Depletion of Mitogen-Activated Protein Kinase Using an Antisense Oligodeoxynucleotide Approach Downregulates the Phenylephrine-Induced Hypertrophic Response in Rat Cardiac Myocytes
Abstract An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 μmol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.
- antisense oligodeoxynucleotides
- p42 and p44 mitogen-activated protein kinases
- atrial natriuretic factor expression
- cardiac hypertrophy
Understanding of the intracellular signaling events leading to cardiac myocyte hypertrophy has developed rapidly since the introduction and characterization of neonatal rat ventricular cardiomyocyte cultures by Simpson and Savion.1 Exposure of cultured cardiac myocytes to suitable agonists induces many of the features of hypertrophy seen in adult ventricular cardiac myocytes in vivo. These include the induction of immediate-early genes,2 the induction of fetal genes (ANF,3 β-MHC,4 and skeletal muscle α-actin5 ) and the upregulation of constitutively expressed contractile proteins (MLC-26 and cardiac muscle α-actin5 ). These changes culminate in an increase in cell size without cell division, an increase in cell protein and RNA content, and an increase in the production and assembly of contractile proteins into sarcomeric units (sarcomerogenesis).6
MAPK, or ERK, is a family of ubiquitously expressed enzymes that are highly conserved and play a central role in the signaling events leading to growth responses in a wide variety of noncardiac cell types.7 8 9 MAPK is a serine/threonine protein kinase, the activation of which requires phosphorylation on both a threonine and a tyrosine residue by a dual-specificity kinase known as MEK.10 One pathway of MEK activation involves its phosphorylation by Raf.11 12 13 The importance of the MAPK cascade is that it may transduce signals from diverse receptor types (receptor protein tyrosine kinases, G protein–coupled receptors) to produce growth responses. The downstream substrates for MAPK have not been fully elucidated but include nuclear transcription factors such as p62TCF/Elk-1,14 as well as cytosolic proteins.8 We have previously demonstrated that the p42 and p44 isoforms of MAPK are activated by acute exposure to the hypertrophic agonists PE, endothelin-1, acidic fibroblast growth factor, and PMA in cultured myocytes.15 16 17 Others have shown activation of MAPK by mechanical stretch of cultured cardiac myocytes, which also produces hypertrophy of these cells.18 19 20 On the basis of these observations and by analogy with what is known of signaling pathways in noncardiomyocytic cells, we postulated that MAPK may integrate signals from multiple receptor systems and thus act as a common distal signaling pathway leading to hypertrophy.15 16 In order to test this hypothesis, we used antisense ODNs to deplete cultured cardiac myocytes of MAPK and assessed the effects of this protocol on the induction of the morphological and transcriptional indices of hypertrophy induced by PE.
Materials and Methods
Sprague-Dawley rats were bred within the National Heart and Lung Institute. Tissue culture products were from Life Technologies Ltd and Sigma Chemical Co, except for the Primaria surface-modified culture dishes, which were from Marathon Laboratories. Lipofectin was from Life Technologies Ltd. Radiochemicals, autoradiography film (Hyperfilm MP), intensifying screens, molecular weight markers, horseradish peroxidase–linked secondary antibodies, and immunoblotting reagents were from Amersham International. SDS-PAGE reagents and reagents for the assay of protein by the Bradford method were from Bio-Rad. Nitrocellulose was from Schleicher & Schuell. A monoclonal antibody (clone Z033) for immunoblotting against p42 and p44 MAPKs was obtained from Zymed. The manufacturer states that the synthetic peptide immunogen was residues 325 to 345 of p44 MAPK. Polyclonal antibody against the VNPKYEQFLE C-terminal oligopeptide sequence of rat PKCδ was from Life Technologies Ltd. Monoclonal anti–β-MHC antibody (Novacastra), biotinylated anti-mouse secondary antibody (Dako), and streptavidin-linked Texas red (Amersham International) were used for immunofluorescent staining. Uvinert mountant was from Merck. RNases were from Boehringer, and RNAzol B was from Biotecx Laboratories Inc. All other laboratory reagents were from Sigma.
