Ethics.

Eight-week-old male C57BL/6 mice were purchased from Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan, and housed in our hospital in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (Frederick, MD) with controlled temperature (24°C), humidity (50% –70%), and light cycle (12/12) for 2 weeks before administration of DOX. Additionally, animals were given ad libitum access to food (FWUSOW Industry Co., Ltd., Taipei, Taiwan.) and autoclaved filtered reverse osmosis water (ELGA MEDICA Pro Water System, West Midlands, United Kingdom). All experimental procedures were approved by the Institute of Animal Care and Use Committee of Kaohsiung Chang Gung Memorial Hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals. The current investigations were restricted to male mice only in order to avoid the possibility that gender specific–effect differences might increase experimental variation and confound statistical analyses.

Pilot Study of DOX-Induced Mouse Cardiomyopathy.

The dose dependency of DOX-induced myocardial damage (i.e., cardiomyopathy) was determined in mice. Male, adult C57BL/6 (B6) mice (n = 24), weighing 25–30 mg (Charles River Technology, BioLASCO Taiwan Co., Ltd.), were grouped to receive stepwise increases of DOX doses i.p. [0 mg (group A), 5 mg (group B), 10 mg (group C), and 15 mg (group D) mg] every other day for 2 weeks, for a total of 7 doses. The regimen and total accumulated dosage of DOX used in the present study was based on previous reports (Olson et al., 2003; Miyagawa et al., 2010) with some modification. Additionally, to minimize animal handling and possible discomfort associated with i.p. injections, DOX was administered every other day for 2 weeks.

The mice were anesthetized with an inhaled anesthesia mixture of isoflurane and oxygen 0.8–1 l/min and placed on a temperature-regulated table (25°C) to maintain body temperature. Isoflurane (2.0%) was vapored at a concentration of 32%–36% in a N₂/O₂ mixture for transthoracic echocardiographic examination. Monitoring of the depth of the anesthesia included testing the rear-foot reflexes before skin incision. The respiratory pattern, mucous membrane color, responsiveness to manipulations, and rear-foot reflexes were closely surveyed throughout the procedure. After echocardiographic examination or DOX administration the mice were returned to the animal center before euthanasia. The mice were euthanized with an overdose of isoflurane (>5.0%, i.e., continue isoflurane exposure until 1 minute after the animal stopped breathing) by day 28 after DOX administration and each heart was harvested for individual study.

The total heart weight to body weight ratio was significantly lower in group D than in the other groups and significantly lower in groups A and B than in group C but it was not different between groups A and B (see Results). Additionally, the LV ejection fraction (LVEF) showed a similar pattern of the heart weight to body weight ratio among the four groups (60% versus 54% versus 50% versus 47.3%, P < 0.001) (see Results). Thus, DOX-induced cardiomyopathy was recreated in the pilot study.

Animal Grouping and Induction of Cardiomyopathy by DOX (15 mg/Every Other Day for 2 Weeks) and Rationale for the Carvedilol Dose in the Current Study.

To elucidate the impact of carvedilol therapy on preserving the LVEF, four additional animals were used and categorized into receiving a higher dose (15 mg/kg/day) and a lower dose (5 mg/kg/day) of carvedilol, respectively. The results of the pilot study showed that the LVEF was better preserved in animals (n = 2) that received 15 mg/kg/day of carvedilol than in animals (n = 2) that received 5 mg/kg/day of carvedilol orally, i.e., by lavage (53.0% versus 49.0%; carvedilol was commenced from day 7 after DOX administration and administered for 4 weeks). Additionally, a previous study has shown that the dosage of carvedilol up to 30 mg/kg daily for rodents was still safe without any side effects (Matsui et al., 1999). Thus, 15 mg/kg/day dose of carvedilol was used in the present study.

In the present study, prior to DOX administration, the mice (n = 30) were equally randomized into group 1 (untreated control, n = 10), group 2 (DOX only, i.p.; n = 10), and group 3 [DOX + carvedilol (15 mg/kg/d by lavage), commenced from day 7 after DOX administration for 28 days, n = 10]. The present study did not provide a carvedilol-treated control group and was based on the spirit of the Guide for the Care and Use of Laboratory Animals, i.e., replace, reduce, and reduce, and the previous study, which had demonstrated that dosage of carvedilol up to 30 mg/kg/day for rodents was safe (Matsui et al., 1999).

