MOUSE CORONARY LIGATION
Male C57Bl/6 mice (25 g) were anesthetized with isoflurane prior to intubation with a 24-gauge catheter. Inhalation anesthesia was then instituted with 1.5% isoflurane using a rodent ventilator (Harvard) at 115 breaths/min. A left lateral thoracotomy incision was placed at the level of the fourth interspace and a 7.0 polypropylene suture was used to ligate the left anterior descending artery (LAD) at approximately 1/3 the distance from the base to the apex of the heart. The left chest was then closed and the animal was recovered after extubation in a light-warmed incubator. Animals were subsequently sacrificed at time points ranging from 1 to 12 weeks after infarction as indicated below; at sacrifice, some hearts were fixed and sectioned for histologic analysis, while others were snap frozen for protein extraction. All procedures conformed with 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), and were approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center.
WESTERN BLOTTING AND ELISA
At the time of harvest of specimens for protein extraction, the left ventricle was dissected from the remainder of the heart. The infarct area was identified visually and dissected from the remainder of the myocardium. In addition, myocardial tissue from the free wall and septum clearly distant from the area of infarction was collected as remote myocardium. Any attempt to accurately isolate the irregular borderzone from these small mouse hearts was felt to introduce a prohibitive sampling error. Specimens were snap frozen in liquid nitrogen. Myocardial tissues were homogenized in a lysis buffer containing 0.13 M KCL, 1 mM EDTA, 1 mM EGTA, 1 mM Na3(VO4), 5 mM NaF, 20 mM HEPES, and Protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN) and centrifuged at 35,000 rpm for 30 min. Supernatants were separated by gel electrophoresis, blotted onto a PVDF membrane (Invitrogen), and detected by phospho-ERK1/2 and phospho-p38 Abs (Cell Signaling, Danvers, MA) and chemiluminescence (Pierce) after incubation with horse-radish peroxidase-labeled secondary antibody according to the manufacturer’s instructions. For ELISA, activities of phospho-ERK1/2, phospho-Akt, phospho-JNK, phospho-p38α and phospho-p38γ were assayed using commercially available ELISA kits (R&D Systems) according to the manufacture’s instructions.
Transthoracic echocardiography was performed in conscious mice using an Acuson Sequoia 512 machine and a 13-MHz probe. A two-dimensional short-axis view of the left ventricle was obtained at the level of the papillary muscles, that is, in myocardium remote to the infarction, and two-dimensional M-mode tracings were also recorded. LV fractional shortening was calculated as (LVDd –LVDs)/LVDdX100, where LVDd = LV diastolic dimension and LVDs = LV systolic dimension [Sahn et al., 1978
; Kanno et al., 2002
Mice were heparinized (500 U/kg i.p.) and anesthetized with pentobarbital sodium (60 mg/kg i.p.). Hearts were rapidly excised, washed in ice-cold arresting solution (120 mmol/L NaCl, 30 mmol/L KCl), and cannulated via the aorta on a 20-gauge stainless steel blunt needle. Hearts were perfused at 70 mmHg on a modified Langendorff apparatus using Krebs–Henseleit solution containing 1 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2
, 1.2 mM MgSO4
, 1.2 mM KH2
, 24 mM NaHCO3
, 5.5 mM glucose, 5.0 mM Na pyruvate, 0.5 mM EDTA, and bubbled with 95% O2
at 37°C. Platinum electrodes were connected to a Grass Instrument stimulus generator and were used to pace hearts at 360 beats/min. LV pressure was measured by placing into the ventricle a fluid-filled balloon that was coupled to a pressure transducer. The balloon volume was gradually increased at 5 ml intervals and pressure curves were recorded until a plateau of developed pressure (systolic–diastolic) was achieved. Pressure measurements were then repeated in the presence of norepinephrine (10 μM). Developed pressures and dP/dt were extrapolated from the pressure tracings [Turnbull et al., 2003
MEASUREMENT OF APOPTOSIS
Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) for detection of apoptotic nuclei was performed according to the manufacturer’s protocol (Roche). Apoptotic indices were calculated by dividing the number of apoptotic nuclei in three thin sections by the total number of nuclei as counterstained with hematoxylin. In situ ligase assay (ApopTag® Peroxidase In Situ Oligo Ligation (ISOL) Kit, Chemicon) was used to verify the apoptotic index measured by TUNEL assay. Negative controls were performed by omitting the enzyme TdT (for TUNEL) or the T4 DNA ligase (for the ligase assay); positive controls were performed by pretreating the sections with 10 U/ml DNase I for 20 min at 37°C.
HISTOLOGY AND HISTOCHEMISTRY
Mouse hearts were arrested in diastole, and embedded in paraffin or frozen medium. For Sirius red staining, paraffin sections were stained for 1 h in saturated picric acid with 0.1% Sirius Red F3BA (Sigma). After staining, slides were washed twice in acidified water (0.01 N HCl), and dehydrated. Photomicrographic images were then acquired and collagen content (fibrosis) was analyzed using Image J software (NIH); fibrosis is expressed as the percent of total tissue surface area per field stained with Sirius red. To measure cell proliferation, paraffin sections were stained with primary antibody against PCNA using the ABC kit (Santa Cruz Biotechnology), and counterstained with hematoxylin; proliferative index is reported as PCNA-positive nuclei as a percent of total nuclei. Omission of primary antibodies and staining with non-immune IgG served as negative controls. For fluorescence staining, cryo-sections was pre-incubated in phosphate-buffered saline containing 0.3% Triton X-100 for 30 min and then in phosphate-buffered saline containing 2% bovine serum albumin, 0.1% Triton X-100, and 2% serum were treated with primary antibodies at 4°C overnight. Primary antibodies were against phospho-ERK1/2, phospho-p38, and myosin heavy chain (MF20). After washing, secondary antibodies were incubated at room temperature for 1 h. Fluorescent-tagged secondary antibodies were applied at 1:200 dilutions (Molecular Probes). Nuclei were counterstained with diamidino-2-phenylindole (DAPI). After additional washes, the sections were mounted in Vectashield (Vector Laboratory). Images were captured using the SPOT imaging system (Diagnostic Instruments, Inc.) and Nikon E400 microscopy (Nikon, Inc.).
CARDIAC MYOCYTE AND NON-MYOCYTE ISOLATION AND CULTURE
Adult myocytes and non-myocytes were isolated from C57Bl/6 male mice as described [Zhou et al., 2000
]. Hearts were digested with 4 ml/min Langendorff perfusion of collagenase. Myocytes and non-myocytes were separated from the digested pellet by differential centrifugation, and were plated in 35 mm dishes in MEM plus 10% FCS with 2% CO2
at 37°C. Myocytes were cultured in MEM containing 1 mM BDM overnight, and were then stimulated for 5 min with norepinephrine (2 μM) prior to harvest and preparation of cell lysate. In a separate experiment, myocytes were stimulated for 10 min with 1 μM epinephrine (EPI) with or without the pretreatment of beta-blocker (1 μM propranolol (PROP)) for 10 min. Non-myocytes were serum starved overnight prior to norepinephrine stimulation.
Values are reported as mean ± SEM. Comparisons among groups were made using ANOVA, followed by Neuman–Keuls post-hoc testing. P-values < 0.05 were considered statistically significant, with Bonferroni correction where appropriate.