2-1. Animals and Experimental model
8- to 12-wk-old male C57BL/6J mice were purchased from National Laboratory Animal Center (NLAC) Taipei, Taiwan. The animals were maintained on a standard laboratory diet and tap water, and exposed to a 12/12 h light–dark cycle. Ambient temperature and humidity during the study were maintained at about 22 and 65%, respectively. The animals℃ were allowed to adapt to the laboratory housing conditions forat least 1 week before starting the experiment. The mouse model of Dox-induced cardiotoxicity was referred to previous studies (Delgado et al., 2004; Fisher et al., 2005). The animals were randomized into 1 of 5 groups and the heart of animals were isolated at week 1, 3, 5, 9, and 12 after the first injection;
besides, each of the groups was subdivided into Dox administration and sham control groups (Figure 1). In the Dox group, the mice were administered with 4 mg/kg i.p. of Dox
hydrochloride weekly for five weeks to reach the accumulated dosage of 20 mg/kg.
Immediately before each infusion, lyophilized Dox hydrochloride was reconstituted by the addition of sterile saline (0.9% NaCl) solution to create a solution with a concentration of 1 mg/mL of Dox. The mice in sham control group received an equivalent volume of saline in place of Dox. All of the animals were observed daily and weighted once a week to record the physical condition. The experimental protocol conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and was approved by the animal welfare committees of the National Chiao Tung University.
2-2. Electrocardiography
Before sacrifice, animals were anesthetized with i.m. injection containing a combination of 50 mg/kg of Zoletil 50® (Virbac, Carros, France) and 15 mg/kg of Rompun® (Bayer,
Leverkusen, Germany). Lead II ECG was introduced by insertion of negative lead at the right shoulder and the positive lead toward the lower left chest. The electrodes were connected to an ECG module Biopac MP150 (Biopac Systems, Goleta, CA, USA), and data were recorded at 2000 Hz for 5 min per animal. The resulting ECG was analyzed by using acqknowledge software (Biopac Systems).
2-3. Tissue sampling and processing
The mice were sacrificed after 1, 3, 5, 9 and 12 weeks following the first injection with Dox or vehicle. After recording the ECG signal, the chest was opened and the heart was perfused with 20 mL ice-cold PBS at the flow speed of 4 mL/min. The remaining buffer was carefully eliminated before the heart was weighted, then the left ventricles were excised from the isolated hearts and tissue samples were stored at -80 ℃until analysis. For pathological examination, the tissues were immersed in 4% paraformaldehyde for 24 hrs then dehydrated and embedded in paraffin wax. Tissue sections were processed by Haematoxylin & Eosin (HE) stain.
2-4. RNA isolation
Total cellular RNA of the left ventricles was extracted as recommended by the
manufacturer of TRIzol™ (GIBCO BRL, Rockville, MD, USA). Briefly, the TRIzol method consists of the addition of 1 mL of the TRIzol reagent to each homogenized tissue (about 100 mg). The mixture was vigorously agitated for 30 sec and incubated at room temperature for 5 min. After this procedure, 200 μL chloroform was added to the tube, and the solution was centrifuged at 12,000 × g for 15 min. The aqueous phase was transferred to a clean tube, precipitated with 500 μL isopropyl alcohol, and centrifuged at 12,000 × g for 15 min. The
resulting RNA pellet was then washed with 1 mL of 75% cold ethanol and centrifuged at 7,500 × g at 4 for 5 min. The pellet was dried at room temperature, resus℃ pended in 20 μL of diethylpyrocarbonate-treated water, and stored at −80 . ℃ RNA was quantified by measuring absorbance at 260 nm and 280 nm and electrophoresed on a 1% denaturing agarose gel. The integrity and relative amounts of RNA were evaluated using ultraviolet visualization of ethidium bromide-stained RNA.
2-5. Reverse transcription-polymerase chain reaction
For cDNA synthesis, 3 μg RNA was supplemented in a total reaction volume of 20 μL with 1× reverse trascriptase (RT) buffer, 1 mM dNTPs, 2.5 μM oligo-dT (Toyobo, Osaka, Japan), 20 U RNase inhibitor (Toyobo), and 100 U ReverTra Ace® (Toyobo). After incubation for 50 min at 42℃, the mixture was incubated for 5 min at 99℃ to denature the products. The mixture was then chilled on ice. PCR primers for RT-PCR analysis were shown in Table 5.
