Supporting Information Lehmann et al. 10.1073/pnas.1409026111 SI Materials and Methods Metaiodobenzylguanidine-PET. All chemicals were purchased from fl/fl – 124 Experimental Animals. Conditional ETA KO mice (ETA ) Sigma Aldrich unless otherwise noted. I was purchased from were described previously (1). ETAfl/fl were crossed with trans- Eckert & Ziegler. Poly-(3-{dibutyl[2-(3-and-4-vinylphenyl)ethyl] genic mice overexpressing Cre recombinase under the control of stannyl}benzylguanidinium acetate)-codivinylbenzene, a tin pre- cursor anchored to a polystyrene matrix, was obtained from the dbh promoter, DBH-Cre (2), to obtain mice lacking ETA only in SNs (ETAfl/fl;DBH-Cre, abbreviated as SN-KO) or under Molecular Insight Pharmaceuticals. High-performance liquid chro- the control of the myh6 promoter, αMHC-Cre (3), to obtain mice matography gamma was performed using an Agilent 1100 HPLC fl/fl equipped with a Raytest (Germany) Model Gabi radioisotope de- lacking ETA only in CMs (ETA ;αMHC-Cre, abbreviated as CM-KO). Control littermates (Ctrl. LM) consisted of ETAfl/fl, tector. Analytical reversed-phase HPLC (RP-HPLC) analyses were + + + + + ETA /fl,ETA / ,andETA / ;DBH-Cre littermates for SN- performed using a Chromolith Performance RP-18 endcapped + + + + + column (3 × 100 mm). The column was eluted in a linear gradient KO and ETAfl/fl,ETA/fl, ETA / , and ETA / ;αMHC-Cre (0–100%) of acetonitrile [0.1% trifluoroacetic acid (TFA)] in water littermates for CM-KO. All these mouse lines and Dahl salt- (0.1% TFA) for 5 min; flow rate of 2 mL/min; and room temper- resistant (DR), Dahl salt-sensitive (DS), Sprague–Dawley, and 124 ature absorbance λ = 214 nm. [ I]Metaiodobenzylguanidine Wistar rats (Charles River Laboratories) were housed under (MIBG) synthesis was conducted as follows: 3.2 mg of the resin standard conditions and maintained on commercial mouse or rat was swollen for 5 min in 50 μL ethanol, and 25 μL of water was chow and water ad libitum. The environment was maintained at added; 120 MBq [124I] in a volume of 35 μL was added, followed – 22 °C with a 12-h light 12-h dark cycle. For preparation of by 25 μL of a solution of hydrogen peroxide in acetic acid. The neonatal CMs or SNs, pups were killed between days 1 and 3 pH value of the reaction mixture was pH 4. The reaction mixture postnatal. Male DR and DS (9 wk of age; mean weight, 200 g; was stirred at a temperature of 85 °C for 20 min. At this time, M&B) were fed on a high-salt diet [8% (vol/vol) NaCl] for 40 d. HPLC analysis revealed a complete conversion, and 20 μLof Treated DS rats received darusentan (30 mg/kg/d; DS + DA in a saturated solution of methionine in water was added to quench the drinking water (was changed twice the day) with onset of the the reaction. The resin was separated by filtration over a sterile diet. Water and drug intake was controlled on a daily basis. filter and washed with 0.9% NaCl. The mixture was neutralized with 35 μL of 1 M NaOH, and the pH was adjusted to 7.4 with Transverse Aortic Constriction. Transverse aortic constriction (TAC) phosphate buffer. Radio-HPLC revealed the purity (>95%) of was performed in 9- to 10-wk-old male SN-KO and CM-KO and the product. Mice were measured 7 wk after TAC surgery 1 h control littermates from the same strain as described previously after i.v. injection of 10.73 ± 0.69 MBeq of [124I]-MIBG. The (4, 5). Briefly, the animals were anesthetized with ketamine anesthetized animals [2% (vol/vol) sevoflurane; Abbott] were (120 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.). The mice were placed in an Inveon small animal PET scanner (Siemens). Re- orally intubated with 20-gauge tube and ventilated (Harvard gion of interest (ROI) analysis of the acquired images was per- Apparatus) at 120 breaths/min (0.2-mL tidal volume). The aortic formed using Siemens imaging software. The resulting image constriction was created via a lateral thoracotomy through the data were normalized to the administered activity to parame- second intercostal space. A suture (Prolene 7-0) was placed terize the microPET images in terms of %ID/g (percentage of around the transverse aorta between the brachiocephalic and left injected dose per gram of heart corrected for radioactive decay carotid artery. The suture was ligated against a 27-gauge needle. to the time of injection). The uptake profiles were fitted to The needle was removed, leaving a discrete stenosis. Afterward, a one-compartment model (realized with the PMOD software) the chest was closed. Integrity of aortic banding was confirmed to estimate the influx rate constant (K1, milliliters of medium per by inspection of the surgical constriction and by visualization of grams of cells per min), and the volume of distribution (Vd). marked differences in the caliber of the right and left carotid arteries in TAC (6). A sham procedure in which the thoracic Heart Tissue Preparation. Mice were killed by cervical dislocation, aorta was not banded was also performed. Mice were killed from and hearts were removed and weighed promptly. Relative heart weight was calculated as ratio to body weight. The left ventricles about 7 wk after surgery. were transversely dissected, and parts of the left ventricles were Echocardiography. For measurements of chamber dimensions and quickly frozen in liquid nitrogen for protein and RNA isolation calculation of fractional shortening or ejection fraction, we used and analysis. Some hearts were arrested in diastole and com- a Tracing software from VisualSonics on the parasternal long axis pletely fixed for 48 h in 4% (vol/vol) paraformaldehyde dissolved × − in 0.1 M PBS (pH 7.4), embedded in paraffin, longitudinally cut via the following formula: 100 [(LVEDV LVESV)/LVEDV)], μ where LVEDV is left ventricular end-diastolic volume and LVESD into 5- m sections, and stained for histomorphometry. is left ventricular end-systolic volume. To quantify the stenosis Primer for RNA Analysis. The primers and probes that were used are after TAC surgery, echocardiographic data were achieved at heart as follows—rat: myh6 sense 5′-tgcagaagaaactgaaggaaaa-3′ and rates between 450 and 550 beats/min, and the evaluation was per- antisense 5′-gctcggcctctagctcct-3′; myh7 sense 5′-caccaacaaccc formed with short-acting isoflurane anesthesia. Arterial blood ctacgatt-3′ and antisense 5′-agcacatcaaaggcgctatc-3′; th sense pressure of awake animals and left ventricular/intraventricular 5′-tctccctgaggggtacaaaa-3′ and antisense 5′-gaattttggcttcaa pressure were recorded as previously described in detail (7). Left atgtctca-3′;mouse:ednra sense 5′-tgtgagcaagaaattcaaaaattg-3′ ventricular end-diastolic pressure (LVEDP), left ventricular con- and antisense 5′-atgaggcttttggactggtg-3′; nppa sense 5′-cacaga + – tractility [( )dP/dtmax], and left ventricular relaxation [( )dP/dtmax] tctgatggatttcaaga-3′ and antisense 5′-cctcatcttctaccggcatc-3′; were calculated using chart software package (ADInstruments col3a1 sense 5′-tcccctggaatctgtgaatc-3′ and antisense 5′-tgagtcg Pty. Ltd.). Transthoracic echocardiography was performed as aattggggagaat-3′; myh7 sense 5′-cgcatcaaggagctcacc-3′ and an- previously described in detail (8). The investigator conducting tisense 5′-ctgcagccgcagtaggtt-3′; gapdh sense 5′-gggttcctataaa- the echocardiography was blinded to the treatment status. tacggactgc-3′ and antisense 5′-ccattttgtctacgggacga-3′; th sense 5′- Lehmann et al. www.pnas.org/cgi/content/short/1409026111 1of9 cccaagggcttcagaagag-3′ and antisense 5′-gggcatcctcgatgagact-3′.For nM ET1 for 18–24 h. Murine CMs were isolated from neonatal SERCA (atp2a2) we used a taqman universal PCR mastermix ETAfl/fl mice as described previously (14). Pups were decapi- (Applied Biosystems). tated, and ventricular tissue was carefully dissected and disso- ciated in 0.41 mg/mL collagenase (Sigma) and 0.30 mg/mL Isolated Heart Perfusion. Wistar rats were anesthetized with thio- pancreatin (Sigma) in Ads buffer (116 mM NaCl, 20 mM pental (100 mg/kg, i.p.). The hearts were rapidly cut out and Hepes, 0.8 mM NaH2PO4,1g/literglucose,5.4mMKCl,and rinsed in ice-cold buffer, and the aorta was cannulated for per- 0.8 mM MgSO4, pH 7.35). CMs were plated on laminin/poly-L- fusion according to the Langendorff method. Within one ex- lysine–coated plates, and cells were treated with adenovirus – periment, 8 12 spontaneously beating hearts were perfused expressing Cre-recombinase (a kind gift by Oliver Müller, Uni- simultaneously at a constant coronary flow and a constant tem- versity Hospital, Heidelberg, Germany) for 12 h with 12 multi- perature of 37.5 °C. The perfusion medium was a modified Krebs- plicity of infection (MOI). For CM-SN coculture, neonatal rat Henseleit solution (125 mmol/L NaCl, 16.9 mmol/L NaHCO3, SNs were plated 1 d after preparation of CMs. After 3 d, medium 0.2 mmol/L Na2HPO4, 4.0 mmol/L KCl, 1.85 mmol/L CaCl2, 1.0 was changed to serum-free medium for 24 h. For hypertrophic mmol/L MgCl2, 11 mmol/L glucose, and 0.027 mmol/L EDTA). experiments, cells were then treated with 10 nM ET1 for 18–24 h The buffer was gassed with 95% (vol/vol) O2 and 5% (vol/vol) 3 before measurement of fixation. CO2, and the pH was adjusted to 7.4. Cardiac [ H]-norepi- nephrine (NE) uptake was determined as described previously Plasmids and Reagents. CMs were transduced with adenoviruses 3 μ (9). Briefly, a bolus of [ H]-NE (1 mL, 3 Ci, 100 pmol NE; harboring FLAG-HDAC4 (Ad-HDAC4), and FLAG-HDAC5 Amersham-Buchler) was injected into the perfusion system and (Ad-HDAC5) were infected for 24 h as described previously (13), proportionally distributed to the hearts and blank channels. 3 starved for 4 h, and stimulated with 10 nM ET1 for 12 h.