MSID#: CIRCULATION AHA/2007/752618 2
Expanded Materials and Methods
Construction of Expression Plasmid, Adenovirus, and Transgenic Mice
The carboxyl-terminal 63 amino acids of human Gαi2 (Gαi2 293–355), was amplified by polymerase chain reaction (PCR) and ligated in frame into the EcoRI and XhoI sites of pCMV-HA vector (Clontech, Mountain View, CA) to generate the construct which we term GiCT, which possesses an N-terminal hemaglutinin (HA) tag for detection of the approximately 10 kDa peptide. Human cDNA was used for GiCT amplification; however, at the amino acid level residues 293-355 share 100% similarity for murine as well as rat Gai2. Recombinant GiCT adenoviruses were generated utilizing the Adeasy XL Adenoviral Vector System (Stratagene, La Jolla, CA).
For transgenic mouse generation, this HA-GiCT construct was ligated into a tetracycline (Tet)-off controlled expression system using the cardiac specific α-myosin heavy chain (α-MHC) promoter described by Sanbe and colleagues1. Transgenic mice were generated by pronuclear injection of this construct into mouse zygotes at the Transgenic Mouse Facility at Fox Chase Cancer Center. Founder mice possessing this transgene were then crossed with transgenic mice possessing an α-MHC driven tetracycline transactivator (tTA) construct1. Mice possessing both the GiCT and tTA transgenes will express the GiCT construct in a Tet-off controlled manner – these mice are referred to as Tg-GiCT mice; whereas mice mice possessing only the tTA transgene are referred to as nTG mice and are used as control mice. The stable tetracycline analog doxycycline (DOX) inhibits tTA transactivation, and was administered to mice at 300 mg/kg of mouse diet (Bio-Serv, Frenchtown, NJ) as described to induce GiCT expression in a cardiac-specific manner in Tg-GiCT mice2.
Immunoblotting
Cells and tissue were lysed in ice-cold RIPA buffer (50MMol/L Tris-HCl, 135mMol/L NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, supplemented with 1mMol/L PMSF, 10µg/ml Leupeptin, 20µg/ml Aprotinin, and 1% (v/v) phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich, St. Louis, MO)). The lysates were centrifuged at 13,000 rpm for 20 minutes and protein concentration was determined using BCA Protein Assay Kit (Pierce, Rockford, IL). Equal amounts of protein were then heated at 95°C for 3 minutes in 6X protein loading buffer, and then electrophoresed through 10-20% polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey Blocking Buffer (Li-COR, Lincoln, NE), and then incubated with primary antibodies detecting total p42/44 extracellular signal-regulated kinase (ERK1/2) (Cell Signaling Technologies, 9102), phospho- p42/44 ERK (Cell Signaling Technologies, 9106), hemaglutinin (Covance, MMS-101P), cleaved caspase-3 (Calbiochem, AP-1027), GAPDH (Chemicon, MAB-374), phospho- Akt (Cell Signaling Technologies, 4051), total Akt (Cell Signaling Technologies, 9272), and Gαi2 (Sigma-Aldrich, G4915) at 4°C overnight. Protein levels were then detected using the appropriate Alexa Fluor 680nm- (Molecular Probes; 1:20.000) and IRDye 800nm-coupled (Rockland Inc.; 1:20.000) secondary antibodies using the Odyssey Infrared Imaging System (Li-COR, Lincoln, NE) and quantitative densitometric analysis was performed applying Odyssey version 1.2 infrared imaging software. For phospho-specific antibodies signals were normalized to total antibody signal, and for caspase-3 signals were normalized to GAPDH densitometric levels that were not different between groups.
Cell Culture, Transfection, and p42/44 ERK Phosphorylation
COS-7 cells were grown at 37°C (5% CO2) on 100mm dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus penicillin (100units/ml) and streptomycin (100µg/ml). Cells were transfected with 3µg of β1-AR, α1-AR, or empty vector to assess using Lipofectamine-2000 per manufacturer’s protocol (Invitrogen, Carlsbad, CA). The β1AR receptor couples primarily to Gs, the α1-AR couples primarily to Gq, and the empty vector condition was used to assess endogenous lysophosphatidic acid (LPA) receptors, which couple primarily to Gi. Following 24 hours, cells were split into 6 well dishes, and transfected with 0.5µg of HA-GiCT or vector control for 24 hours. The cells were then serum starved overnight, and then subjected to agonist treatment – isoproterenol (Iso) – 10µMol/L for 5 minutes, phenylephrine (PE) – 10µMol/L for 10 minutes, and LPA –10µMol/L for 4 minutes, which stimulate the transfected β1-AR, α1-AR, and endogenous LPA-R, respectively. Cells were then lysed and immunoblotted for levels of phospho-p42/44 ERK and total p42/44 ERK to assess the Gi selectivity of GiCT for Gi signals versus Gs or Gq. The ~10 kDa GiCT peptide was detected by immunoblotting using a monoclonal HA antibody.
