Cell culture and transfection
10T1/2 cells were grown in MEM Eagles medium in 6-well plates and transiently transfected with 500 ng of empty pcDNA3.1 control or pcDNA3.1-MRTF-A plasmid for 48hrs prior to RNA isolation. CFs grown in DMEM were treated as noted in text for 24–48 hrs followed by RNA or protein isolation. CF proliferation was determined with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) using the manufacturer’s protocol.
For luciferase assays, CFs or COS cultured in 24 well plates were transfected with a total of 300ng of plasmid DNA using FuGENE6 (Roche). 20ng of pCMV-lacZ was used as an internal control and total plasmid amount was kept constant using empty pcDNA3.1. 48 hrs after transfection luciferase and β-galactosidase assays were carried using a luciferase assay kit (Promega).
Col1a2 reporter construction
420 bp of the mouse Col1a2 promoter was amplified using high fidelity Taq polymerase (TAKARA) and the following oligonucleotides containing 5′ KpnI and 3′ XhoI linkers: Col1a2 For: 5′ – GGTACCGACAGCTCCTGCCTTTTCATC –3′; Col1a2 Rev: 5′ – CTCGAGTAAAATAATAAAGCCCAGACC – 3′. The resulting PCR product was cloned into the KpnI and XhoI sites of the pGL3-basic luciferase vector (Invitrogen). Mutation of the CArG element was accomplished using the QuickChangeII site directed mutagenesis kit and the manufacturer’s protocol (Stratagene). The oligonucleotides used for PCR amplification are as follows: Col1a2 mutCArG For: 5′ – CCTAAAGTGCTTACACACGTGGCAAGGGCG – 3′; and Col1a2 mutCArG Rev: 5′ – CGCCCTTGCCACGTGTGTAAGCACTTTAGG – 3′. All constructs were sequence verified.
CFs were infected with a flag-tagged MRTF-A adenovirus at an MOI of 10 and were treated with TGFβ-1 (10 ng/ml) and/or Y-27632 (10 μM) 24 hrs prior to fixation with cold methanol. Indirect immunofluorescence was performed with a mouse monoclonal Flag M2 antibody (Sigma) or Cy3-conjugated anti-SMA antibody (Sigma, clone 1A4, 1:200). Confocal images were captured using a Zeiss LSM-510 microscope.
Antibodies directed against SMA (Sigma), SM22 (Abcam), Collagen I (Abcam), and tubulin (Sigma) were used to determine protein levels by Western blot. Non-denaturing PAGE was performed to detect Collagen I protein by Western blot.
Histology and immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5-μm intervals. Hematoxylin and eosin and Masson’s Trichrome staining were performed using standard procedures. SMA staining was performed on paraffin embedded sections using a Cy3-conjugated anti-SMA antibody (Sigma, clone 1A4, 1:200). Nuclei were visualized using Dapi in vectashield mounting medium (Vector laboratories). SMA positive vessels and myofibroblasts were counted in the BZ of three WT and three MRTF-A−/− animals and represented as the average +/− SEM. Proliferation and cell death was detected using a phospho-Histone-H3 antibody or the In situ cell death detection kit (Roche) and manufacturer’s protocol, respectively.
Total RNA was isolated using Trizol reagent (Invitrogen) according to manufacturer’s protocol. 2 μg of RNA was used to generate cDNA using Superscript III (Invitrogen) following manufacturer’s protocol and detected using Taqman primer and probesets.
