J Mol Cell Cardiol 32, 1553–1566 (2000) doi:10.1006/jmcc.2000.1191, available online at http://www.idealibrary.com on
Isoenzyme-Specific Protein Kinase C and c-Jun N-terminal Kinase Activation by Electrically Stimulated Contraction of Neonatal Rat Ventricular Myocytes James B. Strait1 and Allen M. Samarel1,2 The Cardiovascular Institute and the Departments of 1Physiology and 2Medicine, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153, USA (Received 15 March 2000, accepted in revised form 15 May 2000) J. B. S A. M. S. Isoenzyme-Specific Protein Kinase C and c-Jun N-terminal Kinase Activation by Electrically Stimulated Contraction of Neonatal Rat Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2000) 32, 1553–1566. Previous studies from our laboratory and others indicate that contractioninduced mechanical loading of cultured neonatal rat ventricular myocytes produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo, and that these changes occur via the activation of serine-threonine protein kinases. These may include the extracellular regulated protein kinases (ERK1 and ERK2), the c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and one or more isoenzymes of protein kinase C. In this study, we assessed whether one or more of these kinases are activated by stimulated contraction, and whether activation was isoenzyme-specific. Low-density, quiescent cultures of neonatal rat ventricular myocytes were maintained in serum-free medium, or electrically stimulated to contract (3 Hz) for up to 48 h. ERK and JNK activation was assessed by Western blotting with polyclonal antibodies specific for the phosphorylated forms of both kinases. PKC activation was analysed by subcellular fractionation, detergent extraction, and Western blotting using isoenzyme-specific monoclonal antibodies. Stimulated contractile activity produced myocyte hypertrophy, as indicated by increased cell size, a 15±5% increase in total protein/DNA ratio, and induction of ANF and MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by >two-fold. Subcellular fractionation revealed a time-dependent increase in PKC, and to a much lesser extent PKC, in a Triton X-100-soluble membrane fraction within 5 min of the onset of stimulated contraction. PKC was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes. 2000 Academic Press
K W: Hypertrophy; Heart; Signal transduction; Mitogen activated protein kinases; ERKs; Mechanical load.
Introduction The chronically increased systolic and diastolic wall stress which accompanies hypertension, valvular and congenital heart diseases, and myocardial infarction results in adaptive cardiac hypertrophy, but the development of cardiac hypertrophy poses
an independent risk factor for subsequent cardiovascular morbidity and mortality.1 It is also clear that cardiomyocytes respond to increased hemodynamic load per se, or to circulating or locally produced growth factors released in response to this increased hemodynamic load, by accelerating the transcription, translation and assembly of
Please address all correspondence to: Allen M. Samarel, The Cardiovascular Institute, Building 110, Room 5222, Loyola University Medical Center, 2160 South First Avenue, Maywood, Illinois 60153, USA. E-mail:
[email protected]
0022–2828/00/081553+14 $35.00/0
2000 Academic Press
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cardiomyocyte proteins to meet the increased functional demands imposed on the overloaded myocardium. Although there are substantial data demonstrating that hemodynamic overload in vivo causes myocardial growth, it remains unclear exactly how mechanical stimuli are converted to biochemical signals resulting in downstream changes in protein transcription, translation, and assembly. Furthermore, the complexity of the in vivo setting of cardiac hypertrophy presents several limitations in determining contributory mechanisms. Therefore, our group and others have turned to the use of cultured neonatal and adult ventricular myocytes to study these fundamental processes in a controlled in vitro setting. Fortunately, cardiomyocytes in culture respond to both neurohormonal and mechanical stimuli and undergo hypertrophy in a manner similar to intact myocardial tissue.2 One approach to studying mechanochemical signal transduction is to experimentally manipulate the process of excitation-contraction coupling in cultured cardiomyocytes. Previous studies from our laboratory and others indicate that spontaneously beating, or electrically stimulated neonatal3–6 and adult7,8 cardiomyocytes undergo hypertrophy as compared to quiescnt, non-contracting cells. Contraction-induced mechanical loading of cultured neonatal rat ventricular myocytes (NRVM) produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo. These include increased cell size,4–6 increased total protein, MHC and actin/DNA ratios,3,4,6,9 and up-regulation of the MHC4,10 and ANF5,11 genes. Based on pharmacological studies, these changes occur at least in part via Ca2+ and PKC-dependent signal transduction pathways.4,5,6,12 However, cardiomyocytes express several different PKC isoenzymes that are localized to different regions of the cell.13 It is currently not known which PKC isoenzymes are activated in response to contraction-induced mechanical loading. In addition, other serine-threonine protein kinses have been implicated in mediating various aspects of the hypertrophic response. These include the extracellular regulated protein kinases (ERKs) and the c-Jun N-terminal kinases (JNKs).14 Therefore, in this study, serum-free NRVM cultures were maintained in the quiescent state, or electrically stimulated to contract in order to generate an intrinsic mechanical load. The development of hypertrophy and the activation of ERKs, JNKs, and PKC isoenzymes were then examined.
