Aquaporin-4 in the Heart: Expression, Regulation and Functional Role in Ischemia

SUPPLEMENTARY METHODS

“Aquaporin-4 in the heart: expression, regulation and functional role in ischemia”

Arkady Rutkovskiy, MD, PhD1,2,3, Kåre-Olav Stensløkken, PhD 3, Lars Henrik Mariero, MS1,2,3, Biljana Skrbic, MD 1,2, Mahmood Amiry-Moghaddam, MD, PhD4, Vigdis Hillestad, MS1,2, Guro Valen, MD, PhD 2,3, Marie-Claude Perreault, PhD 3,Ole Petter Ottersen, MD, PhD4, Lars Gullestad, MD, PhD 2,5, Christen P. Dahl, MD, PhD2,5, Jarle Vaage, MD, PhD 1,6

1Institute of Clinical Medicine,2Center for Heart Failure Research, 3Department of Physiology and 4Anatomy, Institute of Basic Medical Sciences,University of Oslo;5Departments of Cardiology and 6Emergency Medicine and Intensive Care, Oslo University Hospital, Oslo, Norway.

Corresponding author: Arkady Rutkovskiy, Department of Physiology, Institute of Basic Medical Sciences, PB 1103, Blindern, 0371 Oslo, Norway. Telephone +47-22-85-12-54, Fax +47-22-85-12-49 e-mail

Isolation of cardiomyocytes

We used a modified technique from O’Connell et al. [4]All reagents were bought from Sigma unless otherwise stated. WT mouse hearts were isolated, mounted on a Langendorff apparatus and purged of blood with a perfusion buffer containing (mMol) NaCl 120.4; KCl 14.7; KH2PO4 0.6; Na2HPO4 0.6; MgSO4*7H2O 1.2; Na-HEPES 10; NaHCO3 4.6; Taurine 30; Butanedione Monoxime 10; Glucose 5.5 before perfusion with 2.4 mg/ml Collagenase II (Worthington Biochemical, Lakewood, NJ) in perfusion buffer to digest the extracellular matrix. Halfway through the digestion, CaCl2 was added to the perfusion buffer to achieve a calcium concentration of 40 µM. Digested myocardial tissue was cut from the perfusion apparatus, mechanically disrupted and suspended in the perfusion buffer with the addition of 12.5 µM CaCl2 and 5% fetal calf serum (FCS). Cardiomyocytes were purified by serial centrifugations for three minutes at 20 g gradually increasing calcium from 12.5 µM to 1.2 mM. Non-cardiomyocytes present in the supernatant from the first low-speed centrifugation were transferred to a separate tube. The tube was centrifuged for 5 minutes at 2000 rpm, and the cells were resuspended in the Minimum Essential Medium with Hank’s balanced salt solution (Gibco–BRL) containing 5 % FCS, penicillin/streptomycin (100 U/ml:100 mg/ml) and 2 mM L-glutamine. The suspension was plated on non-coated six-well plates. Cardiomyocytes were resuspended in Minimum Essential Medium with Hank’s balanced salt solution and 10% fetal calf serum, butanedione monoxime – 10 mM, penicillin 100 units/ml and glutamine 2 mM and plated on six-well plates coated with laminin (1µg/cm2) (Gibco – BRL). The cells were incubated for four hours at 370C and 2 % CO2 and harvested using a cell scraper.

Immunogold electron microscopy

Isolated hearts were briefly perfused with Krebs-Henseleit Buffer (KHB) (mmol/l: NaCl 118.5; NaHCO3 25; KCl 4.7; KH2PO4 1.2; MgSO4*7H2O 1.2; glucose*1H2O 11.1; CaCl2 1.8) to remove blood and subsequently perfused with fixative (4 % paraformaldehyde with 0.1% glutaraldehyde) at 70 mmHg for 20 minutes. The rinsed hearts were immersed in the same fixative for 12 hours at 4ºC. The fixative was subsequently diluted 1:10 with phosphate buffer for storage. The hearts were cut into 1 mm sections. One mm cubic transmural tissue fragments from the left anterior ventricular wall were cryoprotected, quick-frozen in liquid propane (-1700C) and subjected to freeze substitution [3]. Specimens were then embedded in methacrylate resin (Lowicryl HM20) and polymerized by UV light at <0°C for 35 hours. Ultrathin 200 nm sections were incubated in antibodies against AQP1 (Alpha Diagnostic, dilution 1:800) or AQP4 (Millipore inc., dilution 1:50) then with anti-rabbit secondary antibody (RPN422, Amersham) coupled to 15 nm gold particles and examined with a Tecnai 12 electron microscope at 60 kV.

