RNA extraction and quantitative reverse transcriptase (RT)-PCR.
Total RNA was extracted from cells or tissues using an RNeasy Mini kit (Qiagen) or Trizol reagent (Invitrogen). Total RNA of human heart was purchased from Ambion. cDNA synthesis and kinetic real-time PCR were performed as described previously (33
). Primers used were as follows: mouse MURC (mMURC) forward primer (5′-ACAGTCACACAGCAATACGGGCTA-3′) and mMURC reverse primer (5′-TTCTCGGGCAGGCTTCTGTCTTTA-3′); mouse atrial natriuretic peptide (ANP) forward primer (5′-AACCTGCTAGACCACCTGGA-3′) and mouse ANP reverse primer (5′-TGCTTTTCAAGAGGGCAGAT-3′); mouse brain natriuretic peptide (BNP) forward primer (5′-CTGAAGGTGCTGTCCCAGAT-3′) and mouse BNP reverse primer (5′-CCTTGGTCCTTCAAGAGCTG-3′); mouse serum deprivation response (mSDPR) forward primer (5′-ATGAGGAAGCCCTGGAAGAT-3′) and mSDPR reverse primer (5′-CCCAGATGATGCTTTCTGGT-3′); mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward primer (5′-TTGTGATGGGTGTGAACCACGAGA-3′) and mouse GAPDH reverse primer (5′-CATGAGCCCTTCCACAATGCCAAA-3′); mouse α-myosin heavy chain (αMHC) forward primer (5′-GAGGACCAGGCCAATGAGTA-3′) and mouse αMHC reverse primer (5′-GCTGGGTGTAGGAGAGCTTG-3′); mouse β-myosin heavy chain (βMHC) forward primer (5′-TCGATTTGGGAAATTCATCC-3′) and mouse βMHC reverse primer (5′-CGCATAATCGTAGGGGTTGT-3′); mouse sarcoplasmic reticulum Ca2+
ATPase 2 (SERCA2) forward primer (5′-CTGTGGAGACCCTTGGTTGT-3′) and mouse SERCA2 reverse primer (5′-CAGAGCACAGATGGTGGCTA-3′); mouse transforming growth factor β1 (TGF-β1) forward primer (5′-TTGCTTCAGCTCCACAGAGA-3′) and mouse TGF-β1 reverse primer (5′-TGGTTGTAGAGGGCAAGGAC-3′); mouse TGF-β2 forward primer (5′-CAGCGCTACATCGATAGCAA-3′) and mouse TGF-β2 reverse primer (5′-CCTCGAGCTCTTCGCTTTTA-3′); mouse TGF-β3 forward primer (5′-GATGAGCACATAGCCAAGCA-3′) and mouse TGF-β3 reverse primer (5′-ATTGGGCTGAAAGGTGTGAC-3′); mouse procollagen type I α1 (Col1a1) forward primer (5′-GAGCGGAGAGTACTGGATCG-3′) and mouse Col1a1 reverse primer (5′-GCTTCTTTTCCTTGGGGTTC-3′); mouse Col1a2 forward primer (5′-CCGTGCTTCTCAGAACATCA-3′) and mouse Col1a2 reverse primer (5′-GAGCAGCCATCGACTAGGAC-3′); mouse Col3a1 forward primer (5′-GTCCACGAGGTGACAAAGGT-3′) and mouse Col3a1 reverse primer (5′-GATGCCCACTTGTTCCATCT-3′); rat MURC (rMURC) forward primer (5′-ACTGAAGATGAAGACCAGGACGCA-3′) and rMURC reverse primer (5′-TGTTAACAACGTAGCCCGTGTTGC-3′); rat ANP forward primer (5′-ATACAGTGCGGTGTCCAACA-3′) and rat ANP reverse primer (5′-CGAGAGCACCTCCATCTCTC-3′); rat BNP forward primer (5′-GGAAATGGCTCAGAGACAGC-3′) and rat BNP reverse primer (5′-CGATCCGGTCTATCTTCTGC-3′); and rat GAPDH forward primer (5′-ATGGGAAGCTGGTCATCAAC-3′) and rat GAPDH reverse primer (5′-GTGGTTCACACCCATCACAA-3′).