RNA Isolation and Semi-Quantitative Polymerase Chain Reaction
Total RNA isolation was performed using the Ultraspec RNA Isolation System (Biotecx, Houston, TX), according to the manufacturer’s protocol. Following RNA isolation, cDNA was synthesized from 1µg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Quantitative PCR was carried out on cDNA diluted 50 fold using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) and 100 nM of the following gene-specific oligonucleotides – 28s, Gαi2, Gαs, Gαq, and HA-GiCT, sequences available upon request. Quantitation was established by comparing 28s rRNA, that was not different between groups, for normalization. For each run, saturation of amplification cycles were controlled by the use of MyiQ software (version 1.0), and, subsequently, a melting curve generated by heating the product to 95°C, cooling to and maintaining at 55°C for 20 seconds, then slowly (0.5°C/s) heating to 95°C in order to determine the specificity of PCR products, which were then confirmed by gel electrophoresis.
cAMP Production Assay
To demonstrate the ability of Tg-GiCT mice to inhibit Gi signaling in the heart, mouse cardiac myocytes were isolated using a standard retrograde perfusion method from Tg-GiCT and nTg mice as described previously. Myocytes were plated at a density of 25,000 cells / cm2 in modified α-MEM followed by treatment with 0.5mMol/L IBMX to inhibit phosphodiesterases. The cells were then stimulated with the Gi-coupled agonist carbachol (CCh) for 5 minutes, the Gs-coupled agonist Iso for 10 minutes, or pretreated with CCh for 5 minutes followed by 10 minutes of Iso stimulation. cAMP accumulation was quantified using a cAMP ELISA based assay as per manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO). cAMP production was normalized to baseline cAMP levels which were not different between groups (n=4 mice per group, each condition was performed in triplicate).
Transthoracic Echocardiographic Analysis
Transthoracic two-dimensional echocardiography in mice anesthetized with an intraperitoneal dose of Avertin (8µl/g) with spontaneous respiration was performed with a 12-MHz probe as described previously3. M-mode echocardiography was carried out in the parasternal short axis in Tg-GiCT and nTg mice at 6-8 weeks of age to assess heart rate (HR), left ventricular (LV) end-diastolic diameter (LVEDD), LV septal and posterior wall thickness (LVSWT and LVPWT, respectively), and subsequently fractional shortening (FS) and ejection fraction (EF).
Ischemia / Reperfusion Injury Model
Surgical procedures were carried out according to National Institutes of Health Guidelines on the Use of Laboratory Animals and all procedures were approved by the Thomas Jefferson University Committee on Animal Care. The ischemia / reperfusion (I/R) injury model was performed as previously described with minor modifications4. Briefly, 8-10 week old Tg-GiCT and nTg mice were anesthetized with 2% isoflurane inhalation. The heart was exposed through a left thoracotomy at the level of the fifth intercostal space. A slipknot was made around the left anterior descending coronary artery (LAD) at the level of the left auricle with a 6-0 silk suture. After the slip knot was tied, the heart was immediately placed back into the intrathoracic space followed by evacuation of pneumothoraces and closure of muscle and the skin suture via a previously placed purse-string suture. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the LAD but was not tied. Following 30 min of ischemia, the slipknot was released and the myocardium was reperfused for either 1.5 – 3 hours to determine myocardial apoptosis, 24 hours to assess myocardial infarct size, or 72 hours to assess cardiac function and mRNA expression.
Hemodynamic Analysis of Cardiac Function
Hemodynamic analysis was conducted utilizing the same mode of anesthesia in sham and I/R operated Tg-GiCT and nTg mice at 72 hours following surgical intervention as described previously3. Briefly, a 1.4 French micromanometer-tipped catheter (Millar instruments, Houston, TX) was inserted into the right carotid artery and then advanced into the LV as described previously. Hemodynamic parameters, including heart rate (beats/min-1), LV end-diastolic pressure (LVEDP), mean arterial blood pressure (MABP), and maximal (LV +dp/dtmax.) and minimal (LV +dp/dtmin.) first derivative of LV pressure rise and fall, respectively, were recorded in closed-chest mode at baseline and in response to progressive doses of isoproterenol (100, 500, 1000, 5000, and 10,000 pg in 100 µl saline).
Determination of LV Infarct Size and Area at Risk
LV infarct size and area at risk was performed as previously described with slight modifications4. Briefly, following a 24 hour reperfusion period, the ligature around the LAD was re-tied through the previous ligation and 0.2 ml 2% Evans blue dye was injected. The dye was circulated uniformly and distributed in the heart to areas perfused by the non-ligated coronary arteries. The heart was then excised, and LV was isolated and sliced into five 1.2 mm-thick sections in the short axis of the heart. The sections were then incubated in PBS containing 2% triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, St. Louis, MO) at room temperature for 15 min and then digitally photographed. The areas were assessed as follows: the area not at risk (ANAR) or non-ischemic region consists of the Evan’s blue positively staining regions, the area at risk (AAR) consists of the Evan’s Blue negatively staining region – the summation of the TTC-stained positively staining and TTC-negatively staining regions, the infarct area (IA) consists of the pale-appearing TTC-negatively staining region. These regions were quantified using the computer-based image analyzer SigmaScan Pro 5.0 (SPSS Science, Chicago, IL). Myocardial infarct size was expressed as a percentage of the AAR (IA / AAR) and the AAR was expressed as the percentage of total LV (AAR / (AAR + ANAR)).