In vitro Study for Identifying the Impact of Carvedilol on Protecting the Cardioblasts from DOX-Induced Toxicity.

For the in vitro study, H9C2 cells (cardiomyoblast) were purchased from the American Type Culture Collection Manassas, VA. The cardiomyoblast characteristics of H9C2 cells were verified by immunofluorescent (IF) staining to detect the specific expressions of cardiac troponin T and cardiac sarcomeric α-actinin. Additionally, H9C2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in a T-75 culture flask at 37°C with 5% CO₂. For passage, cells at 70% confluence were enzymatically dissociated with 0.25% trypsin/EDTA and subcultured to new flasks with fresh medium.

To determine the impact of carvedilol against the DOX-induced DNA damage, H9C2 cells were first cultured in Dulbecco’s modified Eagle’s medium culture, and then cocultured with 1) stepwise increases of DOX (0, 20, 100, and 500 nM) for 24 hours; 2) stepwise increases of carvedilol (0, 2, 5, and 10 μM) for 24 hours; or 3) DOX (100 nM) and carvedilol (10 μM) for 24 and 48 hours, respectively. The cells were then collected for individual study.

Functional Assessment by Echocardiography.

The procedure and protocol for transthoracic echocardiography was based on a previous report (Chua et al., 2014). Transthoracic echocardiography (Vevo 2100, Visualsonics Toronto, Ontario, Canada.) was performed in each group prior to and on day 35 after DOX treatment by an animal cardiologist blind to the experimental design. M-mode standard two-dimensional left parasternal/long axis echocardiographic examination was conducted. The LV internal dimensions [i.e., LV end-systolic diameter (LVESd) and LV end-diastolic diameter (LVEDd)] were measured at the mitral valve and papillary levels of the left ventricle, according to the American Society of Echocardiography (Morrisville, NC) leading-edge method using at least three consecutive cardiac cycles. The LVEF was calculated as follows: LVEF (%) = [(LVEDd³ − LVESd³)/LVEDd³] × 100%.

Specimen Collection.

Mice in each group were euthanized by day 35 after DOX treatment, and the heart in each mouse was rapidly removed and then immersed in cold saline. The LV tissues were isolated and divided into three parts for 1) cryosections [embedded in compound (Tissue-Tek, Sakura, Netherlands]; 2) paraffin sections (fixed with 10% formalin); and (3) protein examination (stored at −80°C refrigeration before using), respectively.

Western Blot Study.

The procedure and protocol for western blot analysis were based on previous reports (Chen et al., 2014a,b). In brief, equal amounts (50 μg) of protein extracts were loaded and separated by SDS-PAGE using acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Amersham Biosciences, Freiburg, Germany.). Nonspecific sites were blocked by incubation of the membrane overnight in blocking buffer (5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20). The membranes were incubated for 1 hour at room temperature with the indicated primary antibodies [Bax (1:1000, Abcam Cambridge, MA); cleaved poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling Danvers, MA); caspase 3 (1:1000, Cell Signaling Danvers, MA); Smad3 (1:1000, Cell Signaling); transforming growth factor (TGF)-β (1:500, Abcam Cambridge, MA); Smad1/5 (1:1000, Cell Signaling); bone morphogenetic protein (BMP)-2 (1:1000, Abcam); phosphorylation of histone H2AX (γ-H2AX) (1:1000, Cell Signaling); α-MHC (1:300, Santa Cruz); β-MHC (1:1000, Santa Cruz Santa Cruze, CA); brain natriuretic peptide (BNP) (1:800, Abcam); Akt (1:1000, Cell Signaling); Ku-70 (1:1000, Cell Signaling); and cytosolic (1:2000, BD San Jose, CA) and mitochondrial (1:2000, BD San Jose, CA) cytochrome C]. Horseradish peroxidase–conjugated anti-rabbit IgG (1:2000, Cell Signaling) was used as a secondary antibody for 1-hour incubation at room temperature. The washing procedure was repeated eight times within 1 hour. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences) and exposed to Biomax L film (Kodak Rochester, NY). For the purpose of quantification, ECL signals were digitized using the Labwork software (UVP Upland, CA).

