Methods
Culture of Atrial Myocytes and Fibroblasts and Transient Transfection
Both atria from week-old neonatal Wistar rats were cut into chunks and were subjected to trypsin (0.125%) digestion in a balanced salt solution. The disaggregated cells were collected by centrifugation at 300 ×g for 10 min. The cell pellet was re-suspended in serum-containing medium (Ham’s F-12:DMEM;1:1 with 20% fetal bovine serum and 1% penicillin-streptomycin), plated onto a Petri dish and kept for 2.5h in a 5% CO2 atmosphere at 37°C to allow non-cardiomyocytes to attach to the dish. Non-cardiomyocytes (mostly fibroblasts) attached to the bottom of the dish were subsequently incubated with DMEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin for an additional 3 days. The suspended non-attached cardiomyocytes in the medium were collected and cultured for additional 3 days (Ham’s F-12:DMEM;1:1 with 20% fetal bovine serum and 1% penicillin-streptomycin). After 3 days, the cells were transferred to a serum-free medium (Ham’s F-12:DMEM;1:1 for myocytes and DMEM for nonmyocytes) for 24 h and then used for the experiments.
Due to the low transfection efficiency in neonatal atrial myocytes (~10%), we used HL-1 atrial myocytes for all other in vitro atrial myocyte experiments requiring transient transfection. The HL-1 atrial cell line was derived from adult mouse atria,1 which were obtained from Louisiana State University in New Orleans, LA, USA. The culture and maintenance of HL-1 atrial myocytes were as previously described.1
Transient transfection of atrial fibroblasts was carried out using LipofectAMINE (Invitrogen) and HL-1 cells using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection efficiency based on the GFP fluorescence was 50%-70% for atrial fibroblasts and 60%-80% for HL-1 cells.
FLAG-tagged vectors encoding the wild-type STAT3, constitutively active STAT3 (STAT3C) and dominant negative STAT3 (STAT3 Y705F) were obtained from the Rockefeller University in New York, USA.2 Constitutively active Rac1 (RacV12) and dominant negative Rac1 (RacN17) were kind gifts from TS Jou (Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine). The STAT3 luciferase reporter (TransLucent Reporter Vector) was obtained from Panomics (Redwood City, CA, USA). Twenty-four hours after transfection, the cells were subjected to [3H]Proline incorporation, real-time RT-PCR, Western blot, and dual-luciferase (Promega) analyses.
Rat Model of Continuous AngII Infusion
AngII (2 mg · kg–1 · d–1) was infused for 6 hours, 3 days and 14 days into Wistar rats (weight 300±20 g) with a subcutaneously implanted osmotic minipump (3 days and 14 days)(Alzet Co) or a single injection (6 hours). In addition to normal diet, the rats were also fed with pure water (vehicle), losartan (10 mg · kg–1 · d–1) or simvastatin (5 mg · kg–1 · d–1)3 via orogastric tube everyday during AngII infusion. The hearts were rapid excised after AngII treatment, and retrogradely perfused in a Langendorff apparatus with HEPES-buffered Tyrode solution. The perfusate was oxygenated and maintained at 37±0.2ﾟC . After perfusion for 5 min, both atria and the left ventricle were excised for subsequent studies.
The experimental protocol of neonatal rat and adult rat studies described in this report conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine.
Human Atrial Samples
Consecutive patients with valvular heart disease who underwent mitral or aortic valve replacement were recruited. Patients taking angiotensin converting enzyme inhibitors (ACEIs) and/or ARB were excluded. Finally, there were a total of 6 patients with chronic persistent AF and 6 without history of AF recruited. All patients underwent echocardiography before operation. Samples of right atrial appendages were obtained during open heart surgery. The study protocol was approved by the Institutional Review Board and the study subjects gave the informed consent.
Protein Extracts, Western Blot Analyses and Immunoprecipitation
Preparations for protein extracts and Western blot analyses have bee previously described.4 The extract of cytosolic and cytoskeleton proteins were performed according to the manufacturer’s instructions (Chemicon Compartment Protein Extraction Kit, Millipore, MA, USA).
The primary antibodies used in the present study included rabbit polyclonal anti-JAK1 (Santa Cruz), goat polyclonal anti-p-JAK1 (Santa Cruz), rabbit polyclonal anti-JAK2 (Santa Cruz), rabbit polyclonal anti-p-JAK2 (Upstate), rabbit polyclonal anti-TYK2 (Santa Cruz), goat polyclonal anti-p-TYK2 (Santa Cruz), mouse monoclonal anti-STAT3 (Santa Cruz), mouse monoclonal anti-p-STAT3 (Santa Cruz), rabbit polyclonal anti-STAT1 (Cell Signaling), mouse monoclonal anti-p-STAT1 (Santa Cruz), rabbit polyclonal anti-Rac1 (Santa Cruz), goat polyclonal anti-AngII (Santa Cruz), rabbit polyclonal anti-PAK1 (Santa Cruz) and mouse monoclonal anti-FLAG (Sigma). Peroxidase-conjugated secondary antibodies (Santa Cruz) were used for detection of the primary antibody.
