(NR3C1) Locus Was Modified by Standard Gene Targeting Procedures
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SUPPORTING INFORMATION MATERIALS AND METHODS CardioGRKO mice. The mouse GR (NR3C1) locus was modified by standard gene targeting procedures. In brief, the targeting vector contained loxP sites inserted upstream of exon 3 and downstream of exon 4 and included a neo selection cassette flanked by Frt sites. C57BL/6 embryonic stem cells with homologous recombination were identified by a PCR-based screen and confirmed by Southern blot and PCR analyses. Positive clones were transfected with Flp recombinase and neo deletion was confirmed by PCR. Mice harboring the modified GR allele were derived by blastocyst (albino B6) injection. For all mice, genotypes were determined by PCR using DNA isolated from tail biopsies. To evaluate the tissue specificity of Cre-mediated recombination, DNA was isolated from various tissues of cardioGRKO mice and subjected to PCR. Primers for the floxed GR allele were forward primer 5’- GGATTATAGGCATGCACAATTACGGC-3’ and reverse primer 5’- CTTCTCATTCCATGTCAGCATGTTCAC-3’. Primers for the null GR allele were the forward primer above and reverse primer 5’-CCCATCCAATGTTGTTGGCAGAG-3’. Adrenalectomized mice were maintained on laboratory chow and 0.9% NaCl ad libitum. All experiments were approved and performed according to the guidelines of the animal care and use committees at the University of North Carolina at Chapel Hill and at NIEHS/NIH. Data presented are from studies done on age-matched littermates and, unless indicated, were performed on male mice. Real-time PCR. Total RNA was isolated from the whole hearts of control and cardioGRKO mice using Qiagen RNeasy mini Kit (GmbH, Germany) according to manufacturer's instructions. All primer sets for PCR were purchased from Applied Biosystems (Foster City, CA). PCR was performed with Taqman® probe-based detection system and quantified by the ABI Prism 7900HT Sequence Detection System 1 (Applied Biosystems, Foster City, CA). The abundance of each gene was normalized to the housekeeping gene peptidylprolyl isomerase B (cyclophilin B) that is not regulated by glucocorticoids. Immunoblotting. Hearts from control and cardioGRKO mice were lysed in RIPA buffer (25 mM Tris- HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with complete mini protease inhibitor cocktail (Roche, Indianapolis, IN). Protein concentrations were determined and equal amounts of lysates were separated on 4-12% bis-tris gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in 1x TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1 % Tween-20) for 1 hour at RT and incubated with primary antibodies overnight at 4ºC. Membranes were then washed with 1x TBST and incubated with appropriate secondary antibodies for 1 hour at RT. After additional washes with 1xTBST, membranes were developed by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). The RyR2 antibody was purchased from Abcam (Cambridge, MA) and the GAPDH antibody was purchased from Cell Signaling (Danvers, MA). The MR antibody was generously provided by Dr. Celso E. Gomez-Sanchez (1). Generation of the anti-GR antibodies 57 and 59 has been described previously (2). Histological analysis. Hearts from control and cardioGRKO mice were perfused with PBS and fixed with freshly prepared 4% paraformaldehyde. Samples were embedded in paraffin, cut in 5 μm sections, and stained with hematoxylin-eosin (HE) or Masson’s Trichrome as previously described (3). For immunohistochemistry, samples were deparaffinized, rehydrated, and endogenous peroxide blocked with 3% hydrogen peroxide. Citrate buffer (Biocare Medical, CA) was used for antigen retrieval. After blocking, samples were incubated overnight with anti-GR antibody 59 followed by a donkey anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) (2). Labeling was done with Vector RTU (Vector Laboratories, Burlingame, CA). Slides were developed with diamino- benzidine and counterstained with hematoxylin. 