These were gifts from Dr K.R. Chien (Department of Medicine, University of California at San Diego).
The 5′ flanking region bp −638 to +62 of the rat ANF gene, which confers inducibility to PE, fused to the firefly LUX gene in the pSVOALΔ5′ expression vector.3
This fusion gene, with the constitutively active RSV incorporated into pSVOALΔ5′, was used as a positive control.
The GAL reporter gene under the control of the constitutively active human CMV promoter (pON249) was cotransfected in all experiments to allow correction for variations in transfection efficiency or nonspecific transcriptional activation.
The antisense ODN was a 17-mer (5′-GCCGCCGCCGCCGCCAT-3′) directed against the initiation of translation site of rat p42 MAPK mRNA. An identical sequence is present in rat p44 MAPK mRNA. This ODN has been used successfully to downregulate both isoforms of MAPK in 3T3 cells.21 Sense (5′-ATGGCGGCGGCGGCGGC-3′) and random sequence (5′-CGCGCGCTCGCGCACCC-3′) controls were used. All bases were phosphorothioate-protected. ODNs were synthesized at the University of Southampton using an automated DNA synthesizer (Applied Biosystems 391), replacing the standard iodination bottle with tetraethylthiuram disulfide in acetonitrile for the stepwise thioation of the phosphite linkages. The thioation wait step was increased to 15 minutes. After cleavage and deblocking in concentrated ammonium hydroxide at 55°C for 18 hours, the phosphorothioate ODNs were purified on OP cartridges (Applied Biosystems), dried down, and resuspended in sterile water.
Neonatal rat ventricular myocytes were isolated and cultured on individual dishes (60-mm diameter), 12-well plates (22-mm diameter), or 8-well chamber slides (10×10 mm) using previously described methods.17 Plating densities (350 to 600 cells per square millimeter) were chosen to produce cells that were almost confluent after overnight incubation (37°C, 95% O2/5% CO2, and high humidity) in medium supplemented with 10% horse serum and 5% fetal calf serum; at which time all experiments were initiated. Cells were thereafter cultured in maintenance medium (DMEM/medium 199 [4:1] containing 100 U/mL of both penicillin and streptomycin) and exposed to agents as indicated.
Cardiomyocytes were plated on eight-well chamber slides (Labtek), which had been precoated with 1% gelatin and 20 μg/mL laminin in sterile PBS. After treatment, the chamber slides were fixed in freshly prepared 3% paraformaldehyde (pH 7.4) for 10 minutes and permeabilized in 0.3% Triton X-100 for a further 10 minutes. Nonspecific binding sites were blocked in 1% BSA/0.3% Triton X-100 for 10 minutes. Slides were incubated at 37°C with monoclonal antibody against β-MHC (1/50 concentration, 1 hour), then with biotinylated anti-mouse IgG (1/200 concentration, 30 minutes), and finally with streptavidin-linked Texas red (1/200 concentration, 15 minutes). Coverslips were mounted using Uvinert mountant, and the slides were viewed by epi-illumination on a Zeiss Axioskop fluorescence microscope. Multiple views of each well were photographed on Kodak T-MAX 400 black and white film at ×400 magnification. Prints were produced from the developed films under standardized printing conditions to ensure uniform magnification. A graticule scale was also printed for calibration. Planimetry was performed using VIDS III planimetry software, which automatically calculated cell area from a manual tracing of the cell outline. The areas of at least 30 cells from each treatment group were measured.