Methods Reagents PC-1 tissue culture medium was obtained from BioWhittaker, Walkersville, MD, USA. Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Gibco BRL, Grand Island, NY, USA. Medium 199, Ca2+-free, Mg2+-free Hanks Balanced Salts (modified) (HBSS), acid soluble calf skin collagen, and antibiotic/antimycotic solution were obtained from Sigma Chemical Co., St Louis, MO, USA. Four-well rectangular multidishes (24×67 mm/well) and Permanox chamber slides were obtained from Nunc (Naperville, IL, USA). [32P]ATP and [32P]dCTP were purchased from Amersham, Arlington Heights, IL, USA. Monoclonal antibodies to PKC, PKC, and PKC were obtained from Signal Transduction Laboratories, Lexington, KY, USA. Rabbit polyclonal antibodies to ERK1 and ERK2, and JNK1, JNK2 and JNK3 were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA, and New England Biolabs, Beverly, MA, USA, respectively. Rabbit polyclonal antibodies to the phosphorylated forms of ERKs, and JNKs were obtained from Promega, Madison, WI, USA. Horseradish peroxidaseconjugated goat anti-rabbit and goat-anti-mouse IgGs were from BioRad, Hercules, CA, USA. All other reagents were of the highest grade commercially available and were obtained from Sigma and Baxter S/P, McGaw Park, IL, USA.
Ventricular dissociation and cardiac myocyte isolation Animals used in these experiments were handled in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Council of the American Physiological Society. Ventricular myocytes were isolated from the hearts of 2-dayold Sprague–Dawley rats by collagenase digestion, as previously described.4 Released cells were collected by centrifugation, resuspended in PC-1 medium, and plated at a density of 1000 cells/ mm2 onto collagen-coated tissue culture dishes or chamber slides, and left undisturbed in a 5% CO2 incubator for 14–18 h. Unattached cells were removed by aspiration, washed twice in HBSS, and the attached cells were maintained in a solution of DMEM/Medium 199 (4:1) containing antibiotic/ antimycotic solution and 10 nifedipine to inhibit spontaneous contractile activity. After 24 h, media were replaced with nifedipine-free DMEM/Medium 199 prior to initiating the electrical pacing protocols. Thereafter, media were changed daily.
