The animals were kept under standard animal room conditions (temperature 21 ± 1°C; humidity 55 ± 5%) with food and water unlimited. All experimental protocols were approved by the Experimental Animal Ethic Committee of Harbin Medical University, China. Use of animals followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).
Construction of M3-mAChR overexpression TG mice model
TG mice were generated by using a construct in which the α-myosin heavy chain (α-MHC) promoter drove the exclusive expression of M3-mAChR in cardiomyocytes . TG mice and their wild-type (WT) littermates of either sex, 8–12 weeks old, with a body weight of 20–25 g, were used. Age-matched WT and TG mice were randomly divided into 4 groups: (1) vehicle-infused WT mice (WT-CTRL); (2) Ang II-infused WT mice (WT-Ang II); (3) vehicle-infused TG mice (TG-CTRL); (4) Ang II-infused TG mice (TG-Ang II). Ang II (0.6 mg/kg per day) or normal saline (NS) was injected subcutaneously for 14 days as described previously .
Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg) (Sigma, St Louis, MO, USA). Then, transthoracic echocardiography was performed with an echocardiographic system equipped with a 10.0-MHz phase-array transducer (GE Vivid 7, GE, USA). Left ventricular diameter and wall thickness were measured using M-mode tracings as described previously .
Hemodynamic measurements, heart weight (HW) measurement and histological analysis
After treatment with Ang II, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg), and then put on a heated platform for body temperature and electrocardiography (ECG) measurements. Pressure-volume control unit FV896B PV catheter (Scisense advancing micro-sensor technology, London, Ontario, Canada) was inserted through the right carotid artery into the aorta for mean arterial blood pressure (MAP) measurement . Next, the hearts were quickly excised and washed with cold (4°C) PBS buffer. The ratio of whole heart weight to body weight (HW/BW) and left ventricular weight to body weight (LVW/BW) was measured. Ventricle tissue was then equally divided into three parts. One part was fixed with 4% paraformaldehyde and then stained with hematoxylin and eosin (HE). The other two parts and atrium tissues were frozen in liquid nitrogen and stored at -80°C for subsequent analysis.
Transverse aortic constriction
The pressure-overload cardiac hypertrophy model was induced by transverse aortic constriction (TAC) as described previously . Adult mice (BDF1, 24 ± 2 g), were anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg). After successful endotracheal intubation, the cannula was connected to a volume cycled rodent ventilator (UGO BASILE S.R.L. Italy).The transverse aorta was isolated from annexed tissue, and the artery was partially ligated immediately with 7–0 silk around a 25-gauge blunted needle, which was subsequently removed. Sham operated mice underwent the same procedure, except that the transverse aorta was not partially ligated. The chest was closed and the animals were kept ventilated until the recovery of autonomic breath, and then raised for 14 days.
Preparation of neonatal rat ventricular myocytes (NRVMs)
NRVMs were isolated from 1-day-old neonatal Wistar rat hearts and differentially plated to remove fibroblasts . Ang II (Sigma, St Louis, MO, USA) or isoproterenol (ISO) (Sigma, St Louis, MO, USA) was added to the culture medium at a final concentration of 0.1 μM or 10 μM for 48 h to induce hypertrophy [18, 22]. The cells were then harvested to determine M3-mAChR expression during cardiac hypertrophy.
Construction of M3-mAChR overexpression TG-H9c2 cell model
pcDNA3.1 (+) human M3-mAChR vector was constructed and transfected into rat H9c2 cardiomyoblasts plated on BioFlex plates. After 48 h, cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 500 μg/mL G418 at 37°C in 5% CO2 and 95% air, at a relative humidity of 95%. The cells were split 1 to 3 at sub-confluence (70%). Before each experiment, cells were seeded at a density of 5 × 104 cells/cm2. H9c2 cells were cultured in serum-free DMEM for 12 h before treatment with or without 0.1 μM Ang II for 48 h. To quantify the cell surface area, the H9c2 cells were stained with acridine orange. The relative surface area of the cells was calculated from the number of pixels by using Image-Pro Plus (version 5.0.1).
Western blot analysis
Total proteins (~60 μg) were extracted from cells or tissues as described previously , fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membrane was incubated at 4°C overnight with the primary antibodies against M3-mAChR, M2-mAChR (Alomone Labs, Jerusalem, Israel); Ang II type 1 receptor (AT1 R) (Sigma, St Louis, MO, USA); c-Jun N-terminal kinases (JNK), phospho-JNK (p-JNK), extracellular regulated protein kinase (ERK), phospho-ERK (p-ERK) and p38 mitogen-activated protein kinase (MAPK) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-p38 (p-p38) (Cell Signaling Technology, Boston, USA), followed by incubation with the secondary antibody Alexa Fluor 800 rabbit anti-mouse IgG (H + L) or Alexa Fluor 800 goat anti-rabbit IgG (H + L) (Invitrogen, Carlsbad, USA). The images were captured on the Odyssey Infrared Imaging System (LI-COR, Upland, CA, USA) and band intensity (area × OD) was quantified using Odyssey v1.2 software with GAPDH as loading control.
Real-time quantitative RT-PCR analysis
Total RNA was extracted from the tissues using Trizol reagent (Invitrogen, USA). First-strand cDNA was synthesized by a reverse transcriptase kit (Invitrogen, USA) according to the manufacturer’s instructions, and used as the template for Quatitative RT-PCR analysis on a ABI 7500 fast Real Time system (Applied Biosystems, Foster City, CA, USA), with GAPDH used as an internal control . The primer sequences were as follows: GAPDH, 5′-AAGAAGGTGGTGAAGCAGGC-3′ (forward), 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse); β-myosin heavy chain (β-MHC), 5′-CCAGAAGCCTCGAAATGTC-3′ (forward), 5′-CTTTCTTTGCCTTGCCTTTGC-3′ (reverse); Atrial natriuretic peptide (ANP), 5′-CTCCGATAGATCTGCCCTCTTGAA-3′ (forward), 5′-GGTACCGGAAGCTGTTGCAGCCTA-3′ (reverse); M3-mAChR, 5'-CATCATCGGCAACATCCT-3' (forward) and 5'-GAGGTCACAGGCTAAGTTC-3' (reverse).
Data were expressed as means ± SEM and analyzed with SPSS 13.0 software. Comparisons between two groups were made using Student’s t-test. Comparisons among multiple groups were performed using analysis of variance (ANOVA) followed by Bonferroni post test. Differences were considered to be significant at P < 0.05.