Appropriate volumes of 4× final concentration ODN in antibiotic- and serum-free DMEM were vortex-mixed with an equal volume of DMEM containing 80 μg/mL lipofectin and stored at room temperature for 15 minutes. Myocytes were washed three times in DMEM, and the ODN/lipofectin mixture was added (750 μL for each individual dish, 200 μL for each 22-mm-diameter well, or 100 μL for each chamber slide well). An equal volume of DMEM was immediately added. The final concentration of lipofectin was 20 μg/mL for cells on individual dishes or 12-well plates and 10 μg/mL for cells on chamber slides. Myocytes were incubated for 8 hours at 37°C in 95% O2/5% CO2, with gentle agitation of the plates every 2 hours. Medium was then replaced with the same volume of liposome-free maintenance medium containing the same concentration of ODN and supplemented with either 10% fetal calf serum (which was heat-inactivated to minimize nuclease degradation of ODNs) or the appropriate hypertrophic agonist where indicated.
Myocytes were washed twice in ice-cold PBS and scraped into ice-cold extraction buffer (20 mmol/L β-glycerophosphate, 20 mmol/L NaF, 2 mmol/L EDTA, 0.2 mmol/L sodium vanadate, 10 mmol/L benzamidine, 25 μg/mL leupeptin, 50 μg/mL phenylmethylsulfonyl fluoride, and 0.3% [vol/vol] mercaptoethanol, pH 7.5). A one-third volume of SDS sample buffer (0.33 mol/L Tris/HCl, 10% [wt/vol] SDS, 13% [vol/vol] glycerol, and 0.1 mol/L dithiothreitol containing 0.13 mg/mL bromophenol blue) was added to each sample, and proteins were denatured by boiling for 5 minutes. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes using a semidry transfer cell (Bio-Rad). Equal protein loading of lanes was ensured by prior Bradford protein assay22 of fresh samples, by Coomassie staining of preliminary gels, and by Ponceau staining of nitrocellulose immediately following protein transfer. Nonspecific binding sites were blocked with 5% fat-free milk powder in PBS containing 0.05% (vol/vol) Tween-20 (PBS/Tween) for 1 hour. Membranes were then exposed overnight at 4°C to monoclonal anti-MAPK primary antibody diluted 1/5000 or to anti-PKCδ antibody diluted 1/250 in the blocking solution. After three washes in PBS/Tween, the nitrocellulose membrane was exposed for 1 hour at room temperature to the secondary antibody (horseradish peroxidase–linked immunoglobulin, donkey anti-mouse for anti-MAPK, and donkey anti-rabbit for anti-PKCδ) diluted 1/5000 in PBS/Tween containing 1% fat-free milk powder. Finally, membranes were washed three times in PBS/Tween, developed by the enhanced chemiluminescence method, and exposed to film. In an earlier publication involving immunoblotting with the Zymed monoclonal anti-MAPK antibody, we have shown that there is a linear relationship between the intensity of immunostaining and the quantity of MAPK applied to the gel.21
Assay of MAPK Activity
PMA-stimulated myocyte extracts were subjected to SDS-PAGE using gels that had been formed in the presence of the MAPK substrate myelin basic protein (0.5 mg/mL).15 Gels were washed with 20% (vol/vol) propan-2-ol in 50 mmol/L Tris-HCl (pH 8.0) to remove SDS and then in 5 mmol/L 2-mercaptoethanol in 50 mmol/L Tris-HCl (pH 8.0). Proteins were further denatured by washing the gels in 6 mol/L guanidine HCl and then renatured by washing in 50 mmol/L Tris-HCl (pH 8.0) containing 0.04% (vol/vol) Tween 40 and 5 mmol/L 2-mercaptoethanol at 4°C overnight. After equilibration at 20°C for 1 hour in 40 mmol/L HEPES, 2 mmol/L dithiothreitol, and 10 mmol/L MgCl2, pH 8.0, in situ phosphorylation of myelin basic protein was performed in 40 mmol/L HEPES, 0.5 mmol/L EGTA, 10 mmol/L MgCl2, 2 μmol/L cAMP-dependent protein kinase inhibitory peptide (TTYADFIASGRTGRRNAIHD, Bachem), and 40 μmol/L [γ-32P]ATP (5 μCi/mL, 25 μCi per gel), pH 8.0, at 20°C for 3 hours. After extensive washing in 5% (wt/vol) trichloroacetic acid and 1% (wt/vol) disodium pyrophosphate, gels were dried and autoradiographed.
