INTRODUCTION

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PROLONGED EXPOSURE to ₁-adrenergic agonists produces a variety of hypertrophic alterations in neonatal rat ventricular myocytes (NRVM), including increased cell size, sarcomeric protein assembly, and specific changes in gene expression that are typical of hemodynamic overload in vivo. ₁-Adrenergic receptor activation also acutely activates cell signaling cascades that involve the heterotrimeric G protein G_q (18, 30), mitogen-activated protein kinases (MAPK) (12, 26, 41), and the small GTPases Ras (18, 40) and Rho (30, 42). However, it is unclear exactly which steps in the signal transduction pathways are necessary and/or sufficient for the induction of various aspects of the hypertrophic phenotype. Whereas either Ras or G_q activation by the ₁-adrenergic agonist phenylephrine (PE) was sufficient for the induction of atrial natriuretic factor (ANF) gene transcription (a molecular marker of the NRVM hypertrophy), only Ras activation was necessary and sufficient to elicit the structural reorganization of cytoskeletal proteins into sarcomeres (18, 40). In another study, Rho, a stimulator of stress fiber formation in nonmuscle cells (28), was neither necessary nor sufficient for PE-induced sarcomeric assembly in NRVM (42). In contrast, inactivation of Rho A protein by ADP ribosylation in embryonic chick ventricular myocytes caused sarcomeric disruption in the absence of other exogenous stimuli (43).

In contrast to studies with ₁-adrenergic agonists, we and others have found that mechanical load in the form of either electrically stimulated or spontaneous contractile activity induces NRVM growth in the absence of exogenous agonists (14, 21-24, 31). The hypertrophic response was very similar to that observed in response to ₁-adrenergic receptor activation, in that the intrinsic mechanical load generated during excitation-contraction coupling was sufficient to elicit the transcriptional activation of the "fetal" genes, ANF (10, 24) and -myosin heavy chain (MHC) (25, 27, 31), as well as to promote the assembly of newly synthesized contractile proteins into sarcomeres (3, 24, 31, 32, 35). Of particular interest to us was the observation by Kimura et al. (16) that ₁-adrenergic receptors couple directly to Ca²⁺ influx via voltage-gated, L-type Ca²⁺ channels, resulting in a marked increase in beating frequency. Earlier observations by Simpson (38) indicated that ₁-adrenergic stimulation was sufficient to stimulate quiescent, low-density NRVM cultures to contract. These observations suggested that at least some of the phenotypic alterations produced by ₁-adrenergic stimulation were secondary to mechanical events generated after receptor activation. In the present study, we have quantitatively analyzed the role of Ca²⁺ influx and contractile activity on specific features of the hypertrophic phenotype induced by PE exposure. Data are presented to indicate that PE-induced contractile activity is required to elicit sarcomeric assembly, but not the activation of the MAPKs ERK1 and ERK2 or upregulation of ANF gene expression in response to ₁-adrenergic receptor activation.

	METHODS

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Reagents. PC-1 tissue culture medium was obtained from BioWhittaker (Walkersville, MD). DMEM was obtained from GIBCO BRL (Grand Island, NY). Medium 199, Ca²⁺-free and Mg²⁺-free Hanks' balanced salts (modified) (HBSS), acid-soluble calf skin collagen, and antibiotic/antimycotic solution were obtained from Sigma Chemical (St. Louis, MO). Permanox chamber slides and slide wells were obtained from Nunc (Naperville, IL). Tissue culture plates were obtained from Costar (Cambridge, MA). [³²P]ATP, [³²P]dCTP, and [³⁵S]methionine were purchased from Amersham (Arlington Heights, IL). Fura 2-AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). 4-(2-Aminoethyl)-benzenesulfonyl fluoride was obtained from Boehringer Mannheim (Basel, Switzerland). Rabbit polyclonal antibodies to ERK1 and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad (Hercules, CA). All other reagents were of the highest grade commercially available and were obtained from Sigma and Baxter S/P (McGaw Park, IL).