PCR reactions contained 4 μL cDNA, 2 μL each primer (10 μM), 5 μL 10 × PCR buffer, 2 μL 10 mM dNTP, 1 μL of 5 U/μL Taq polymerase (Violet Bioscience, Hsinchu, Taiwan) and 34 μL distilled water in a total volume of 50 μL. General thermal cycler (MiniCycler™; MJ Research, Waltham, MA, USA) conditions were as follows: 1 cycle of 5 min at 94 , 24~38 ℃ cycles of denaturation at 94 for 30~60 sec, annealing at 55~60 for 30~60 sec, and ℃ ℃ elongation at 72 for 30~60 sec, and 1 cycle of 15 min at 72 . The resulting PCR products ℃ ℃ were visualized on 2% agarose gels stained with SYBR Safe™ (Invitrogen, Carlsbad, CA, USA). The stained image was recorded by an image analyzer (DGIS-8 Digital Gel Image System; Topbio, Czech), and the band intensity was quantified using densitometric analysis by Scion image™ (National Institutes of Health, Bethesda, MD, USA). The relative mRNA expression of the collagen, ANP, BNP, and TIMPs were calculated as ratios to
glyceraldehyde-3-phophate dehydrogenase (GAPDH) expression.
2-6. Quantitative real-time PCR
SYBR Green quantitative real-time reverse transcription-PCR (RT-PCR) was performed to detect the mRNA expression level of genes MMP-2, MMP-9 and GAPDH (as an internal control). The specific forward and reverse primers were designed with Primer Express software (Applied Biosystems, Foster, CA, USA) (Table 5). For each selected gene, the primer sets were tested for quality and efficiency to ensure optimal amplification of the samples. Real-time RT-PCR was performed at 1, 1/4, 1/16, 1/64, 1/128, and 1/512 dilution of the synthetic cDNAs from RT-PCR to define relative fold changes and optimal range.
Real-time RT-PCR reaction contained 12.5 μL SYBR Green PCR master mix (Applied
Biosystems), 400 nM forward primer, 400 nM reverse primer, 6 μL cDNA, and distilled water into a total 25 μL volume. All PCR reactions were carried out in triplicate with the following conditions: 2 min at 50℃, 12 min at 95℃, followed by 40 cycles of 15 sec at 95℃, and 1 min at 60℃, in an optical 8-tubes strip (Applied Biosystems) in the ABI 7000 Sequence Detection System (Applied Biosystems). A PCR reaction without cDNA was performed as a
template-free negative control. According to the instructions of Applied Biosystems, the expression of each gene was quantified as ΔCt (Ct of target gene − Ct of internal control gene) using GAPDH as the control and applying the formula 2−ΔΔCt to calculate the relative fold changes (Livak and Schmittgen, 2001).
2-7. Protein extraction and electrophoresis
The frozen left ventricular tissues of mice were homogenized in ice-cold lysis buffer (by a ratio of volume:mass = 5:1) containing 50 mM Tris-HCl (PH 7.5), 150 mM NaCl, 10 mM CaCl2, 0.02% NaN3, 0.5 mM PMSF, 5% glycerol and 1% (v/v) Triton X-100. The
homogenates were then centrifuged at 12,000 × g at 4 for 30 min, and the supernatants ℃ were collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Equal amounts of proteins (20 μg/lane) were separated on 10% polyacrylamide gels by SDS-PAGE.