AQP4 KO mouse heart sections were used as negative controls for AQP4 antibody, and WT mouse brains as positive. The latter were also used as negative controls for AQP1, while kidney was used as positive control. Magnifications of 18000x and 25000x were used. Quantification was performed as described by Bergersen et al. [1] For the quantification of AQP4 immunogold labeling, digital images of sections from eleven WT mouse hearts were acquired, 30 images for each of the three membrane types: myocyte/myocyte, intercalated disc, myocyte/endothelial cell (a total of 90 pictures per heart). To quantify AQP1 in AQP4 KO hearts, 30 pictures from each heart were taken. Density of gold particles per µm of membrane was quantified with IMGAP software created at the Centre of Molecular Biology and Neuroscience, University of Oslo, and developed in collaboration with SIS (Soft Imaging Systems, Münster, Germany). Two types of filters (100 and 23.5 nm) were used. Gold particles were counted automatically within the user-defined regions of interest. Quantitative results from images were averaged and one value per membrane type was obtained for each individual heart. The quantification was performed by a person blinded to the experimental groups.

Isolated heart perfusion.

The procedure has been performed as described previously. [5,2] Mice weighing 25 ± 3 g were injected intraperitoneally with sodium pentobarbital 60 mg/kg and 500 IU Heparin (Leo Pharma A/S, Denmark). The aorta was cannulated and the heart retrogradely perfused at 37ºC with oxygenated KHB at 70 mmHg. A polyethylene balloon was inserted into the left ventricle for isovolumetric pressure measurements. The balloon was filled with distilled water until left ventricular end-diastolic pressure (LVEDP) reached 5-10 mmHg. The hearts were not paced. Hearts with left ventricular systolic pressure (LVSP) ≤ 60 mmHg, coronary flow ≤ 1 or ≥ 4 ml/minute, heart rate ≤ 220 beats/minute after 20 minutes of stabilization, time delay > 3 minutes from heart excision to the start of perfusion, or irreversible arrhythmias during stabilization were excluded from the study. LVSP and LVEDP were measured using PowerLab system (AD Instruments Pty Ltd, Australia). Developed pressure, dPdt min, dPdt max and rate-pressure product were calculated. Coronary flow was measured by timed collections of the coronary effluent. Global ischemia was achieved by clamping the inflow tubing for 35 minutes. Reperfusion (clamp release and perfusion at 70 mmHg) lasted for two hours.

Immunoblots

Frozen heart tissue was homogenized in lysis buffer and protein content was determined on the BCA assay (Pierce, USA). Twenty micrograms of protein per lane were separated on a 10% sodium SDS-polyacrylamide gel and transferred onto a hybond-P membrane (Amersham Biosciences Europe, Germany). For the analysis of human material, the gels were supplemented with 2M Urea. The membrane was blocked for one hour (5% heated, filtered skimmed milk in TBS-T) and incubated overnight with primary antibodies. For an overview of primary antibody sources and dilutions, see table S1. The membranes were subsequently washed and incubated for two hours with the secondary antibody (goat anti-rabbit 1:2500. Southern Biotech, England). The proteins were visualized with ECL Plus (Amersham Biosciences) and band intensity was measured with ImageQuant software (Amersham Biosciences) The membranes were subsequently stained with the non-specific protein dyeСommassieBlue (Bio-RadLaboratories, USA) and scanned with CanonScanLide 35 scanner (CanonEuropeNV, TheNetherlands) to account for protein loading.

