Generation of recombinant adenoviruses encoding for Calsarcin-1
The adenovirus (AdV) encoding full length human calsarcin-1 cDNA was generated using the Adeno-X kit (Clontech) according to the manufacturer´s protocol. Briefly, the HA-tagged cDNA was subcloned in the pShuttle vector. After cleavage with I-CeuI and PI-SceI the insert was cloned into the adenoviral backbone. Amplification and titration of the virus was carried out in HEK 293 cells. The eGFP enconding adenovirus was a kind gift from Dr. P. Most, University of Heidelberg. Neonatal rat cardiomyocytes were infected with a multitude of infection (moi) of 20.
Isolation and culture of neonatal rat ventricular cardiomyocytes (NRVCMs)
Hearts from 2 days old Wistar rats (obtained from Charles River, Sulzfeld, Germany) were harvested and minced in 1xADS buffer. Up to six digestion steps were carried out with pancreatin (Sigma, Germany, 0.6 mg/ml) and collagenase type II (Worthington, 0.5 mg/ml) in sterile 1xADS buffer containing 120 mmol/l NaCl, 20 mmol/l HEPES, 8 mmol/l NaH2PO4, 6 mmol/l glucose, 5 mmol/l KCl, 0.8 mmol/l MgSO4, pH 7.4. Cardiomyocytes were purified from fibroblasts using a Percoll (Amersham, Germany) gradient centrifugation step. Finally, NRVCMs were resuspended and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, penicillin/streptomycin and L-glutamine (PAA, Austria) 1.
RNA isolation and purification
Total RNA from rat neonatal cardiomyocytes was isolated with the RNeasy kit from Qiagen (Germany) following the manufacturer´s instructions. RNA from mouse hearts was isolated using the TRIzol method (Invitrogen, Germany) according to the manufacturer’s protocol. In both cases, DNase digestion and purification of RNA was carried out on the RNeasy columns (Qiagen, Germany).
Quantitative Real time PCR
DNase-digested total RNA of each condition was transcribed to cDNA using the Supercript III first strand kit (Invitrogen, Germany). Real time PCR primer sequences are shown in supplementary table 1. 18S rRNA served as an internal standard. For quantitative real-time PCR, the Platinum SYBR Green qPCR SuperMix-UDG system (Invitrogen) was used in the ABI Prism 7700 Sequence Detection System (Perkin Elmer Applied Biosystems). Each PCR amplification was carried out using the following conditions: 2 minutes at 95°C, followed by a total of 40 temperature cycles (15 seconds at 95°C, 15 seconds at 57°C and 1 minute at 72°C).
Immunoblotting
NRVCM were harvested and lysed in completed RIPA buffer containing 10 mmol/l Tris, 15 mmol/l EDTA pH 7.5, 1% NP 40 (v/v), 0,5 % Sodium deoxycholate (w/v), 0,1 % SDS (w/v) (Sigma, Germany), protease inhibitor cocktail tablets (Roche, Germany), as well as phosphatase inhibitor cocktail I and II (Sigma, Germany). After up to three brief freeze-and-thaw cycles and a centrifugation step whole cell lysate was obtained.
Heart samples of animals were harvested, immediately transferred into completed RIPA buffer and homogenized using an Ultra-turraxTM tissue separator (IKA, Germany). Whole cell extracts and muscle homogenates were resolved by SDS-PAGE, transferred to an Immobilon FL membrane (Millipore), and immunobloted with the respective primary antibodies (monoclonal-HA (Sigma, Germany) 1µg/ml, polyclonal calsarcin-1 1:10002, MCIP-13. After staining with the secondary antibodies IRDye 680 Goat Anti-Mouse IgG or IRDye 800CW Goat Anti-Rabbit IgG (Li-Cor Biosciences, USA), respectively, proteins were visualized with a LI-COR infrared imager (Odyssey). Quantitative densitometric analysis was performed using Odyssey version 1.2 infrared imaging software. Signals were normalized to tubulin unless stated otherwise.
Conventional western blotting was carried out using the WestranTM PVDF membrane (Schleicher Schüll, Germany). Application of the first antibody (anti-Calsarcin-1 (1:10002), or anti-HA monoclonal (1µg/ml, Sigma, H-9658)) was followed by incubation with an HRP-coupled secondary antibody (1:10000) (Santa Cruz). Visualization was achieved using a chemoluminescence kit (Amersham).