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Electrical pacing Platinum electrodes attached to a sterilized culturedish top were immersed into the culture medium of the four-well multidish. Cells were then maintained under control conditions, or stimulated to contract (paced) by a 5-ms square-wave pulse (3.4 V/cm) delivered with alternating polarity using a custombuilt cell stimulator.6 Pacing leads connected the platinum electrodes of the two center wells of the CO2 incubator to the externally mounted cell stimulator. The two outer wells were not electrically stimulated and thus served as controls for each experiment. Cellular composition For the quantitative analysis of total cellular protein and DNA content, 0.2 mol perchloric acid (1 ml) was added and the cells were then quantitatively scraped from the dishes and collected by centrifugation (10 000 g, 10 min). The precipitate was redissolved by incubation (60°C, 20 min) in 250 l of 0.3 mol KOH. Aliquots were then used for analysis of total protein by the Lowry method using crystalline human serum albumin as standard, and for DNA using 33258 Hoecht dye and salmon sperm DNA as standard, as previously described.4 Data are the means of duplicate wells from each treatment group for each cell isolation, and are expressed a g total protein/g DNA. mRNA analysis Total cellular RNA was isolated by the method of Chomszynski and Sacchi.15 RNA was quantified by absorbance at 260 nm and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. Total RNA (10 g per lane) was then separated by denaturing agarose gel electrophoresis, subjected to alkali pre-treatment, transferred to nylon membranes by capillary action, and cross-linked by u.v. irradiation. MHC, MHC, and ANF mRNA, and 18S rRNA were detected by hybridization to 32 P-labeled, oligodeoxynucleotide or cDNA probes specific for each transcript, as previously described.4,11,16 MHC and ANF promoter activities MHC and ANF promoter-luciferase reporter constructs were used to assess promoter activities in
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transient transfection assays of quiescent and electrically paced NRVM. The MHC expression plasmid was generated by PCR amplification using as template a 5-kb genomic clone of the rat MHC gene. The promoter consisted of−354/+34 bp [relative to the transcriptional start site (tss)] of 5′ upstream regulatory sequences ligated into the luciferase expression plasmid pLUC (p354MHC-luc).10 p3003ANF-luc expression plasmid consisted of 3003 bp of upstream regulatory sequences of the rat ANF gene linked to the gene encoding firefly luciferase, and was kindly provided by Dr Andrew Thorburn, University of Utah, and Dr Kenneth Chien, University of California, San Diego. In all transient transfection experiments, the constitutively active Rous sarcoma virus long terminal repeat ligated to the bacterial -galactosidase reporter gene plasmid (pRSV-lacZ) was used to normalize for DNA transfection efficiency. Luciferase expression plasmid (5 g) and pRSV-lacZ (1 g) were combined with 20 l of lipofectamine reagent in 1 ml of DMEM/F12 medium. The cells were incubated with this transfection medium for 6 h, after which time the cells were washed and maintained in standard growth medium or subjected to electrical pacing. After 48 h, luciferase and galactosidase activities were measured in cellular extracts as previously described.10,11 Relative light units were measured using an Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, Ann Arbor, MI, USA) and a luminometer (Berthold, model LB9501).
Subcellular fractionation and Western blotting Subcellular fractionation was performed as described by Jiang et al.17 Briefly, cultured NRVM were washed with phosphate-buffered saline (PBS), and 200 l of homogenization buffer (2 m EDTA, 2 m EGTA, 10 g/ml aprotinin, 10 g/ml leupeptin, 500 Na orthovanadate, 1 m PEFABloc, 20 m Tris-HCl, pH 7.5) was added. Cells were then frozen in a dry-ice–methanol bath, thawed on ice, scraped into a plastic tube, and sonicated. The cell homogenate was then centrifuged (100 000g; 60 min, 4°C), and the supernatant fraction (Cyt) was stored at −80°C. The pellet was resuspended by sonication in 2 m EDTA, 2 m EGTA, 1% Triton X-100, 10 g/ml aprotinin, 10 g/ml leupeptin, 500 Na orthovanadate, 1 m PEFABloc, 20 m Tris-HCl, pH 7.5, and re-centrifuged (10 000 g; 10 min). The resulting supernatant fraction (P1 fraction) was then stored at −80°C. The pellet from the second centrifugation was resuspended in 150 m NaCl,
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10% glycerol, 1.5 m MgCl2, 1 m EGTA, 1% Na deoxycholate, 1% Triton X-100, 0.1% SDS, 10 g/ ml aprotinin, 10 g/ml leupeptin, 500 Na orthovanadate, 1 m PEFABloc, pH 7.5, sonicated, centrifuged at 10 000 g for 5 min, and the supernatant fraction (P2 fraction) stored at −80°C. Protein concentration in each subcellular fraction was measured by the BCA-Bradford method. In some experiments, equal amounts of total protein (40 g) from each fraction were separated by SDSpolyacrylamide gel electrophoresis. In other experiments, an equal percentage of the total protein fraction was applied to the gels to control for the non-uniform distribution of total protein in each subcellular fraction. Following transfer to nitrocellulose membrane by electroblotting, the amount of PKC present in each fraction was determined by probing with PKC isoenzyme-specific monoclonal antibodies. Antibody binding was visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL, USA), and quantified by laser densitometry.