For immunoprecipitation, cell lysates were incubated with 1 µg/mL of the anti-Rac1 or anti-STAT3 antibody overnight at 4°C. Immunocomplexes were collected by incubating with 50 µL of protein A for 2 hours. Immunoprecipitates were washed 4 times with ice-cold lysis buffer, and the pellets were re-suspended in 2x sample buffer containing 50 mmol/L Tris (pH 6.8), 2% SDS, 2% β-mercaptoethanol, 2% glycerol, and bromphenol blue. The samples were then subjected to SDS-PAGE and immuno-blotted with anti-p-STAT3 or anti-Rac1 antibody. The proteins were visualized by enhanced chemiluminescence (Amersham).
Measurement of AngII Concentration and Activated Rac1 assay
Ang II concentration was measured by ELISA, as previously described.7 The detection of active, GTP-bound Rac1 was performed using a PAK-PBD pull-down assay as previously described.5
RNA Extraction and Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total cellular RNA was isolated and was reverse transcribed. The single-strand cDNA was amplified by real-time quantitative RT-PCR (ABI-Prism 7900 Sequence Detection System, Applied Biosystems) using SYBR Green dye. For mouse and rat procollagen type I alpha-1 (COL1A1), the forward primer was 5’-GAGCGGAGAGTACTGGATCG-3’ and the reverse primer 5’-GTTCGGGCTGATGTACCAGT-3’ (142 bp). For mouse and rat atrial natriuretic peptide (ANP; NPPA), the forward primer was 5’-GGGGGTAGGATTGACAGGAT-3’ and the reverse primer 5’-CAGAGTGGGAGAGGCAAGAC-3’ (273 bp), and forward 5’-ATACAGTGCGGTGTCCAACA-3’ and reverse 5’-AGCCCTCAGTTTGCTTTTCA-3’ (268 bp), respectively. For mouse and rat GAPDH, the forward primer was 5’-TTGCCATCAACGACCCCTTC-3’ and the reverse primer 5’-TTGTCATGGATGACCTTGGC-3’ (408 bp).
Incorporation of [3H]-Leucine and [3H]-Proline
Twenty-four hours after transfection of STAT3 and constitutively active STAT3 (STAT3C) vectors, HL-1 atrial myocytes and neonatal atrial fibroblasts were plated in 96-well micro-titer plates. The plates were incubated at 37°C in a humidified incubator with 5% CO2 and 95% air for 48 h. The cells were then pulsed with [3 H]-leucine (1 μCi/well in 10 μl TCM medium) for HL-1 atrial myocytes, and [3 H]-proline for neonatal atrial fibroblasts. After 24 h, the cells were harvested using a semi-automated sample cell harvester (FILTERMATE 196 PACKARD). Radioactivity was determined using a Direct Beta Counter (MATRIX 96, PACKARD). Values were corrected for background radioactivity and presented as mean ± SD (n =6).
References
1. Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ Jr. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA. 1998;95:2979-2984.
2. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE Jr. Stat3 as an oncogene. Cell. 1999;99:239.
3. Bayorh MA, Ganafa AA, Eatman D, Walton M, Feuerstein GZ. Simvastatin and losartan enhance nitric oxide and reduce oxidative stress in salt-induced hypertension. Am J Hypertens. 2005;18:1496-1502.
4. Lin CC, Lin JL, Lin CS, Tsai MC, Su MJ, Lai LP, Huang SK. Activation of the calcineurin-nuclear factor of activated T-cell signal transduction pathway in atrial fibrillation. Chest. 2004;126:1926-1932.
5. Dudley SC Jr, Hoch NE, McCann LA, Honeycutt C, Diamandopoulos L, Fukai T, Harrison DG, Dikalov SI, Langberg J. Atrial fibrillation increases production of superoxide by the left atrium and left atrial appendage: role of the NADPH and xanthine oxidases. Circulation. 2005;112:1266-1273.
Figure legends
Online Figure 1. Angiotensin II (AngII) induces translocation of p21-activated kinase (PAK) from the cytosolic fraction to the cytoskeletal fraction in atrial fibroblasts, but not in atrial myocytes. The upper panels show representative immnuoblotting and the lower panels show the quantification by densitometry. A and B, Effect of AngII (1 µmol/L) for indicated times on the protein level of PAK in cytosolic and cytoskeletal fractions in atrial myocytes and atrial fibroblasts, respectively. C and D, Effect of AngII (1 µmol/L) and simvastatin (1 µmol/L, 24h pretreatment) on the protein level of PAK in cytosolic and cytoskeletal fractions in atrial myocytes and atrial fibroblasts, respectively. The ratio of cytoskeletal PAK to cytosolic PAK was calculated for comparisons. Data were expressed as mean ± SD. *Post-hoc P<0.05; ＃Post-hoc P<0.05 vs AngII-treated cells. Csk=cytoskeletal fraction; Cyt=cytosolic fraction; IB=immunoblotting.
Online Figure 1.