Immunofluorescence experiments
Neonatal rat cardiomyocytes were fixed in 4% paraformaldehyde (Sigma) in PBS, permeabilized with 0.3% Triton X-100 (Sigma, T9284) and blocked for 1 h with 2% BSA (Sigma) in PBS. Sections from heart muscle (8µm) were fixed for 10 min in ice-cold acetone, followed by a washing step with PBS. Sections were blocked with 2% BSA (Sigma) in PBS for 1h. The monoclonal antibody against sarcomeric α-actinin (Sigma) was used at a dilution of 1:200, the polyclonal anti-Calsarcin-1 antibody (PNAS) at 1:150. Secondary antibodies (fluorescein coupled anti-rabbit antibody, Vector Laboratories, Burlingame, CA, USA, and Cy3 coupled anti mouse antibody, (Dianova:115-165-003) were used at 1:200 for one hour. Cell surface areas of cardiomyocytes were determined applying the ImageJ software, NIH, USA.
Generation of Calsarcin-1 transgenic mice
A cDNA encoding mouse hemagglutinin (HA)-tagged Calsarcin-1 was cloned into a plasmid containing the heart-specific -myosin heavy chain (MHC) promoter and human growth hormone (hGH) poly(A)+ signal4, which was a kind gift from Jeffrey Robbins, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH, USA. DNA isolation and linearization were carried out using standard techniques. DNA injections into pronuclei of mice with mixed FVB 129BL/6 background were performed in the transgenic core facility of the University of Heidelberg, Germany. Genomic DNA was isolated from mouse tail biopsies and analyzed by PCR with primers specific for the hGH poly(A)+ signal: (forward: 5’- GTC TAT TCG GGA ACC AAG CTG GAG TG-3’, reverse: 5’- ACA GGC ATC TAC TGA GTG GAC CCA AC-3’). Generation of transgenic animals as well animal handling were performed according to the institutional guidelines of the University of Heidelberg as well as the state of Baden-Württemberg.
Echocardiography
Echocardiography of mice was carried out using an ATL HDI 5500 device and a 10 Mhz probe as described previously5. Briefly, mice were anesthetized using Avertin (2.5%, 15-20 µl/g body weight) and placed onto a warmed mat. Three independent M-mode measurements per animal were obtained by a genotype-blinded examiner. The following parameters were obtained in a short axis at the level of the papillary muscles: End systolic and end diastolic chamber diameter, interventricular septum and posterior wall thickness, as well as left ventricular fractional shortening {FS% = [(LVEDD - LVESD)/LVEDD] 100}.
Angiotensin II Administration
Animals were anesthetized with Isoflurane 3% vol/vol and alzetmini pumps model 2002 Durect Corporation, USA) releasing either angiotensin II (500 ng/kg/min) or vehicle alone (0.9 % NaCl),were implanted subcutaneously. Bloodpressure was measured in conscious animals using a visitech 2000 blood pressure measuring apparatus according to the manufacturer´s guidelines. 14 days after implantation of the mini pumps, cardiac catheterization was performed as described below.
Cardiac catheterization
Invasive assessment of cardiac hemodynamics was conducted as described previously 6. Briefly, mice were anesthetized with thiopental (100 mg/kg, injected intra peritoneally), intubated and artificially ventilatedusing asmall animalventilator (Hugo Sachs, Germany). A micromanometer catheter (Millar Instruments, USA) was introduced into the right carotid artery and retrogradely advanced into the left ventricle. Subsequently, intraventricular pressures as well as dp/dt were recorded.
At the end of the experiment animals were sacrificed in deep anesthesia by exsanguination and organs were harvested to determine organ weightsand to perform further biochemical and molecular analyses.
Statistical analyses
All results are shown as the mean +/- standard error of the mean (SEM) unless stated otherwise. Real time PCR data analyses were carried out using the ΔΔct method7. Statistical analyses of the data were carried out using one or two way ANOVA followed by Student-Newman-Keuls post-hoc tests. If appropriate, Student’s t-test was employed (two sided, assuming similar variances). P values <0.05 were considered statistically significant.
References
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