Transfections and Reporter Gene Assays
A calcium phosphate coprecipitation method was used to transfect plasmid constructs into cardiac myocytes, which had been cultured on 60-mm-diameter gelatin-coated dishes.23 Plasmids were diluted in 0.25 mol/L CaCl2, and an equal volume of 50 mmol/L N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (pH 6.9), 280 mmol/L NaCl, and 1.5 mmol/L Na2HPO4 was added. After 20 minutes, cells were transfected with this suspension (1 mL per dish). Myocytes were standardly transfected with 15 μg ANF/LUX and 4 μg CMV/GAL per dish. After an overnight incubation, cells were washed twice in maintenance medium containing 10% horse serum and twice in serum- and antibiotic-free DMEM before being subjected to liposomal transfection of ODNs. The following day, PE was added to medium to a final concentration of 100 μmol/L. Cells were incubated for a further 48 hours, washed twice with PBS, and extracted on ice with 0.1 mol/L potassium phosphate (pH 7.9), 0.5% Triton X-100, and 1 mmol/L dithiothreitol for 15 minutes. For the assay of GAL, 100 μL of cell extract (diluted 5 to 10 times for PE-treated cells) was incubated with 200 μL GAL assay buffer (6.6 mmol/L O-nitrophenyl-3-d-galactopyranoside, 0.1 mol/L sodium phosphate [pH 7.3], 1.5 mmol/L MgCl2, and 75 mmol/L β-mercaptoethanol) at 37°C until a yellow color change was visible. The reaction was terminated by the addition of 500 μL of 0.5 mol/L Na2CO3 and the A410 (absorbance at 410 nm) was measured. LUX activities (20 μL cell extract) were assayed in 0.5 mL of 100 mmol/L tricine (pH 7.8), 10 mmol/L MgSO4, and 2 mmol/L EDTA containing 5.5 mmol/L ATP and 75 μmol/L luciferin. Light emitted was measured using an LKB Wallac 1219 Rackbeta liquid scintillation counter with the photomultipliers set out of coincidence. For all assays, untransfected cell extracts served as blanks.
Cardiac myocytes were extracted in 100 μL ice-cold perchloric acid (0.56 mol/L) and centrifuged at 10 000g for 10 minutes at 4°C, and the ATP content of the supernatant fractions was measured by LUX assay.24
RNase Protection Assay
An antisense riboprobe to ANF mRNA was produced from the entire coding sequence of rat ANF cDNA cloned into the Pst I site of pGEM-1 (from Dr K.R. Chien, Department of Medicine, University of California at San Diego).3 This template was linearized by digestion with Xho I. A radiolabeled probe was then generated by in vitro transcription using T7 DNA-dependent RNA polymerase and [α-32P]GTP (specific activity, 410 Ci/mmol). This produced a 141-nt antisense probe protecting a 95-nt fragment of rat ANF mRNA.3 An antisense riboprobe to GAPDH was generated using a 316-bp fragment of the rat GAPDH gene derived from exons 5 to 825 inserted into the Sac I–BamHI sites of the pTRIPLEscript transcription vector. The plasmid was linearized by digestion with Sty I. In vitro transcription produced a probe that protected 134 nt of rat GAPDH mRNA.
Total RNA was extracted from cultured myocytes by the guanidinium thiocyanate method26 using RNAzol B and stored at −70°C in ethanol. For analysis of mRNA, precipitated total RNA from each well was dissolved in 25 μL of hybridization buffer (80% formamide, 40 mmol/L PIPES, 400 mmol/L NaCl, and 1 mmol/L EDTA, pH 8). Hybridization was performed overnight at 60°C with ANF and GAPDH probes (2.5×105 cpm of each). RNase protection assays were carried out by digestion with a mixture of RNase A (40 μg/mL) and RNase T1 (2 μg/mL). Protected RNA fragments were then resolved on a denaturing 8% polyacrylamide gel, which was dried and subjected to autoradiography at −70°C followed by densitometric quantification of bands. Preliminary experiments had shown that the integrated absorbance was linear across the range of radioactivity assayed in these experiments. Results were expressed as ANF mRNA–to–GAPDH mRNA ratios.
