Download MATERIALS AND METHODS

02-1043R3 Online Methods Supplement
MATERIALS AND METHODS
The investigation was performed in accordance with the Guide for the Care and Use
of Laboratory Animals (NIH publication No. 85-23, revised 1996). Male 16 to 18 week-old
Wistar rats with free access to standard rat chow and tap water were randomly assigned to
one of the following groups: 1) Control (Ctrl, n=10): receiving a daily injection of 0.9%
saline i.p.; 2) Iso (n=12): receiving 5.0 mg/kg (±)isoproterenol hydrochloride (Sigma)
dissolved in 0.3 ml saline i.p. once daily,1 and 3) Iso+BIIB (n=8): receiving 3.0 mg/kg/day
BIIB723 (Boehringer-Ingelheim) in the drinking water plus the daily injection of Iso. The
solution of BIIB723 was prepared every day according to the daily records of water
consumption and body weight (BW), the latter measured every 2 days. Systolic blood
pressure (SBP), by indirect tail-cuff method, and heart rate (HR) were determined weekly
during treatment, which lasted 30 days. At the end of treatment, animals were euthanized
under deep ether anesthesia, the hearts removed and subjected to further analysis. The
ventricles were blotted and weighed (HW) and HW normalized by BW (HW/BW ratio, in
mg/g) was used as hypertrophy index. Only one rat, from the Iso-treated group, died during
the protocol.
Measurement of pHi: One papillary muscles isolated from the left ventricle (LV) of each
heart, isometrically contracting at 0.2 Hz were used for pHi determinations with the
epifluorescence approach previously detailed.2 Briefly, muscles loaded with 10 mol/L
BCECF-AM (Molecular Probes) were superfused (4 mL/min) with bicarbonate-free
(HEPES)-buffered solution (pH ~7.4).10 NHE activity was estimated in terms of steady pHi
values. To measure intrinsic buffer capacity (i), muscles were acid loaded by transient (10
min) exposure to 20.0 mmol/L NH4Cl followed by washout with Na+-free HEPES buffer
02-1043R3 Online Methods Supplement
(NaCl was equimolar replaced with N-methyl-D-glucamine). i was calculated as i=
+
4 ]i
i.
Acute in vitro NHE-
Echocardiographic examination: Rats were monitored echocardiographically under light
anesthesia (35 mg/kg pentobarbital sodium i.p.) a day or two before sacrifice. Twodimensional targeted M-mode tracings were recorded through the anterior and posterior LV
walls (paper speed 50 mm/sec) during 10 cycles with a 7 MHz transducer. Anterior and
posterior end-diastolic thickness (AWT and PWT, respectively), end-systolic wall thickness
and LV internal dimensions were measured using the American Society of
Echocardiography leading-edge method from at least 3 cardiac cycles.3 These tracings were
analyzed with an off-line analysis system (Scion Image) by a single observer blinded to the
experimental group. LV mass (LVM) was calculated from the formula: LVM = 1.047 X
[(EDD + AWT + PWT)3– EDD3] where 1.047 is muscle specific gravity and EDD is the
LV end internal diastolic diameter.4 Endocardial fractional shortening (FS) was calculated
as: (EDD – ESD) / EDD x 100 where ESD is the LV internal systolic diameter. In our
hands, the correlation coefficient between the estimation of LVM by echocardiographic
examination and the LVW at necropsy was r = 0.92 (n=21, P<0.001).
Histomorphometric studies: After the isolation of a papillary muscle, the LV was fixed in
10% buffered formaldehyde for 24 h and embedded in paraffin. Serial sections of 4 µm,
obtained at different levels were stained with hematoxylin-eosin for cell morphometry or
picrosirius red (Direct Red 80, Aldrich) for collagen quantification. Sections were analyzed
under the microscope (magnification x250), and the fields were digitized. An image
analysis system (Imaging Technology, Optimas 5.2) was used to measure cell parameters.
Cardiomyocytes cross-sectional area (CSA), collagen volume fraction (CVF), and the
02-1043R3 Online Methods Supplement
reference area (RA) were determined in each group for an average of 10 micrographs. To
determine CSA, only round to ovoid cells with visible nucleus were considered and each
cell was individually traced. No less than 250 cardiomyocytes from each heart were
examined. CVF was calculated as the sum of stained collagen areas divided by the total RA
x100, with perivascular collagen excluded. Since the greatest degree of fibrosis in the
treated groups was confined to the region lying below the endocardium, the LV wall was
halved into a subendocardial (inner half) and subepicardial (outer half) region and CVF
separately quantified in both regions.
Western blotting: Heart tissue was homogenized in 7 volumes (w/v) of ice cold
homogenization buffer containing (in mmol/L) 20 Tris pH 7.4, 300 sucrose, 1 DTT, 4
EGTA, 5 EDTA, and HaltTM protease inhibitor cocktail kit (Pierce Endogen). The
homogenate was centrifuged at 5000 x g for 15 min at 4 ºC. The supernatant was
centrifuged at 100000 x g for 60 min at 4 ºC. The resulting membrane fraction was
resuspended in homogenization buffer followed by brief sonication. Cardiac membranes
were denatured in NuPAGE 4X LDS sample buffer (Invitrogen) and equal amounts of
protein (45 g/lane) were subjected to PAGE on 4-12 % Bis-Tris gels (Invitrogen)
according to manufacturer’s instructions. Separated proteins were transferred to
nitrocellulose membranes. Nonspecific binding sites of nitrocellulose sheets were then
blocked for 1 hour at room temperature with 10% nonfat milk in TBS. Immunoblot analysis
was performed with rabbit polyclonal antibody for NHE-1, 1/2000 (a kind gift from Dr
Donowitz, The Johns Hopkins University, Baltimore, MD) overnight at 4 C. After
washing with TBS 0.1% Tween 20, membranes were probed with the secondary antibody
couple to peroxydase (anti rabbit IgG 1/5000; Amersham Pharmacia) for 1 hour at room
02-1043R3 Online Methods Supplement
temperature. Blots were washed several times and then treated with the ECL-detection
reagent (Amersham Pharmacia) and exposed to X-ray films. The autoradiographies were
quantified by densitometry. A membrane corresponding to a gel were one sample of each
experimental group was loaded following exactly the same protocol than that described
above, was incubated with secondary antibody alone to confirm the specificity of the
primary antibody.
Statistics: Results are expressed as mean ± SEM. Statistical analysis of results was
performed using Student’s t test or one-way analysis of variances (ANOVA) followed by
Student-Newman-Keuls test, as appropriate. Significance level was set at P<0.05.
REFERENCES
1. Mészaros J, Khananshvili D, Hart G. Mechanisms underlying delayed
afterdepolarizations in hypertrophied left ventricular myocytes of rats. Am J Physiol
Heart Circ Physiol. 2001;281:H903-H914.
2. Ennis IL, Alvarez BV, Camilión de Hurtado MC, Cingolani HE. Enalapril induces
regresion of cardiac hypertrophy and normalization of pHi regulatory mechanisms.
Hypertension. 1998;31:961-967.
3. Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding
quantitation in M-mode echocardiography: results of a survey of echocardiographic
measurements. Circulation. 1978;58:1072-1083.
4. Litwin SE, Katz SE, Weinberg EO, Lorell BH, Aurigemma GP, Douglas PS. Serial
echocardiographic-Dopler assessment of left ventricular geometry and function in
rats with pressure-overload hypertrophy. Chronic angiotensin-converting enzyme
inhibition attenuates the transition to heart failure. Circulation. 1995;91:2642-2654.