Measurements of Blood Pressure and Heart Rate-Systolic blood pressure and heart rate were measured using a noninvasive computerized tail-cuff system (BP98A Softron Corp., Tokyo, Japan) at 2 weeks after pump implantation. Unanesthetized mice from each group were placed in a holding device mounted on a thermostatically controlled warming plate, maintained at 37 °C. Systolic blood pressure and heart rate were measured on two consecutive days, and at least 10 readings were taken for each measurement.
Echocardiographic Analysis-Transthoracic echocardiography was performed using a 15-MHz imaging transducer (Aplio 80 Toshiba Medical Systems Co. Ltd., Japan). Mice from each group were anesthetized by peritoneal injection with 20 mg/kg of 2.5% pentobarbital. The left hemithorax of each mouse was carefully shaved, and M-mode images of the left ventricle were recorded. Left ventricular anterior wall thickness (AW), left ventricular end-diastolic dimension (LVDd), left ventricular end-systolic dimension (LVDs), and left ventricular posterior wall thickness (PW) were measured. All measurements were performed using the leading edge-to-leading edge convention adopted by the American Society of Echocardiography. Percent fractional shortening (FS), left ventricular mass (LVM), and relative wall thickness (RWT) were calculated as follows: FS = ((LVDd - LVDs)/LVDd) x 100, LVM = 1.05 x ((LVDd + AW + PW)3/1000 - LVDd3/1000 + 0.6) and RWT = (AW + PW)/LVDd.
Determination of Heart-to-Body Weight Ratio and Histological Analysis-At the end of pump infusion, mice from each group were anesthetized, and the hearts were excised. The weight of the whole heart (HW) was measured, and the ratio of HW to body weight (HW/BW) was calculated. The whole heart was resected and placed in 20% neutral buffered formalin overnight. After fixation, samples were embedded in paraffin. Then 3-µm sections were cut and stained. The cross-sectional areas of LV myocytes were measured on the mid free wall of the LV from sections stained with hematoxylin/eosin. Suitable cross-sectional areas were defined as having nearly circular capillary profiles and nuclei. Approximately 100 cells were counted in each section, and the average area was used for analysis. To calculate the ratio of the interstitial fibrosis area in the left ventricular area, excluding perivascular fibrosis, samples were stained with Masson-Trichrome and 10 fields were randomly selected from 3 individual sections. Measurements of both the cross-sectional area, and collagen fraction volume ratio were performed using Image J 1.29, a free software program.
Western Blot Analysis-Activities of extracellular signal-regulated kinase1/2 (ERK1/2), ERK5, also referred to as big mitogen-activated protein (MAP) kinase 1, and Smad2 were measured by Western blot analysis as described previously (14, 15). For evaluation of activated ERK1/2, ERK5, and Smad2, phosphospecific ERK1/2 antibody, phosphospecific ERK5 antibody, and phosphospecific Smad2 antibody (Cell Signaling Technology, Beverly, MA) were used, respectively. For Western blot analysis, protein extracts (30 µg for ERK1/2 and ERK5 and 150 µg for Smad2) from the hearts of WT and ARKO mice were boiled for 5 min in Laemmli sample buffer and then run on SDS-PAGE. The protein extracts were then transferred to a nitrocellulose membrane (HybondTM-ECL, Amersham Biosciences). The membrane was blocked for 1 h at room temperature with 5% bovine serum albumin in phosphate-buffered saline/Tween 20. The blots were incubated overnight at 4 °C with antibodies for phosphospecific ERK1/2, phosphospecific ERK5, and phosphospecific Smad2, followed by incubation for 1 h with anti-rabbit secondary antibody (horseradish peroxidase-conjugate). Immunoreactive bands were visualized using enhanced chemiluminescence with ECL reagent (Amersham Biosciences) treatment and exposure to Hyperfilm-ECL. The intensity of the bands was measured using Image J version 1.29.
Northern Blots Analysis-Procedures were performed as previously described (16). In brief, total RNA was isolated from both the right and left ventricles with TRIzol (Invitrogen). RNA concentrations were measured spectrophotometrically at 260 nm, and samples were stored in diethyl dicarbonate-treated water at -80 °C. Approximately 20 µg of total RNA from each sample was fractionated on 1% formaldehyde-agarose gels and transferred to Hybond nylon membranes (Amersham Biosciences) by capillary action in a high salt solution (20 x SSC). Blots were prehybridized in a hybridization solution for 1 h at 42 °C, followed by overnight hybridization with digoxigenin-labeled specific oligonucleotide probes (DIG Northern Starter Kit, Roche Applied Science). Blots were washed twice in 2 x SSC/0.1% SDS at room temperature for 5 min and then washed twice in 0.2 x SSC/0.1% SDS at 68 °C for 15 min before exposure to x-ray film. Evaluation of mRNA levels encoding A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP), -myosin heavy chain (MHC), -myosin heavy chain (MHC), and collagen types I and III was performed. Quantification of mRNA levels was estimated after correction for loading differences by measuring the amount of 28 S rRNA. The intensity of the bands was also measured using Image J version 1.29. Oligonucleotide primers for ANP, BNP, MHC, MHC, and collagen types I and III are shown in Table I.
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