or without 3 min pretreatment of 100 μM nifedipine. FRET was measured using a Zeiss LSM 510 LIVE microscope with a 40×/1.2 water immersion objective and excitation of 405 nm. YFP signal was corrected for mTurquoise bleedthrough and used as FRET intensity. Transfecting HEK cells with GαqmTq allowed for determination of donor bleed through into FRET channel. Bleed through coefficient, b, was calculated as ImTq/IFRET. In experiments, FRET channel intensities were then corrected as: FRETcorrected = IFRET – b*ImTq. FRET ratios were finally calculated as ImTurquoise / IFRETcorrected. Increase in FRET ratio corresponds with increased Gq-protein dissociation.
Statistical analysis
Statistical analysis was performed using Matlab, one way Anova analysis and subsequent multiple comparison tests based on studentized range distribution. Quantified data is shown as mean value +/− SEM.