Supporting Information

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Table 1

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The BNP-116-luc and the ANF-638-luc plasmids were generous gifts from Dr. C Glembotski (San Diego State University, San Diego, CA) (44, 45). The KLF15-3'Flag expression plasmid (pcDNA3.1-KLF15Flag) was created by PCR amplification of the KLF15 ORF using specific primers that included a FLAG sequence at the 3' end, and BglII and XhoI restriction sites at the 5' and 3' ends, respectively. The cDNA fragment was cut with BglII and XhoI and subcloned into the pCDNA3.1 vector digested with BamHI and XhoI (24). The KLF15 expression-adenovirus was prepared by the Harvard Gene Therapy Initiative (HGTI). The GATA4- and the MEF2A expression plasmids, pCDNA3.1-GATA4 and pCMV-MEF2A, respectively, were generous gifts from Dr. G. Huggins (Tufts New England Medical Center, Boston, MA) and Dr. E. N. Olson (UT Southwestern Medical Center, TX). The cDNA probes for Northern analyses were designed from various expression plasmids that were obtained from the following sources: The expression plasmid for ANF was a kind gift of Dr. M.T. Chin (Harvard Medical School, Boston, MA); that for bMHC was from Dr. M. Buckingham (L'institut Pasteur, France); the BNP plasmid was from Dr. S. Izumo (Harvard Medical School, Boston, MA).

Using Trizol reagent (Gibco-BRL), total RNA was isolated from isolated NRVM, NRVF as well as portions of ventricle, lung, liver and kidney from adult rats and mice. 10 mg RNA was fractionated on denaturing formaldehyde gels and transferred to a nylon membrane. The membranes were probed with [³² P]-labeled cDNA probes specific for the indicated genes, as experiments required.

Luciferase reporter plasmids (-638 ANF-luc or -116 BNP-luc) were cotransfected with the CMV-b-gal plasmid. 24 h after transfection, the culture medium was replaced with serum-free medium before treatment with PE (50 mM) for an additional 24 h. H9c2 cells were plated in a 6-well plate in DMEM at a density of 50,000 cells/well. The cells were cotransfected with 200 ng of the reporter plasmids (-638 ANF-luc, -116 BNP-luc), 200 ng each of expression plasmids (KLF15, GATA4, MEF2A) and 100 ng of pCMV-b-gal to normalize the reporter activity. The total amount of DNA in all experiments was kept constant using pCDNA3.1. Reporter activity was measured using the

Luciferase Assay System (Promega) on a luminometer, according to the manufacturer's protocol. b-galactosidase assays were performed by conversion of o-nitrophenyl b-D-galactopyranoside (ONPG). Each sample was tested in triplicate and each experiment was repeated at least 3 times.

Probe design: Primers for KLF15 were designed from the published rat KLF15 cDNA sequence (GenBank accession # NM_053536) using Primer3 (Whitehead Institute for Biomedical Research). Forward primer 5'-CAG CTT CTG GTC AAC ATC CA-3'. Reverse primer 5'-GAA GTT CTG CTG CTG GGT TC-3'. These primers amplified a 205 bp sequence (nucleotides 1023 to 1227) that was cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced to assure its identity (ABI Prism 310, Molecular Genetics Maastricht University). The plasmid DNA was purified, linearized and RNA probes [both sense (negative) and anti-sense (positive probe)] were obtained by RNA labeling by in vitro transcription of DNA with Digoxigenin (DIG), according to the protocols by Roche (Roche Applied Science, DIG Application Manual). In situ hybridization (ISH): Four micrometer sections were used from paraffin-embedded hearts of Sprague-Dawley rats (n = 6). The sections were deparaffinized, washed with PBS, permeabilized with 25 mg/ml proteinase K (Ambion, Austin, TX) for 20 min at 37°C, rinsed with cold PBS for 5 min, postfixed with 4% paraformaldehyde (Merck) in PBS, acetylated with 0.25% acetic anhydride (Merck) in 0.1M, pH 8.0 triethanolamine (Riedel-de Haën, GmbH, Seeize, Germany) and rinsed in PBS. Subsequently the slides were dehydrated and covered with prehybridization mix: 10 mM DTT (Sigma), 1 mg/ml tRNA (Roche), 2x SSC (Roche), 100 mg/ml salmon testis DNA (Sigma), 1x Denhardt's solution (Sigma) and 50% vol/vol deionized formamide (Ambion) for 1h at 46°C. The hybridization mixture was prepared as described above for the prehybridization mix with the addition of 7% dextran sodium salt (DSS, Sigma) and the respective anti-sense and sense probe (1 ng/100 ml hybridization buffer). The mixture was added to each slide and allowed to hybridize overnight at 46°C. The slides were washed and unbound probe was removed with 20 mg/ml RNase A (Sigma). The hybridized probe was detected with sheep anti-DIG antibody conjugated with alkaline phosphatase and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche). The sense probe was used as a negative control. In addition, RNase treatment (100 mg/ml) for 1h at 37°C performed previous to the postfixation with paraformaldehyde was used to confirm the specificity of the ISH method.