MAPK Western blotting NRVM were washed once with PBS, and 300 l of lysis buffer (50 m Na pyrophosphate, 50 NaF, 50 NaCl, 5 m EDTA, 5 m EGTA, 100 Na orthovanadate, 10 g leupeptin/ml, 10 g aprotinin/ml, 1 m PEFABloc, 0.01% Triton X-100, 10 m HEPES, pH 7.4) was added. Cells were then frozen on a dry-ice–methanol bath, thawed on ice, and then scraped into a plastic tube. Samples were then sonicated once, centrifuged (16 000 g; 30 min), and the supernatant fraction was stored at −80°C. MAPK activation was assessed by separating equal amounts of cellular protein (30–80 g as determined by BCA-Bradford method) by SDSpolyacrylamide gel electrophoresis and Western blotting. ERK1/2 activation was assessed by gelshift analysis, as previously described.18,19 In some experiments, the phosphorylation state of the separated ERKs was independently assessed using polyclonal antibodies specific for the phosphorylated forms of the ERK1/2. Similarly, JNK1/JNK2/JNK3 phosphorylation state was assessed using polyclonal antibodies specific for the phosphorylated forms of the enzymes. JNK protein levels were assessed using polyclonal antibodies recognizing both phosphorylated and unphosphorylated forms of the enzymes. Primary antibody binding was visualized using ECL and quantified by laser densitometry.
Data analysis Results were expressed as means±... Normality was assessed using the Kolmogorov–Smirnov test, and homogeneity of variance was assessed using Levene’s test. Data were compared by analysis of variance (ANOVA) on Ranks followed by Dunn’s Test for multiple groups, or by paired t-test, or Wilcoxon Signed Rank Test, for two groups, where appropriate. Data were analysed using the SigmaStat Statistical Software Package, Ver. 1.0 (Jandel Scientific, San Rafael, CA, USA).
Results Stimulated contractile activity induces NRVM hypertrophy In order to establish that stimulated contractile activity resulted in NRVM hypertrophy, we examined a number of features of the cardiomyocyte hypertrophic phenotype in non-contracting (control) NRVM, and NRVM stimulated to contract for up to 48 h. In preliminary experiments, we altered voltage amplitude and pacing frequency to ensure that approximately 75% of the cells contracted immediately in response to electrical stimulation at a frequency which produced 1:1 capture. We found that pacing at a rate of 3 Hz at 3.4 V/cm provided these conditions. Thereafter, all experiments were performed using this pacing regimen. As seen in Figure 1(a), NRVM subjected to the pacing protocol for 48 h were clearly larger, and formed more cell-to-cell contacts than the quiescent, control cells. This increased cell size was accompanied by a statistically significant, 15±5% increase in the level of total cellular protein normalized to DNA [Fig. 1(b)]. mRNAs encoding ANF and MHC also increased, whereas the level of MHC mRNA decreased in response to electrical pacing [Fig. 1(c)]. This pacing-induced “switch” in MHC isoenzyme predominance is characteristic of hemodynamic overload of the adult rat heart in vivo,20 and has been observed in response to a variety of hypertrophic stimuli that activate PKCs in vitro.4,16,21,22 The increase in MHC and ANF mRNA levels resulted from an increase in MHC and ANF promoter activity, as assessed in transient transfection experiments. As seen in Figure 2, normalized luciferase activities corresponding to MHC and ANF promoter activities were increased three- and 13fold, respectively. Overall, these data are consistent with previously published studies indicating that
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Figure 1 Stimulated contraction induces cardiomyocyte hypertrophy. Panel A depicts Hoffman modulation contrast images of non-contracting (CONTROL) cardiomyocytes, and cardiomyocytes stimulated to contract at 3 Hz for 48 h (PACED). Images were obtained using a 40×-objective mounted on an inverted microscope and captured using a video camera interfaced to a personal computer. Panel B depicts total protein/DNA ratios from CONTROL and PACED NRVM. Data are means±... from six different cell isolations. ∗ P