Serial analysis of gene expression (SAGE) was performed as described previously (33
). The SAGE libraries were constructed essentially following the I-SAGE long kit protocol (Invitrogen) using total RNA extracted from adult mouse hearts. Double-stranded cDNAs were digested with NlaIII, and the restriction enzyme was replaced by MmeI after linker ligation. Ditags produced from 400 PCRs were isolated, cleaved with NlaIII, and cloned into pZErO. All sequence files were processed using SAGE2000 version 4.5 software. The extracted tags were further processed to determine the identity of associated genes through several stringent filters using the CGAP website (http://cgap.nci.nih.gov/SAGE
The corresponding cDNA fragments for human MURC (hMURC), mMURC, and rMURC were cloned by PCR from human heart cDNA, mouse heart cDNA, and rat cardiomyocyte cDNA templates, respectively. PCR was performed using the following primers: hMURC forward primer (5′-ATGGAACATAATGGGTCTGC-3′) and hMURC reverse primer (5′-TTACGATGAGTGCTTTAAATCTAAC-3′); mMURC forward primer (5′-ATGGAACACAACGGATCAGCT-3′) and mMURC reverse primer (5′-CTATTTGTAGTCTGAGGACTGCTTTAGCTCCA-3′); rMURC forward primer (5′-ATGGAACACAATGGATCTGC-3′) and rMURC reverse primer (5′-CTATGAGGACTGCTTTAATTCCAAC-3′). The cDNAs encoding mMURC and hMURC with a C-terminal Flag epitope were cloned into pcDNA3 (Invitrogen) to generate pcDNA3-mMURC and pcDNA3-hMURC, respectively. The corresponding cDNA fragment for human SDPR (hSDPR) was cloned by PCR from human heart cDNA template. PCR was performed using hSDPR forward primer (5′-ATGGGAGAGGACGCTGACAGGC-3′) and hSDPR reverse primer (5′-TCACGGCAGTCTGATCCACAT-3′). The cDNA encoding hSDPR with a C-terminal hemagglutinin (HA) epitope was cloned into pcDNA3 to generate pcDNA3-hSDPR. The cDNA encoding hMURC was cloned into pGBKT7 (Clontech) to generate a bait vector, pGBKT7-hMURC. The RNA interference (RNAi) target sequences for rMURC (5′-TTCGAGTAACCAAAGTCGAAA-3′), rat SDPR (rSDPR, 5′-GAAGCAGTGTGTACAGGTGAA-3′), and green fluorescent protein (GFP; 5′-CGTAAACGCCCACAAGTTC-3′) were cloned into the BamHI-EcoRI sites of the RNAi-Ready-pSIREN-RetroQ vector (Clontech) as an inverted repeat with a hairpin loop spacer to generate RNAi-Ready-pSIREN-RetroQ-rMURC, RNAi-Ready-pSIREN-RetroQ-rSDPR, and RNAi-Ready-pSIREN-RetroQ-GFP (used as a control), respectively.
Northern blot analysis.
Total RNA was isolated from tissues with Trizol reagent (Invitrogen). Total RNA was size fractionated by electrophoresis in a 1.3% agarose gel containing 2.2 M formaldehyde and transferred to nylon membranes. A HindIII fragment of pcDNA3-mMURC (nucleotides 1 to 797 of the mMURC open reading frame) was used as a probe.
Production of polyclonal antibody.
Rabbit immunization was conducted by Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan) using synthetic peptides spanning fragments of mMURC with N-terminal acetylation (GERLRQSGERFKKSISC). For immunostaining and Western blot analysis, immunoglobulin G (IgG) was purified from antisera with protein A-Sepharose beads.
Specimens were fixed in 4% paraformaldehyde and stained with rabbit polyclonal anti-MURC antibody, mouse monoclonal anti-α-actinin antibody (Sigma), mouse monoclonal anti-α-smooth muscle actin antibody (Sigma), rat monoclonal anti-mouse CD31 antibody (BD Biosciences), rat monoclonal anti-HA antibody (Roche), mouse monoclonal anti-Flag antibody (Sigma), or rabbit polyclonal anti-Flag antibody (Sigma). Secondary antibodies were conjugated with Alexa Fluor 488, 555, or 594 (Invitrogen), and nuclei were visualized using 4′,6-diamino-2-phenylindole (DAPI; Invitrogen).
Rat neonatal cardiomyocytes, cultured from 1-day-old Sprague-Dawley rats, were prepared as described previously with slight modifications (42
). Briefly, ventricles were digested enzymatically, and cardiomyocytes were purified over a Percoll gradient. The culture medium was changed to serum-free medium after 24 h. Neonatal cardiomyocytes were cultured under serum-free conditions for 24 h before experiments. Adult cardiomyocytes were isolated from the hearts of male C57BL6 mice at 8 weeks of age. Ventricles were minced roughly and subsequently placed into 0.05% trypsin-EDTA (Invitrogen) at 4°C. After overnight incubation, ventricular tissue segments were put into Hanks' balanced salt solution buffer (Invitrogen) containing 0.1% (wt/vol) type 2 collagenase (Worthington Biochemical Corporation) at 37°C for 2 h. Dissociated adult cardiomyocytes were plated on 0.1% gelatin-coated slides and cultured in Dulbecco's modified Eagle's medium-F-12 medium with 5% bovine serum.