Data are presented as mean±SEM values with a minimum of three separate myocyte preparations for each experiment. Statistical significance between two groups was tested using the two-tailed Student’s t test, with P<.05 being taken as significant. Statistical significance between three or more groups was tested by one-way ANOVA. If significant variation was detected between treatment groups, further analysis was performed using the Tukey-Kramer multiple comparison test.
Effect of Antisense ODN on MAPK Protein Content and Activity in Cultured Cardiac Myocytes
Immunoblotting revealed depletion of MAPK protein 48 hours after liposomal transfection of anti-MAPK ODN (Fig 1A⇓). At a concentration of 0.2 μmol/L, antisense ODNs reduced MAPK protein by 82% (Fig 1B⇓). MAPK depletion was 81% at 24 hours and 90% at 72 hours after antisense ODN exposure (results not shown). Exposure of myocytes to lipofectin in the absence of ODNs also had no effect on MAPK content compared with control myocytes (Fig 1A⇓). Sense and random sequence ODNs had no effect on relative MAPK content (Fig 1⇓). Simultaneous immunoblotting for PKCδ and laser densitometry showed no concomitant reduction of this protein in antisense ODN–treated cells (relative PKC abundance, 672±99 arbitrary units for antisense ODN–treated cells versus 613±122 arbitrary units for cells treated with lipofectin alone). In order to assess the viability of antisense ODN–treated cells, ATP contents were measured 48 hours after antisense ODN treatment. Antisense ODN (0.2 μmol/L) did not significantly reduce ATP contents (56±7 pmol/μg protein for antisense ODN–treated cells versus 55±10 pmol/μg protein for cells treated with lipofectin alone) despite reducing MAPK protein content by 90% in parallel samples.
PMA is the most effective stimulator of p42 and p44 MAPKs in cultured cardiomyocytes that we have so far identified.15 16 In order to measure whether MAPK activities were downregulated by antisense ODNs, cells that had been incubated in serum-free medium after liposomal transfection were exposed to 1 μmol/L PMA for 5 minutes. In-gel MAPK assays of PMA-stimulated myocyte extracts revealed that antisense ODN treatment (0.2 μmol/L) inhibited the activities of the p42 and p44 MAPK isoforms by 44% and 60%, respectively (Fig 2⇓). Increasing the antisense ODN concentration to 0.4 μmol/L had no additional effect.
Effect of Antisense ODN on Morphological Changes in Cardiac Myocytes Exposed to PE
Light microscopy of antisense ODN–treated cells revealed inhibition of myocyte growth in serum-supplemented medium. Immunofluorescent staining of β-MHC permitted planimetry of myocytes and assessment of sarcomerogenesis. Treatment with 0.2 μmol/L antisense ODN attenuated the increase in myocyte area and sarcomerogenesis in response to PE (Fig 3⇓, photomicrographs). Similar results were obtained when the phorbol ester PMA was used to induce hypertrophy, and the results of planimetry for both agonists are presented in Fig 3⇓ (graph). Sense ODN had no effect on the development of the hypertrophic morphology (Fig 3⇓).
Effect of Antisense ODN on ANF Promoter Activity and mRNA Abundance
Treatment with antisense ODN inhibited ANF/LUX activity in PE-stimulated myocytes (Fig 4⇓) but had no effect on a constitutively expressed RSV-LUX construct in parallel experiments (ratio of RSV/LUX to GAL, 3242±1189 for 0.2 μmol/L antisense ODN versus 3685±692 for lipofectin alone). Sense and random ODNs were without effect (Fig 4⇓).