2-8. Gelatin zymography
Zymography was performed by using gelatin-containing gels as described previously (Stawowy et al., 2004). Briefly, 20 μg of non-reduced tissue homogenates were mixed with zymography sample buffer, composed of 0.5 M Tris-HCl, pH 6.8, glycerol, 10% (w/v) SDS, and 0.1% bromophenol blue without reductant (mercaptoethanol or dithiothreitol), and stood for 10 min at room temperature, then loaded on each lane of an 10% SDS-polyacrylamide gel containing 0.1 mg/mL gelatin (Sigma, St. Louis, MO, USA), the stacking gel contained 5%
acrylamide mix in 1.5 M Tris, pH 6.8. Following electrophoresis, the gel was washed twice for 30 min in zymogram renaturing buffer (2.5% Triton X-100) with gentle agitation at room temperature to remove SDS, then incubated at 37 for 24~48 hrs in developing buffer (50 ℃ mM Tris-HCl, pH 7.4, 200 mM NaCl, 5 mM CaCl2 ). After coomassie brilliant blue (0.125%
Coomassie Brilliant Blue R250, 50% (v/v) methanol, 10% (v/v) acetic acid) staining prior to destain with destain buffer (25% (v/v) methanol, 7.5% (v/v) acetic acid in ddH2O),
gelatinolytic activities were identified as clear zones against a blue background. Molecular weights of gelatinolytic bands were estimated by the positive control and protein marker.
Gelatinase activities in the gel slabs were quantified using Scion image™ which quantifies both the surface and the intensity of lysis bands after scanning the gels.
2-9. Reverse gelatin zymography
Inhibitory activity of TIMPs was analyzed by reverse zymography as described
previously (Oliver et al., 1997) with a slight modification. SDS-12% polyacrylamide gel was prepared with 1 mg/mL gelatin and 0.2 μg human MMP-2 control (cat. no. cc071; Chemicon, Temecula, CA, USA) in 10 mL running gel solution. It was overlaid with 5% stacking gel.
Samples were mixed with equal volume of 2 × zymography sample buffer, and let the mixture stand for 10 min at room temperature. Applied samples (10 μg total protein/lane) and ran the gel with 1X Tris-glycine SDS running buffer (0.038 mM Tris base, 0.12 mM glycine, and 0.003 mM SDS) according to the standard running condition. After electrophoresis, gel was removed from the glass plate and incubated twice on a rotary shaker for 30 min in 2.5%
Triton X-100. The Triton X-100 solution was decanted and replaced with 50 mL of
developing buffer and the gel was incubated at 37 for 48 hrs. In the gel,℃ protein bands from TIMPs were evident as darkly stained bandsagainst a clear background. Dried gel was scanned as describedabove and quantified using Scion image™.
2-10. Western blot assay
Protein extracts of the left ventricular tissues separated by SDS-PAGE were
electrophoretically transferred to PVDF membranes (Perkin Elmer Life Sciences, Inc., Boston, MA, USA) by semi-dry electroblotting (HoeferTM; Amersham Biosciences, Uppsala, Sweden).
Briefly, nonspecific binding sites were blocked by incubating membranes in 5% non-fat milk of PBST (PBS with 0.1% Tween-20) for an hour and the membranes were incubated at 4 ℃ overnight with antibody against MMP-1 (1:3000 dilution of anti-MMP-1 mouse mAb, cat. no.
IM35; Calbiochem, Little Chalfont, UK) and GAPDH (1:2000 dilution of anti-GAPDH goat pAb, cat. no. sc20357; Santa Cruz, Santa Cruz city, CA, USA) as internal control. The probed
blots were washed several times with PBST. Antibody binding of incubated horseradish peroxidase-conjugated donkey anti-goat IgG (1:2500 dilution, cat. no. sc2020; Santa Cruz) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2500 dilution, cat. no. sc2005;
Santa Cruz) in a condition of 4 for 4 hrs ℃ were visualized by Enhanced Luminol Chemiluminescence (ECL) Reagent (NEN, Boston, MA, USA) and by exposing the
membranes to X-Ray film (Super Rx Medical X-Ray Film; Fujifilm, Kanagawa, Japan). The bands were detected at the expected size. The band intensity was quantified using
densitometric analysis by imaging software Scion image™. The amount of MMP-1 was expressed relative to the amount of GAPDH in respective samples.
2-11. Statistical analysis
All data were expressed as mean ± standard deviation (SD). The difference between sham control and Dox groups was evaluated with the Student’s t test. Statistical significance was considered if P < 0.05.