Ethics statement. All experiments were approved by the State Office for Nature, Environment and Customer Protection (LANUV) North Rhine Westphalia: AZ 84-02.05.50.15.029) and performed in accordance with the European Community guidelines (Directive 2010/63/EU).
II. Animals and tissue isolation. Wilde type (WT) and heterozygote (MYPT1-T696A/+) animals, all with C57BL/6N background, were bred and kept in the Center for Molecular Medicine Cologne and randomly allocated to the different sets of experiments. Young adult (y-FA; 20-25 weeks, n=18 WT and n=17 MYPT1-T696A/+) or old mice (o-FA; ≥ 90 weeks, n=8 WT and n=9) were sacrificed by decapitation after an isoflurane anesthesia. Legs were cut out from the body and after gentle pelt removal, were transferred in a petri dish, containing ice-cold HEPES-buffered low Ca2+ physiological saline solution (HPSS, see solutions). Then, the part between external iliac and gracilis artery was isolated, cut into ~2 mm rings and left for 10 minutes in ice-cold HPSS for equilibration.
III. Wire myography with intact preparations
Arterial rings for these measurements were mounted on Wire Myograph System 620A (Danish Myotechnology) in HPSS-solution using 40µm stainless steel wire and then low Ca2+-HPSS was replaced by bicarbonate buffered physiological salt solution (PSS; 1.6 mmol/l Ca2+) under continuous aeration with carbogen (95% O2/5% CO2). After that, the temperature was gradually increased until it reached 37°C and then the preparations were stepwise stretched to IC90 (90% of the internal diameter reached at 100 mmHg) as described previously in [18]. This diameter was kept during the whole experiment and pH-value was controlled by placing a pH-electrode into the organ bath. If not indicated otherwise, all drugs were administered directly into the organ bath. To determine passive tone under Ca2+ - free conditions, preparations were washed several times with PSS containing 2 mmol/l EGTA for 10-15 minutes at the end of all experiments.
1. Vascular reactivity of WT and MYPT1-T696A/+ FAs to the stable thromboxane A2- analogue, U46619. In these experiments after normalization and 20 minutes equilibration in PSS, preparations were treated either by vehicle (1% H2O) or 100 µmol/l of pan-NOS inhibitor, Nω-Nitro-L-arginine-methylester-hydrochlorid (L-NAME). Then, the arteries were challenged by cumulative application of U46619 in a concentration range of 0.001-3 µmol/l with each concentration step applied for 10 minutes. After that, vessels were washed out several times until force reached initial values. Tone was calculated as absolute force in mN without subtraction of initial tone reached after stretching to IC90.
2. Vascular reactivity of WT and MYPT1-T696A/+ FAs to acetylcholine. After the concentration-response curves to U46619, preparations were submaximally stimulated by administration of 0.3 µmol/l U46619 for 15-20 minutes. After force stabilization, the reactivity to cumulatively increased acetylcholine concentrations (0.001-10µmol/l) was tested. Results were calculated as % force reached prior to administration of acetylcholine.
3. Vascular reactivity of WT and MYPT1-T696A/+ FAs to the NO-donor, DEA-NONOate and the direct soluble guanylate cyclase (sGC) activator cinaciguat. To avoid endogenous release of NO, all vessels were pretreated with 100 µmol/l L-NAME followed by submaximal stimulation with 0.3 µmol/l U46619 for 15-20 minutes. After force stabilization, the reactivity to cumulatively increased concentrations of DEA-NONOate (0.001-10µmol/l) or cinaciguat (0.0001-10µmol/l) was tested. Results were calculated as % force of the force reached in response to U46619 prior to application of DEA-NONOate or cinaciguat.
IV. Wire myography with a-haemolysin permeabilized preparations
These experiments were performed with minor modifications as in Lubomirov et al., 2023. Briefly, FAs were isolated in HPSS-solution as described previously and left for 10 minutes in this solution at room temperature. Then, HPSS was replaced by Ca2+-free PSS (2 mmol/l EGTA) for another 10 minutes and permeabilized by staphylococcus aureus a-toxin (40 units per µl; final volume 50 µl) in EGTA-buffered relaxing solution pCa >8 (see solutions). Then, the permeabilized preparations were mounted in a myograph in relaxing solution and stretched to IC90 under continuous bubbling with air. To deplete Ca2+-stores, the arteries were treated for 20 minutes with the Ca2+-ionophor A23178, 10 µmol/l and stimulated by replacement of the pCa >8 solution by solutions with increasing pCa (7.0, 6.6, 6.2, 5.8 and 4.3), 10 minutes each. The solutions with intermediate pCa concentrations (7.0, 6.6, 6.2, 5.8) were obtained by mixing relaxing and contracting solutions in the appropriate ratio. All experiments in these series were carried out at room temperature (23°C).