Materials and methods - qPCR

The tissue samples were crushed in liquid nitrogen, and RNA was extracted using RNeasy Mini Kit (Qiagen inc.) with a supplementary phenol/chloroform purification step (Sigma) and treated with DNase (Qiagen cat. no79254). RNA concentration was measured with NanoDrop spectrophotometer at a wavelength of 260 nm. RNA integrity was assessed in Agilent 2100 Bioanalyzer (Agilent technologies). cDNA was synthetized in accordance with First Strand cDNA synthesis protocol, with the use of Superscript II (Invitrogen) and RNasin (Promega). The protocol included 10 minutes at 250С, 50 minutes at 420С, and 4 minutes at 940С). qPCR was carried out in ABI7900 (PE Applied biosystems). The sequences of primers used in the study are listed in Table S2. The reactions were run in MicroAmpoptical plates (PEAppliedBiosystems), the mixtures contained 5 µlcDNA, 10 µlUniversal TaqMan MasterMix (PEAppliedBiosystems, CAS # 67-68-5) or SYBR green Master Mix, primers up to a final concentration of900 nMol, and probes up to 200 nMol. The totalmixturevolumewas 25 µl, and all reactions were run in duplicates. The cycle included 2 minutes at 500С, then 10 minutes at 950С, followed by 40 х (15 seconds at 950С, 1 minute at 600С).

Optical recording and cardiomyocyte membrane integrity measurement

Cardiomyocytes were isolated from six AQP4 KO and six WT hearts and cells from each heart were plated on six 35 mm culture dishes with 2 mm grid (Nalge Nunc int., USA). The dishes were placed in an incubator (Heraeus 6000, Germany) at 370C, 2% CO2 overnight. The cell medium was removed and replaced by 1 ml of 0.5 µmol Calcein-AM (Invitrogen AS, Oslo) solution in buffer (mmol: NaCl 136, KCl 4.7, MgSO4 1.25, CaCl2 1.25, Na2PO4 5, HEPES 20, glucose 25). After 10 minutes of incubation in the dark at room temperature, loaded cells were washed twice with the buffer. Labeled cardiomyocytes were positioned under an epi-fluorescence microscope (Axioscop 2 FS plus, Carl Zeiss, Oberkochen, Germany) equipped with an 100 W halogen lamp and excitation (BP 450–490 nm) and emission (LP 515 nm) filters, and visualized with a 40X water immersion objective (LUMPlanFl, 0.8 NA, Olympus, Norway). The field of view was focused on the area containing 2 - 5 cardiomyocytes with distinct morphology and changes in fluorescence intensity were followed using a CCD camera (Cascade 650, Photometrics, Texas Instruments, USA). To minimize photo-bleaching, low intensity epi-illumination was used. Video images were acquired using image-processing software (Metamorph 5.0, Universal Imaging Corporation, Molecular Devices, USA) at a frame rate of 1 Hz. The cell medium was quickly replaced by distilled water (first series) or hypotonic buffer containing 25 mM NaCl, 1 mM EGTA and 30 mM butanedione monoxime (second series). After the application of hypotonic solution, video recording with a CCD camera was started after 15 seconds, and performed for 1200 seconds (1 frame per second) to monitor Calcein fluorescence quenching, and to register the time of cell bursting. In the first series with distilled water, 48 AQP4 KO and 45 WT cardiomyocytes from three AQP4 KO and three WT were filmed. In the second series with hypotonic buffer 52 AQP4 KO and 27 WT cardiomyocytes from three more AQP4 KO and three WT were filmed. Cell death was evidenced by the simultaneous loss of typical cardiomyocyte morphology that followed membrane rupture and dramatic drop in fluorescence signal (see video suppl. 1). For cardiomyocytes that survived the entire recording session (series 1) or the first 400 seconds (series 2), a region of interest was drawn over the cell images and the mean fluorescence intensity (averaged over all the regions of interest pixels) was measured in all recorded frames. For statistical analysis, the video data were exported to GraphPad Prism.

References

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2. Kaljusto ML, Rutkovsky A, Stenslokken KO, Vaage J (2010) Postconditioning in mouse hearts is inhibited by blocking the reverse mode of the sodium-calcium exchanger. Interact Cardiovasc Thorac Surg 10 (5):743-748. doi:10.1510/icvts.2009.217083

3. Neely JD, Amiry-Moghaddam M, Ottersen OP, Froehner SC, Agre P, Adams ME (2001) Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein. Proc Natl Acad Sci U S A 98 (24):14108-14113. doi:10.1073/pnas.241508198

4. O'Connell TD, Rodrigo MC, Simpson PC (2007) Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol 357:271-296. doi:10.1385/1-59745-214-9:271

5. Stenslokken KO, Rutkovskiy A, Kaljusto ML, Hafstad AD, Larsen TS, Vaage J (2009) Inadvertent phosphorylation of survival kinases in isolated perfused hearts: a word of caution. Basic Res Cardiol 104 (4):412-423. doi:10.1007/s00395-009-0780-1