Male mice were anesthetized with 2,2,2-tribromoethanol (0.25 mg/g of body weight; Aldrich). A midline abdominal incision was used to expose the suprarenal abdominal aorta. The aorta was tied with a 6-0 silk suture against a blunt needle (26 gauge). The needle was immediately removed, leaving the aortic lumen constricted to the diameter of the needle. Sham-operated mice were subjected to the same procedure without the aortic banding. Seven days after surgery, mice were sacrificed and total RNA was extracted from the heart.
Yeast two-hybrid screen.
A Saccharomyces cerevisiae two-hybrid screen was performed using a Matchmaker Gal 4 two-hybrid system 3 (Clontech) and a Matchmaker pretransformed human heart cDNA library (Clontech) according to the manufacturer's instructions.
Replication-defective recombinant adenoviruses and gene transfer.
The cDNAs encoding mMURC with a C-terminal Flag epitope and hSDPR with a C-terminal HA epitope were inserted into a pAxCAwtit cosmid vector in an adenovirus expression vector kit (Dual Version; Takara Bio Inc., Otsu, Japan). RNAi-Ready-pSIREN-RetroQ-rMURC and RNAi-Ready-pSIREN-RetroQ-luciferase (Clontech), which was used as a control, were digested with BglII and EcoRI to obtain the U6 promoter with each of the target sequences, and blunted fragments were inserted into a promoterless pAxcwit cosmid vector with an adenovirus expression vector kit (Dual Version). Recombinant adenoviruses expressing Flag-tagged mMURC (Ad-MURC), HA-tagged hSDPR (Ad-SDPR), LacZ (Ad-LacZ), MURC shRNA (Ad-rMURC shRNA), and Luc shRNA (Ad-Luc shRNA) were generated as described previously (43
). Twenty-four hours after seeding, cardiomyocytes were infected with Ad-MURC, Ad-SDPR, Ad-LacZ, Ad-rMURC shRNA, or Ad-Luc shRNA diluted in the culture medium at a multiplicity of infection (MOI) of 10 or 20 and incubated at 37°C for 1 h. The viral suspension was removed, and cardiomyocytes were cultured with serum-depleted culture medium. Phenylephrine (PE) or Y-27632 was added after infection.
COS cells were plated in 60-mm dishes. The following day, the cells were transfected with 1 μg of pcDNA3-hMURC and/or pcDNA3-hSDPR. The total plasmid amount was adjusted to 2.0 μg with an empty vector plasmid. Cardiomyocytes were plated in 60-mm dishes. The following day, the cells were infected with Ad-MURC and/or Ad-SDPR. The total MOI was adjusted to 20 with Ad-LacZ. Cells were cultured for another 48 h and lysed with a lysis buffer (20 mM HEPES, pH 7.7, 100 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.1 mM Na3VO4, 2 μg/ml aprotinin, 0.7 μg/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Cell lysates were incubated with anti-Flag M2 affinity gel (Sigma) or an anti-HA antibody and protein A-Sepharose beads (GE Healthcare) at 4°C. After the beads were extensively washed with the lysis buffer, the bound proteins were eluted by boiling the beads in sodium dodecyl sulfate (SDS) sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis.
Western blot analysis.
Cell lysates were extracted with a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail (Pierce), 1 mM Na3VO4, and 1 mM NaF. Cell lysates were electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were subsequently incubated with primary antibodies against Flag (Sigma), HA, and MURC. Horseradish peroxidase-conjugated anti-rabbit IgG, anti-rat IgG, and anti-mouse IgG (GE Healthcare) were used as secondary antibodies.
RhoA activation assay.
RhoA activity was determined from protein isolated from adenovirus-infected neonatal cardiomyocytes or heart tissues of transgenic mice using an absorbance-based G-LISA RhoA activation assay biochemistry kit (Cytoskeleton) according to the manufacturer's instructions. Cell protein was isolated on day 2 postinfection using the provided cell lysis buffer. Heart tissue protein of mice at 6 weeks of age was also isolated by homogenizing in cell lysis buffer. Cell or tissue lysates were clarified by centrifugation at 10,000 rpm at 4°C for 2 min, and these extracts were processed rapidly on ice and snap-frozen until the time of assay. Protein concentration was determined according to the manufacturer's protocol, and extracts were equalized to contain total protein concentrations of 1 mg/ml for the assay. Signals were measured at an absorbance of 490 nm using a microplate spectrometer as suggested by the manufacturer.