In our western blot study for the assessment of specific protein expressions, we loaded the lysate in the same SDS/polyacrylamide gel and then transferred it to the polyvinylidene difluoride membrane, followed by hybridization with different antibodies and with the use of β-tubulin as the control for each lane in order to assess the expressions of the proteins, which means the β-tubulin expression of each lane was used to compare the expressions of different proteins on the same lane.

Oxidative Stress Measurement of LV Myocardium.

The procedure and protocol for assessing the protein expression of oxidative stress have been detailed in previous reports (Chen et al., 2014a,b). The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (Billerica, MA). 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out in 6 μg protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out in 10% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes, which were then incubated in the primary antibody solution (anti-dinitrophenyl [DNP] 1:150) for 2 hours, followed by incubation in secondary antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by ECL (Amersham Biosciences), which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using the Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.

IF and Immunohistochemical Staining.

IF staining was performed using the respective primary antibodies for examination of CD31⁺ (1:100, Abcam), γ-H2AX⁺ (1:500, Abcam), Ku-70 (1:100, Abcam), CD90⁺ (1:100, abcam), XRCC1⁺ (1:200, Abcam), and 53BP1⁺ (1:100, Abcam; 1:300, Novus Littleton, CO) cells and actinin-phalloidin (1:500, LuBio Science, Switzerland; 1:500, Life Technologies Carlsbad, CA) in LV myocardium based on recent studies (Rajagopalan et al., 1988; Kim et al., 1989; Arola et al., 2000; Wu et al., 2000). Moreover, immunohistochemical staining was performed for examinations of Sca-1⁺ (1:300, BioLegend San Diego, CA) and C-kit⁺ (1:300, Santa Cruz) cells using the respective primary antibodies as described previously (Sung et al., 2009; Leu et al., 2011; Chen et al., 2014a,b; Chua et al., 2014). Irrelevant antibodies were used as controls in the current study.

Histologic Quantification of Myocardial Fibrosis and Collagen Deposition.

The procedure and protocol have been described in previous reports (Leu et al., 2011; Chua et al., 2014). Masson’s trichrome staining was used to identify fibrosis of the LV myocardium. Three serial sections of LV myocardium in each animal at the same levels were prepared at 4 µm thickness by cryostat (Leica, Singapore). The integrated areas (µm²) of fibrosis in each section were calculated using the ImageTool 3 software, version 3.0 (University of Texas, Health Science Center, San Antonio, TX). Three randomly selected high-power fields (HPFs) (100×) were analyzed in each section. After determining the number of pixels in each infarct and fibrotic area per HPF, the numbers of pixels obtained from three HPFs were summated. The procedure was repeated in two other sections for each animal. The mean pixel number per HPF for each animal was then determined by summating all pixel numbers and dividing by 9. The mean integrated area (µm²) of fibrosis in LV myocardium per HPF was obtained using a conversion factor of 19.24 (where 1 µm² represented 19.24 pixels).

To analyze the extent of collagen synthesis and deposition, cardiac paraffin sections (6 µm) were stained with Picrosirius Red (1% Sirius Red in saturated picric acid solution) for 1 hour at room temperature using standard methods. The sections were then washed twice with 0.5% acetic acid. The water was physically removed from the slides by vigorous shaking. After dehydration three times in 100% ethanol, the sections were cleaned with xylene and mounted in a resinous medium. The HPFs (×100) of each section were used to identify the Sirius Red–positive area in each section. Analyses of collagen deposition area in LV myocardium were identical to the description of the calculations of the infarct and fibrotic areas.

Statistical Analysis.

Quantitative data are expressed as means ± S.D. Statistical analysis was performed by analysis of variance followed by the Bonferroni multiple-comparison post hoc test. The SAS statistical software for Windows, version 8.2 (SAS institute, Cary, NC) was used. A probability value <0.05 was considered statistically significant.