Collagen Synthesis Assay
[3H]-Proline incorporation was performed to determine the effects of MRTF-A over-expression on collagen synthesis. CFs at passage 2 or 3 were cultured in 24-well tissue culture dishes for 48hrs, or until confluent, in DMEM supplemented with 10% FBS under standard culture conditions. CFs were then made quiescent by serum starvation for 24 hrs, infected with 10MOI of adenovirus mediating the expression of MRTF-A or control β-galactosidase, and cultured in SF conditions for an additional 24 hrs. CFs were then stimulated with the addition of 2.5% FBS, TGFβ-1 (10 ng/ml), Y-27632 (10 μM), or a combination of TGFβ-1and Y-27632 for 48hrs in the presence of [3H]-Proline (1 μCi/ml, PerkinElmer Life Sciences). CFs were then washed three times with Dulbelcco’s PBS and protein was precipitated with ice cold 5% TCA for 1hr. The precipitate was then solubilized with 400 μl of 0.2 M NaOH at 37°C for 30 min. Radioactivity was determined by liquid scintillation counting. Each condition was performed in quadruplicate and repeated in 3 independent experiments.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation was performed using the EZ-ChIP kit (Millipore) using manufacturer’s instructions. Briefly, native chromatin from 10T1/2 was crosslinked and immunoprecipitated with antibodies directed against SRF (Santa Cruz), RNA PolII (Millipore), or mouse IgG (Millipore). Col1a2 promoter sequences or GAPDH was detected using PCR amplification. Chromatin was incubated with 2 μg of an immunoprecipitating antibody against endogenous SRF (Santa Cruz), RNA PolII (Millipore), or mouse IgG (Millipore), followed by immunoprecipitation with Protein G beads (Millipore) overnight at 4°C.
Electrophoretic Mobility Shift Assay
EMSA was performed using double stranded oligonucleotides corresponding to the Col1a2 CArG sequence. 4 μl of protein lysate from flag-SRF or empty pcDNA3.1 transfected COS cells was incubated with 32P-labeled oligonucleotide probes in the presence of 1 μl of poly dIdC (1.0 μg/μl) for 20 min. at room temperature. Supershift formation was detected by adding 2μl anti-Flag M2 antibody (Sigma).
Myocardial infarctions were generated using male MRTF-A−/− and WT mice at 12 weeks of age (25–30g) by surgical ligation of the left anterior descending (LAD) coronary artery. Sham operated mice underwent the same procedure without occlusion of the LAD. For determination of infarct size, at least four images of Trichrome stained sections per heart were imported to OpenLab 3.1 and the area of Trichrome staining was measured and taken as a percentage of the total LV area in each section. For the studies designed to measure AAR for infarct, 0.3% methylene blue dye was perfused throughout the animal using direct LV administration immediately following confirmation of myocardial ischemia. Perfusion was carried out until significant staining of cardiac tissue had occurred and no further increase in stained area was apparent for 1 minute. The mouse was then further perfused with 4% paraformaldehyde in saline in order to ensure the proper fixation of tissue and vital dye. The heart was then collected and analyzed for AAR. The heart was photographed in whole mount to document the size of stained (perfused) versus unstained (unperfused) regions. The heart was then histologically dissecting into 3 equal size transverse sections, starting at the site of ligature and progressing towards the apex of the heart. Stained LV tissue was separated from unstained tissue, and weighed. The proportion of unstained versus stained tissue based on dry weight determined the AAR.
Angiotensin II infusion
Ang II (dissolved in 0.01 mol/l acetic acid) was subcutaneously infused at the rate of 0.6 mg/kg/day for 2 weeks using an osmotic minipump (Alzet model 2002; URECT Corp., Cupertino, USA) implanted in each mouse. After 2 weeks Angiotensin II infusion, left ventricles were then fixed in 10% formaldehyde. To determine the extent of collagen fiber accumulation, we randomly selected fields and measured the Masson’s Trichrom stained interstitial fibrosis area in relation to the total left ventricular area using microscopy BIOREVO BZ-9000 (Keyence, Osaka, Japan). Perivascular fibrosis area was excluded in the present study.
Data and statistical analysis
Results are presented as mean +/− SEM unless otherwise stated. Statistical analysis of group differences was performed by Student’s two-tailed t-test with unequal variance and significance between groups of FS% was performed using multiple measures two-factor ANOVA. Significance was considered as P < 0.05.
Mouse mutants and animal care
All experiments utilizing animals were previously approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center. The MRTF-A−/−
mouse line used in this study has been previously reported 19
. Mice were genotyped using previously described PCR strategies.