Protein extraction and Western blotting
Femoral arteries were isolated and mounted on single tungsten wires (25 µm) in HPSS and placed in PSS-filled 1.5 ml reaction tubes. Then, the temperature was increased to 37°C under continuous aeration with carbogen and preparations were equilibrated under these conditions for 30 minutes. Thereafter, arterial rings were stimulated with 0.3 µmol/l U46619 for 15 minutes and acetylcholine was administered for another 5 minutes at concentrations of 0.03 or 0.3 µmol/l. Then, preparations were immersed in reaction tubes filled with 15 % trichlor-acetic-acid in acetone precooled to -80°C by dry ice. Samples were then fixed at -80°C for 3h and rinsed several times with acetone on dry ice. Then, the samples were homogenized in 60 µl Laemmli buffer (see buffers) using tissue grinders (Kimble Chase LLC, Tissue Grinder Micro PKG/6, USA). After 1 h extraction on ice, homogenates were centrifuged at 14,000 rpm and 4°C and the supernatant was subjected to SDS-PAGE using gradient gels with 4-20% acrylamide and tris-glycine running buffer (see solutions). Afterwards, proteins were transferred overnight onto nitro-cellulose membranes (Amersham™ Protran®) in transfer-buffer (SERVA electrophoresis GmbH), at 4°C and constant voltage of 22 V. The quality of the protein transfer was controlled by visualization of the protein bands using Ponceau-S staining for 5 minutes in H2O. Then, the membranes were incubated with respective primary and secondary antibodies (see below section “antibodies and antibody dilution”). The immunoreactive signals were visualized using enhanced chemiluminescence (West Pico or West Dura, Thermo Fisher Sciantific) and detected with the Chemi Premium Imager (VWR, Darmstadt, Germany). Quantification and densitometry analysis was performed using Gel Documentation software from VWR.
Antibodies and antibody dilutions
Anti-phospho-MLC20-S19 (pMLC20-S19), rabbit polyclonal, #600-401-416, dilution 1:1000 (Rockland Immunochemicals Inc. Limerick, PA, USA); anti-phospho-MYPT1-T696 (pMYPT1-T696), rabbit polyclonal (PA5-104617), #PA5-104617, dilution 1:20000; anti-GAPDH, rabbit polyclonal, #G9545, dilution 1:10000 (Sigma-Aldrich/Merck) (Merck KGaA/Millipore, Darmstadt, Germany); anti-MYPT1-total, mouse polyclonal, #612164, dilution 1:5000 (BD Transduction Laboratories, Franklin Lakes, NJ, USA); SM-22, goat polyclonal, #NB600-507, dilution 1:10,000 (Novus Biologicals/ Bio-Techne); anti-phospho-MYPT1-T853 (pMYPT1-T853) rabbit polyclonal, #4563, dilution 1:500; phospho-eNOS (Ser1177) antibody #9571, dilution 1:500; anti-eNOS-total (D9A5L) rabbit monoclonal antibody, #32027, 1:500; MLC20-total (D18E2), rabbit monoclonal antibody, #8505 (Cell Signaling Technology, Inc. Danvers, MA, USA).
Chemicals
Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), Y27632, Cinaciguat and DEA-NONOate were purchased from Merck KGaA, Darmstadt, Germany; U46619 from TOCRIS Bioscience, Abingdon, United Kingdom.
Solutions for wire myography
HEPES buffered physiological salt solution (in mmol/l): 118 NaCl, 5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 0.16 CaCl2, 10 Glucose, and 24 HEPES, pH 7.4 at room temperature.
Physiological salt solution (in mmol/l): 119 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 1.6 CaCl2, 5.5 glucose, 25 NaHCO3, 0.03 EGTA, pH 7.4 at 37°C, equilibrated with carbogen (95% O2, 5% CO2).
Relaxing solution (pCa = −log10[Ca2+] ≥ 8; in mmol/l): 20 imidazole, 7.5 Na2ATP, 10 EGTA, 10 Mg [CH3COO-]2, 10 creatine phosphate, 31.25 potassium methane sulfonate, 5 NaN3, 0.01 GTP, 0.001 leupeptin, 2 dithiothreitol; рН 7.00 adjusted with CaCl2-free KOH (Merck); ionic strength adjusted with potassium methane sulfonate (pH=7.0) to 150 mmol/l.
Contraction solution (pCa = −log10[Ca2+] = 4.3): 20 imidazole, 7.5 Na2ATP, CaCl2,10 EGTA, 10 Mg [CH3COO-]2, 10 creatine phosphate, 31.25 potassium methane sulfonate, 5 NaN3, 0.01 GTP, 0.001 leupeptin, 2 dithiothreitol; рН adjusted at 7.00 by CaCl2-free KOH; ionic strength adjusted with potassium methane sulfonate (pH=7.0) to 150 mmol/l.
Buffers for western blotting
Tris-glycine running buffer for SDS-PAGE (pH 8.3): 25 mmol/l Tris, 0.192 mol/l glycine, SDS 1 % (purchased by SERVA Electrophoresis GmbH, Heidelberg, Germany).
Transfer-buffer: 25 mmol/l Tris, 0.192 mol/l glycine, 10% methanol, 0.05 or 0.005% SDS.
Tris-buffered saline solution with Tween20 (TBST) in mmol/l: 20 Tris, 150 NaCl, 0.05 % Tween20; pH 7.6 at room temperature, 23°C;
2x Laemmli-sample-buffer for SDS-PAGE: 126 mmol/l Tris-HCl (pH 6.8), 20 % glycerin, 4 % SDS, and 0,02 % bromophenol blue20 mmol/l dithiothreitol;
All chemicals used for SDS-PAGE and Western Blotting were purchased from SERVA Electrophoresis GmbH, Heidelberg, Germany.
Statistics
Results are given as mean ± SEM. The values of Fmax (maximal contraction induced by the contractile agonist U46619 at 3 µmol/l) is given as absolute force in mN. Dmax (maximal dilation) is given as percent reduction of vascular tone relative to submaximal precontraction with 0.3 µmol/ U46619 accepted as 100%. If not indicated otherwise, n represents the number of animals. The significance level was set at p<0.05 and was tested with Student’s t-test if two pairs were compared or with 2 way-ANOVA for multiple comparisons followed by Tukey multiple comparisons post-test. All statistic calculations have been performed using GraphPad-Prism software.