Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2 (original) (raw)

Mice. Mice lacking FGF-2 gene expression (FGF-2–/– mice) were generated using homologous recombination in embryonic stem cells by replacing most of the second exon, resulting in the deletion of sequences encoding amino acids 82–93 (A. Foletti and F. Beermann, unpublished results). Depending on the strain, mice carry either one or two renin genes (24). C57BL/6 mice are the prototype of one-renin-gene mice. Therefore, to be more relevant to the human situation, mice were backcrossed for five generations in a C57BL/6 background, and males bearing one renin gene were used at 8 weeks of age.

2K1C hypertension. Briefly, mice were anesthetized using 1.5% halothane in oxygen. The left kidney was exposed, and a clip (0.12–mm opening) was placed on the renal artery, which was isolated by blunt dissection (23). The sham procedure included the entire surgery, with the exception of artery clipping.

Blood pressure. Four weeks after clipping, the right carotid artery was exposed, and a silicon catheter filled with 5% glucose solution containing heparin (300 IU/ml) was inserted into the vessel and tunneled subcutaneously to exit at the back of the neck. Blood pressure was recorded in conscious mice by connecting the catheter to a pressure transducer using a computerized data acquisition system (Notocord Systems, Croissy-sur-Seine, France).

Echocardiography. Left ventricular dimensions were assessed 4–6 weeks after clipping in anesthetized mice (90 mg/kg ketamine, 5 mg/kg xylazine, intraperitoneal) by echocardiography using an ATL HDI 5000 ultrasound machine (Philips Medical Systems, Bothell, Washington, USA) equipped with a 12-MHz phase array linear transducer (L12-5). M-mode images were used for measurement of wall thickness, chamber dimension, and shortening fraction.

Cardiac weight index and cardiac protein homogenates. Cardiac weight index was determined as the ratio of cardiac ventricle weight to body weight. Ventricles were homogenized on ice in 1 ml RIPA buffer (150 mmol/l NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mmol/l NaVO3, 1 mmol/l NaF, 1 μg/ml aprotinin, 1 mmol/l PMSF, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 mmol/l EDTA, and 50 mmol/l Tris, pH 7.5).

Histology. Heart sections from 2K1C wild-type (FGF+/+) mice and FGF-2–/– mice were stained with hematoxylin and eosin.

Plasma renin concentration. Plasma renin concentrations were measured as described (23). Aliquots of plasma samples were incubated for 15 minutes at 37°C in the presence of plasma from nephrectomized rats as a source of renin substrate. The Ang I concentrations produced during the incubation period were determined with a sensitive radioimmunoassay.

Northern blot analysis. Total RNA, purified from frozen tissues using TriPure (Roche Diagnostics Corp., Basel, Switzerland), was subjected to Northern blot analysis. Ten micrograms of total RNA was separated on 1% agarose-formaldehyde gel, transferred to GeneScreen nylon membrane (NEN Life Science Products Inc., Boston, Massachusetts, USA), and hybridized with radiolabeled cDNA probes spanning the mouse renin (kidney RNA) and atrial natriuretic factor (cardiac RNA) sequences. Signals were quantified using an Instant Imager (Packard Bioscience Co., Meriden, Connecticut, USA), and normalized to the GAPDH signal.

Cell culture. Neonatal ventricles were digested at 37°C in 116 mmol/l NaCl, 1 mmol/l NaH2PO4, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4·7H2O, 5.5 mmol/l glucose, and 20 mmol/l HEPES (pH 7.4) containing 0.45 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, New Jersey, USA) and 1 mg/ml pancreatin (Life Technologies Inc., San Diego, California, USA). Cells were washed by centrifugation in a 3:1 mixture of DMEM and Medium 199 (Life Technologies Inc.) supplemented with 10% horse serum (Serotec Ltd., Oxford, United Kingdom), 5% FBS (Serotec Ltd.), 2 mmol/l L-glutamine (Life Technologies Inc.), 10 mg/ml streptomycin, and 100 UI/ml penicillin (Life Technologies Inc.). Cardiomyocytes and nonmyocyte cells (NMCs) were separated by differential plating (45 minutes), and plated at a density of 1 × 105 cells/cm2 on gelatin-coated wells and noncoated wells, respectively. After 24 hours, cardiomyocytes were switched to serum-free medium. NMCs were passaged once before switching to serum-free medium. Cells were stimulated with Ang II, FGF-2, or NMC supernatants, washed with PBS, and lysed in 2× SDS-PAGE loading buffer.

Production of conditioned medium from NMC culture. NMCs were grown to subconfluence in 10-cm dishes, placed in serum-free medium for 24 hours, and stimulated for 24 hours with 100 nM Ang II. FGF-2 concentrations in supernatants were measured by ELISA (Chemicon International Inc., Temecula, California, USA).

MAPK activation. Thirty micrograms of soluble protein was run in a 10% SDS-PAGE gel and transferred onto nitrocellulose (ECL nitrocellulose; Amersham Pharmacia Biotech Ltd., Little Chalfont, United Kingdom). Phosphorylation of MAPK was determined by incubation with specific antibodies for the phosphorylated and nonphosphorylated forms of p38, ERK, and JNK (New England Biolabs, Beverly, Massachusetts, USA) at 4°C in blotto (20 mmol/l Tris, pH 7.6, containing 0.1% Tween-20 and 5% dry milk). Primary antibodies were revealed with anti-rabbit secondary antibodies conjugated to horseradish peroxidase, using a chemiluminescent detection system (Amersham Pharmacia Biotech Ltd.).

Immunofluorescence. Sarcomeres in cardiomyocytes (fixed with 3% paraformaldehyde) were detected using a monoclonal anti–myomesin B4 antibody kindly provided by Jean-Claude Perriard (Swiss Institute of Technology, Zurich, Switzerland) (25), and a confocal unit equipped with an inverted microscope (LSM 410; Carl Zeiss, Jena, Germany).

Protein synthesis. Protein synthesis was determined by adding [3H]phenylalanine (5 μCi/ml) to culture medium, and counting uptake using standard scintillation techniques.

Expression of α-skeletal actin. Protein was detected by Western blot analysis using an anti–α-skeletal actin polyclonal antiserum as described (26).

Statistical analysis. Data are expressed as mean ± SEM. Statistical analysis was performed using the Student-Newman-Keuls test. P values less than 0.05 were considered significant.