Assessment of Myocardial Apoptosis and Apoptotic Signaling
Myocardial apoptosis was assessed by terminal deoxynucleotidyl-transferase mediated dUTP nick end labeling (TUNEL) staining, DNA fragmentation, and caspase-3 cleavage. For TUNEL staining, following 3 hours of reperfusion mice (n = 4-6 for sham groups, n=6-8 for I/R groups) were anesthetized, and hearts were quickly removed, and fixed in 4% paraformaldehyde at 4°C. The hearts were then embedded in paraffin and cut into 6 µm thickness sections. TUNEL staining of sections was carried out according to the in situ cell death detection kit protocol (Roche, Switzerland). Slides were covered with a glass cover slip and counterstained with a DAPI-containing mounting media. The section was visualized under a fluorescence microscope with the DAPI filter (330-380 nm) in the region of the infarct border zone at high power; the same region was then examined using a FITC filter (465-495 nm), and digital images were collected for DAPI, FITC, and merged images for >5 images per section, and >3 sections per animal. Apoptotic cells with green fluorescence were detected using NIS Elements Software (Nikon, Japan) to assess the apoptotic index (number of TUNEL positive nuclei / number of total nuclei), which was confirmed with manual counting.
To assess DNA fragmentation, total genomic DNA was isolated from the area at risk of sham and I/R operated Tg-GiCT and NTg mice (n=4 mice per group) utilizing the Gentra Puregene Tissue DNA Isolation Kit (QIAgen, Valencia, CA). Isolated DNA was visualized on a 1% agarose gel, and DNA laddering is represented by the ~200-500 base pair fragments, which are consistent with the DNA laddering visualized in positive control lane consisting of HeLa cells exposed to 1µMol/L staurosporine for 18 hours.
To assess caspase-3 cleavage in (n=4) sham and (n=4-5) I/R operated mice following 1.5 hours of reperfusion, mice were anesthetized and hearts were rapidly excised and the area at risk of the LV was isolated and snap-frozen. Tissue was processed utilizing standard protocols, and equal amounts of protein were immunoblotted for cleaved caspase-3 (Calbiochem, San Diego, CA) – the antibody detects the ~17kDa caspase-3 large cleavage product, which was normalized to GAPDH (Chemicon, Temecula, CA).
Isolation and primary culture of neonatal rat ventricular cardiomyocytes
Ventricular cardiomyocytes from 1-2-day-old rat neonatal hearts (NRVMs) were prepared as published in detail elsewhere5. NRVMs were cultured in Ham’s F-10 supplemented with penicillin/streptomycin (100 units/ml), 5% fetal calf serum at 37°C in a 95% air/5% CO2 humidified atmosphere for 2 days. On day 2 of culture NRVMs were infected with the indicated adenoviruses at a multiplicity of infection of 100, and experiments were performed within 24 hours of infection.
Mitochondrial Membrane Potential (Δψm)
The maintenance of mitochondrial membrane potential (Δψm) has been previously demonstrated as a key determinant of myocyte survival. Loss of Δψm in response to exposure to oxidative stress, such as hydrogen peroxide (H2O2), which has been implicated in the processes of myocardial infarction and reperfusion injury, is an initial step in the mitochondrial pathway of apoptosis6. To assess Δψm, we utilized tetramethylrhodamine ethyl ester (TMRE, Molecular Probes, Eugene, OR), which accumulates in actively respiring mitochondria in response to the Δψm. Assessment of Δψm was performed utilizing a microplate reader to assess a large population of cells in an unbiased manner as described elsewhere7. Briefly, NRVMs were plated at a density of 65,000 cells / cm2 in Falcon Primaria-coated 24-well tissue culture dishes, and infected with LacZ or HA-GiCT adenoviruses which were allowed to infect for 18 hours. NRVMs were then serum-deprived for 18 hours, following which NRVMs were pretreated with 1μM Zinterol for 15 minutes followed by 100μM H2O2 for 60 minutes. NRVMs were loaded with 100nM TMRE for 20 minutes prior to washing twice in ice-cold PBS and lysis in 1X IP Lysis Buffer (50mMol/L HEPES, 125 mMol/L NaCl, 1mMol/L EDTA, 5% Glycerol, 1mMol/L NaF, 0.5% NP-40, 1mMol/L PMSF, 10µg/ml Leupeptin, and 20µg/ml Aprotinin). TMRE fluorescence was measured using a microplate reader with excitation and emission wavelengths set at 508nM and 580nM, respectively. TMRE fluorescence measurements were performed in triplicate and normalized to protein content; TMRE intensity is then normalized as a percentage change versus Lac-Z basal. Importantly, this method assesses Δψm in living cells as dead cells do not remain adherent, and would therefore not be included in the lysate prepared from the scraped cells. Furthermore, dead cells do not accumulate TMRE, and do not contribute to the protein concentration of the lysate to which the TMRE signal was normalized.