Transfection and reporter assay.
COS cells were plated in six-well plates. The following day, the cells were transfected with 200 ng of ANP luciferase reporter construct (−638 ANP Luc; kindly provided by Kenneth R. Chien, Massachusetts General Hospital, Boston, MA) or no-SRE1/SRE2 mutant in the ANP luciferase reporter construct (kindly provided by Andrew Thorburn, Wake Forest University School of Medicine, NC). The cells were also transfected with 1.0 μg of expression vectors containing hMURC, mMURC, RhoA Val14, hSDPR, GFP shRNA, or rSDPR shRNA, 500 ng of a C3 expression vector, and 200 ng of pTKβ-Gal using FuGene6 reagent (Roche). The total plasmid amount was adjusted to 1.9 μg with an empty vector plasmid. An expression vector containing RhoA V14 (the mutant of Gly to Val at codon 14), which is a point-mutated active form of RhoA, was kindly provided by Yoshimi Takai (Osaka University, Suita, Japan). A C3 expression vector was kindly provided by Seigo Izumo (Novartis Institutes for Biomedical Research, Cambridge, MA). Cells were cultured for another 48 h, lysed with 200 μl of reporter lysis buffer (Promega), and assayed for luciferase activity (by using a Promega assay) and β-galactosidase activity. Luciferase activity was normalized against β-galactosidase activity.
Myofibrillar organization analysis.
Ad-LacZ- or Ad-MURC-infected cardiomyocytes were incubated in serum-free medium. Y-27632 was added after infection. After 96 h of 10 μM Y-27632 treatment, cells were fixed in 4% paraformaldehyde and stained with fluorescein isothiocyanate-conjugated phalloidin (Sigma) for the detection of actin filaments. Ad-Luc shRNA- or Ad-rMURC shRNA-infected cardiomyocytes were incubated in serum-free medium at 37°C for 48 h prior to stimulation with 100 μM PE. After 48 h of PE stimulation, cells were fixed and stained with fluorescein isothiocyanate-conjugated phalloidin.
Generation of transgenic mice.
The cDNA encoding Flag-tagged mMURC was cloned into the third 5′-untranslated exon of αMHC promoter plasmid clone 26 (a generous gift from Jeffrey Robbins, Cincinnati Children's Hospital Medical Center, Cincinnati, OH) (16
), and transgenic mice were generated as described previously (25
). All of the aspects of animal care and experimentation performed in this study were approved by the Institutional Animal Care and Use Committee of Kyoto University.
Echocardiography and electrophysiological analysis.
Echocardiographic examination of mice was performed as described previously (25
). Briefly, mice were anesthetized with 2,2,2-tribromoethanol (0.20 mg/g), and M-mode recordings of the left ventricle (LV) were obtained at the level of the papillary muscles from a parasternal window using a Hewlett-Packard (Andover) Sonos 5500 equipped with a 12-MHz probe. Mice anesthetized with 2,2,2-tribromoethanol (0.20 mg/g) by intraperitoneal injection were analyzed by multilead-surface electrocardiogram (ECG). ECG recordings were performed using an ECG-9902 (Nihon Kohden, Tokyo, Japan).
For left ventricular catheterization with a 1.0-F high-fidelity micromanometer-tipped catheter (model SPR-1000; Millar Instruments), mice were anesthetized with 2,2,2-tribromoethanol (0.25 mg/g), subsequently anesthetized with isoflurane (2.5% [vol/vol] in O2), and placed on a 36.5° table. A microtip catheter was inserted into the LV via the right carotid artery. To determine cardiac contractile and diastolic function, signals for LV pressure and the maximal rates of systolic pressure increase (maximum dP/dt) and isovolumetric relaxation (minimum dP/dt) were recorded at 1,000 Hz and analyzed by using a PowerLab system (AD Instruments).
Total RNA isolated from LV tissue was extracted using an RNeasy Mini kit and treated with the DNase I (Qiagen) according to the manufacturer's instructions. DNA microarrays used in these experiments were produced using the Mouse Genome Oligo 4.0 set of 70-mer oligonucleotides (Operon). Hybridization, processing, and the scanning process were done by Filgen Incorporated (Nagoya, Japan). Scan data images were analyzed using Microarray Data Analysis Tool version 2.0 software (Filgen).
All experiments were performed at least three times. Data are expressed as means ± standard errors and were analyzed by one-way analysis of variance with post hoc analysis. A P value of <0.05 was considered significant.