Primary myocytes were cultured in 6-well plates (1 ´ 10⁶/well) and infected with adenovirus (Ad-GFP or Ad-GFPKLF15) for 48 h. After verifying infection (> 90% GFP positivity) the cells were treated with serum-free medium for 48 h with or without PE (50 mM). The myocytes were subsequently trypsinized and fixed with 2% PFA-PBS. A minimum of 20,000 cells per group were analyzed by fluorescence-activated cell sorter (FACS) (FACScalibur; Becton Dickinson) measuring fluorescein isothiocyanate (FITC) fluorescence and forward scatter (FSC). The data from 3 independent experiments were pooled and analyzed with Cell Quest software (Becton Dickinson). The geometric mean of FSC data are used as a correlate of cell size. Fold change in population cell size was calculated as the ratio of [geometric mean FSC] (treated) / [geometric mean FSC] (control) (33).

The murine KLF15 genomic clone was obtained by hybridization of a mouse 129SVJ lambda library with an EcoRI-BamHI cDNA fragment from the mouse KLF15 coding region. Restriction-mapping of the clone indicated that the majority of the 5'UTR is contained within Exon 1 (Suppl. Figure S3A). Exon 2 contains part of the 5'UTR, translation start site, and entire coding region through the first 1.5 zinc fingers. The remaining 1.5 zinc-fingers and the entire 3'UTR are contained in exon 3 (Suppl. Figure S3A). The targeting vector was generated by inserting a 3 kb SmaI-EcoRI genomic fragment from the second intron into the BamHI (blunt) and EcoRI sites of pPNT followed by simultaneous insertion between pPNT (52) NotI and XhoI sites of a 3.6 kb HindIII XhoI fragment containing a nuclear lacZ gene (53) and a 3.5 kb genomic fragment extending from genomic NotI site to an engineered HindIII site at the KLF15 ATG. The resulting targeting construct was linearized with NotI before electroporation into 129SVJ ES cells. Neomycin resistant transfectants were further selected by growth in G418 (200 mg/ml) and gancyclovir (1 mM). DNA from ES cell clones was digested with BamHI, Southern blotted and hybridized with a 1.5 kb genomic fragment located 3' of the knockout construct. ES cells from two independently derived KLF15 ± clones were microinjected into C57BL/6 donor blastocysts that were implanted into pseudopregnant females. The male chimeras were mated with C57BL/6 females and the agouti offspring were genotyped using Southern analysis.

Paraffin-embedded heart sections from mice one week after AAC were fixed in 4% paraformaldehyde. Multiple sections were stained using H&E and TUNEL, and observed under light microscopy and representative digital images were taken. Immunohistochemistry for cleaved caspase-3 was performed on paraffin sections of mouse heart tissue following the antibody manufacturere's protocol (Cell Signaling Technologies, cat # 9664).

After one week of AAC, mice were killed and their myocytes were isolated as previously described (51), fixed in formaldehyde, and imaged under brightfield microscopy. Thirty consecutive microscopic fields were digitally photographed for each sample. For each animal, area, width and length measurements were performed on 100 consecutive myocytes and quantified using image-analysis software (Sigma, Inc.) Data are based on pooled data from 3 (+/+) vs. 3 (-/-) hearts.

The wild-type BNP-116 oligonucleotide 5'-GATAAATCAGAGATAACCCCACCCCTA-3' (for GATA EMSA) and the wild-type Atr oligonucleotide 5'-CATATATCAGTGATATAAATAGAACCTGCA-3' (for MEF2 EMSA) and their respective antisense oligonucleotides were used to make double-stranded DNA probes. The annealed and radiolabelled oligonucleotides were then used in binding reactions performed at room temperature (49). For supershift studies, the reactions were preincubated with 1 mg of anti-Flag antibody (Sigma Aldrich), 1 mg of the anti-GATA4 antibody (Santa Cruz Biotechnology) or 1 mg of the anti-MEF2 antibody (Santa Cruz Biotechnology) for 30 min on ice. Cold competition experiments were performed using a 100-fold molar excess of unlabelled self-competitor oligonucleotide.

Real-time PCR was carried out on RNA from KLF15+/+ and KLF15-/- mice 1 week after AAC on a Mx3005p machine (Stratagene) using Sybergreen. Apoptotic gene expression was analyzed using the software provided with the instrument. PCR conditions were as follows: Hot start 95°C for 10 min followed by 45 cycles of 95°C for 30 sec, 60° C for 30 sec, 72°C for 1 min, melting 1 cycle 95°C for 1 min and extension 72°C for 9 min. The primers used in the real time PCR were designed using information available at pga.mgh.harvard.edu/primerbank/.