2 Survival analysis. Control and cardioGRKO mice were monitored on a daily basis for morbidity or death until 12 months age. Severely morbid mice were euthanized according to approved animal protocol. Echocardiographic analysis. Trans-thoracic echocardiography was performed on conscious mice using a VisualSonics Vevo 770 ultrasound biomicroscopy system (VisualSonics, Inc., Toronto, Ontario, Canada) using a 30MHx 707B scan head as previously described (3-5). Two dimensional guided M- mode analysis of the left ventricle was performed in a genotype-blinded fashion in the parasternal long- axis at the level of the papillary muscle. The leading edges of the epicardium and endocardium were used to measure anterior wall thickness (IVSTD, IVSTS), posterior wall thickness (PWTD, PWTS), and left ventricular internal diameters (LVEDD, LVESD). LV volume in diastole (LV VolD) was calculated from the equation LV VolD = (7/2.4 + LVEDD) x LVEDD3 x 1000, and LV volume in systole (LV VolS) was calculated from the equation LV VolS = (7/2.4 + LVESD) x LVESD3 x 1000. Left ventricular systolic function was assessed by ejection fraction (EF), calculated from the equation EF % = (LV VolD-LV VolS)/LV VolD x 100, and fractional shortening (FS), calculated from the equation FS % = (LVEDD- LVESD)/LVEDD x 100. M-mode measurements represent 3 average consecutive cardiac cycles from each mouse. Microarray analysis. Gene expression analysis was performed on RNA from the hearts of 2 day (neonatal), 1 month, 2 month, and 3 month old control and cardioGRKO mice using the Agilent Whole Mouse Genome oligo arrays (014868) (Agilent Technologies, Santa Clara, CA) following the Agilent 1- color microarray-based gene expression analysis protocol as described previously (6, 7). Data was obtained using the Agilent Feature Extraction software (v9.5), using the 1-color defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. To determine differentially expressed probes, an error-weighted ANOVA and Benjamini-Hochberg multiple test correction with a p value < 0.01 was performed using Rosetta Resolver System (version 7.0; Rosetta Biosoftware, Kirkland, WA). Significantly regulated genes were analyzed 3 by Ingenuity Pathway Analysis software (Ingenuity Systems) or by Gene Ontology using GATHER (8). For GATHER, inferred annotations and a Bayes factor cutoff of 6.0 were employed. Calcium measurements on isolated cardiomyocytes. Adult cardiomyocytes from control and cardioGRKO mice were isolated using a commercially available kit (Perfusion Adumyts Cardiomyocyte Isolation Kit, Cellutron, Baltimore, Maryland). Fluorescence measurements were performed on single cardiomyocytes loaded with the calcium sensitive indicator, fluo-4 (Molecular Probes, Kd = 345 nM). Briefly, isolated cardiomyocytes were allowed to attach to Elastin-coated wells in complete AW medium. Cells were then incubated in serum-free AW medium containing 6 µM fluo-4/AM at 37° C in the dark for 15 min. Before intracellular calcium measurements were made, cells were washed 3 times and incubated for 15-30 min at room temperature (25° C) in a HEPES-buffered Tyrode’s salt solution (135 mM NaCl; 4 mM KCl; 1.0 mM MgCl2; 20 mM HEPES; 1.0 mM CaCl2 and 10 mM glucose, with pH 7.4 adjusted by NaOH). Calcium measurements were performed on a Zeiss LSM 710 inverted confocal microscope equipped with a 20x 0.4 NA objective. Fluo-4 fluorescence was monitored by exciting the indicator at 488nm, and collecting the emission wavelength at 500-630 nm. Data were collected in either line scan or frame mode, with a temporal resolution that ranged from 0.1 to 0.5 sec, respectively. Changes in intracellular calcium are represented as the change in fluo-4 fluorescence emission intensity (fluorescence intensity units). Cardiomyocytes displaying spontaneous intracellular calcium changes in the form of local calcium transients were selected for study. Intracellular calcium changes were monitored at the single cell level with data collected from a region of interest (ROI) representing an individual cell. Typically, 1 to 4 ROIs were monitored per experiment. The effects of caffeine stimulation on the intracellular calcium signal were monitored by the dilution of a caffeine solution into the medium bathing the cells. Final concentrations of caffeine bathing cells were either 0.25 mM, 0.5 mM, or 10 mM. Electrocardiographic analysis. Electrocardiographs were recorded in conscious mice non-invasively using the ECGenie system (Mouse Specifics) as described previously (9-11). 4 REFERENCES 1. Gomez-Sanchez CE, et al. (2006) Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 147(3):1343-1348. 2. Cidlowski JA, Bellingham DL, Powell-Oliver FE, Lubahn DB, & Sar M (1990) Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors. Mol Endocrinol 4(10):1427-1437. 3. Willis MS, et al. (2007) Muscle ring finger 1, but not muscle ring finger 2, regulates cardiac hypertrophy in vivo. Circ Res 100(4):456-459. 4. Li HH, et al. (2011) The ubiquitin ligase MuRF1 protects against cardiac ischemia/reperfusion injury by its proteasome-dependent degradation of phospho-c-Jun. Am J Pathol 178(3):1043- 1058. 5. Li HH, et al. (2007) Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin- dependent coactivation of Forkhead proteins. J Clin Invest