Antisense ODN (0.2 μmol/L) treatment of myocytes inhibited the PE-stimulated induction of ANF mRNA (Fig 5⇓). The effect of 0.4 μmol/L antisense ODN did not reach statistical significance (n=6). This may be related to the very low signal strength for GAPDH, which may distort the ratio of ANF to GAPDH mRNA. Although lipofectin alone tended to increase the ratio of ANF/LUX to GAL (Fig 4⇑) and ANF mRNA (Fig 5⇓) compared with the control condition, these effects were not statistically significant.
The potential of antisense ODN protocols in cardiac myocytes has not yet been fully exploited. To our knowledge, only two studies have been reported. One of these studied the effects of downregulating endothelin-1 mRNA on angiotensin II–induced hypertrophy,27 and the other showed that downregulation of the Egr-1 transcription factor inhibited certain hypertrophic changes induced by endothelin-1.28 In both of these, high concentrations of ODNs (10- to 100-fold greater than used here) were added directly to the medium. These high concentrations were required to downregulate the target mRNA effectively. We believe that this is the first report of liposomal introduction of ODNs into cardiac myocytes.
Our results demonstrate that suitable antisense ODNs can be used to deplete cardiac myocytes of MAPK and that the depletion is dependent on the ODN being complementary to the target mRNA (Figs 1⇑ and 2⇑). MAPK depletion resulted in reduced expression of ANF after exposure to PE (Figs 4⇑ and 5⇑) and attenuated other hypertrophic changes (increased myocyte area, sarcomerogenesis) after exposure to PE or PMA (Fig 3⇑). There was no evidence of generalized cytotoxicity in cells treated with antisense ODN–treated cells. ATP and PKCδ contents were preserved, and constitutive expression of a transfected RSV/LUX construct was not inhibited.
The p44 MAPK isoform was not consistently detected on immunoblotting (Fig 1⇑). This may be related to inefficient cross-reactivity. p44 MAPK activity is certainly present in cardiac myocytes,15 16 although the activity as measured by in-gel MAPK assays may be ≈25% less than the p42 isoform (Fig 2⇑). These in-gel MAPK assays showed concomitant reduction of PMA-stimulated levels of both p42 and p44 MAPK activities after treatment with antisense ODNs (Fig 2⇑). Moreover, Sale et al21 have shown that the antisense ODN used here depletes both MAPK isoforms in 3T3 cells.
The inhibitory effect of antisense ODN on ANF expression described in the present study is supportive of the experiments of Thorburn et al,29 who reported that PE-stimulated induction of ANF/LUX was inhibited by cotransfection of dominant-negative MAPK. However, unlike the present study, this dominant-negative MAPK did not prevent the PE-induced changes in myocyte morphology. The reasons for this disparate result are unclear. The dominant-negative MAPK is thought to compete for MAPK activators such as MEK, but this may result in less profound inhibition of MAPK than depletion by antisense ODN. Interestingly, the effects on cardiac myocyte morphology and gene expression reported in the present study did not require total depletion of MAPK. We recently reported that the stimulatory effects of a constitutively active MEK on ANF promoter activity were enhanced by cotransfection of wild-type MAPK.30 Taken together, these observations suggest that MAPK may be a limiting component of the distal limb of the signaling pathway.
We conclude that p42/p44 MAPK is required for the transcriptional and morphological changes of hypertrophy induced by PE. The mechanisms that regulate MAPK activity and the precise events downstream from MAPK are still uncertain. The judicious use of liposomally transfected antisense ODNs may provide further information about this and other pathways.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|ERK||=||extracellular signal–regulated kinase|
|lipofectin||=||N-(1,2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA)|
|MAPK||=||mitogen-activated protein kinase|
|MEK||=||MAPK (or ERK) kinase|
|MHC||=||myosin heavy chain|
|MLC||=||myosin light chain|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
|RSV||=||Rous sarcoma virus|
This study was supported by grants from the British Heart Foundation and the Medical Research Council.
- Received January 3, 1996.
- Accepted March 5, 1996.
- © 1996 American Heart Association, Inc.
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