Ventricular dissociation and cardiac myocyte isolation. Animals used in these experiments were handled in accordance with National Institutes of Health "Guide for the Care and Use of Laboratory Animals" [Department of Health and Human Services Publication No. (NIH) 85-23, Revised 1985]. Ventricular myocytes were isolated from the hearts of 2-day-old Sprague-Dawley rats by collagenase digestion, as previously described (31). Released cells were collected by centrifugation, resuspended in PC-1 medium, and plated at a density of 400 cells/mm² onto collagen-coated plastic 35-, 60-, and 100-mm dishes as well as Permanox chamber slides or slide wells. They were left undisturbed in a 5% CO₂ incubator (37°C) for 14-18 h. Unattached cells were then removed by aspiration, and cells were maintained in a 4:1 mixture of DMEM-medium 199 containing antibiotic/antimycotic solution (myocyte growth medium). Medium was changed daily.

Measurement of intracellular Ca²⁺ concentration. Myocytes plated onto Permanox chamber slides were maintained in growth medium with daily medium changes for 2-3 days. The cells were then loaded with the fluorescent Ca²⁺ indicator fura 2 by incubating with fura 2-AM [2 µM in a modified Krebs medium (135 mM NaCl, 5.9 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 11.5 mM glucose, and 11.6 mM HEPES, pH 7.3) supplemented with 0.1% BSA and 0.02% Pluronic F-127 detergent] for 2 h at room temperature, followed by a 1- to 3-h incubation in Krebs medium alone. Fura 2 fluorescence was then measured in individual cells using a video microscopy system composed of a Nikon Diaphot inverted epifluorescence microscope, rotating filter wheel, and an intensified charge- coupled device video camera (Hamamatsu model XC77/C2400). The slide was mounted in a chamber (Warner Instrument) on the stage of the microscope and superfused with media at a rate of 5-10 ml/min. A four-way valve mounted adjacent to the chamber allows rapid switching of solutions from four gravity-fed reservoirs. The field of cells was excited alternately with 340- and 380-nm light, and the average brightness of eight individual cells was recorded to disk. Background fluorescence was determined for each cell at the end of the experiment by quenching the fura 2 fluorescence for 15 min in the presence of 1 µM ionomycin and 6 mM MnCl₂ in Ca²⁺-free medium. After background fluorescence was subtracted, the 340- and 380-nm values were ratioed and calibrated in terms of intracellular Ca²⁺ concentration ([Ca²⁺]_i). This procedure allows simultaneous measurement of [Ca²⁺]_i in eight individual cells with a temporal resolution of ~0.6 s. All [Ca²⁺]_i measurements shown are from experiments conducted at 25°C.

Cell area measurements. Cells were grown on Permanox slide wells and maintained in growth medium with daily medium changes for 2-3 days. During the last 48 h of this period, the cells were treated with medium alone (control) or growth medium containing verapamil (10 µM), PE (50 µM), or PE plus verapamil. The cells were then loaded with fura 2 as described above, except that the concentration of fura 2-AM was 4 µM, and the treatments were present in both the loading medium and subsequent wash medium. The area of the fluorescently labeled cells was then determined by image analysis using Universal Imaging Image 1 software. A binary mask was created by setting a threshold brightness that distinguished the fluorescent cells (illuminated with 380-nm light) from the black background. The area of the mask for each cell was then determined. When a cell was in contact with one or more adjacent cells, the area of the mask was divided by the number of cells. The mean cell area was determined for 120 cells in each of the treatment groups. Absolute area measurements may be slightly underestimated because very thin areas at the cell periphery may not have been detected above the background fluorescence.

Myofibrillar structure. Cells grown on Permanox slide wells were fixed (10 min, room temperature) with 2% (wt/vol) paraformaldehyde in sodium PBS, washed (15 min) in 1% (wt/vol) glycine in PBS, and permeabilized (15 min) with 0.5% (vol/vol) Triton X-100 in PBS. Myocytes were then stained with FITC-conjugated phalloidin to visualize F-actin filaments and myofibrillar structure (35). The phalloidin-stained cells were viewed using a Zeiss model LSM 410 scanning laser confocal microscope. Multiple optical sections ~1 µM thick were taken of each sample to eliminate out-of-focus fluorescence of the intensely stained myocytes.

Cellular composition. For the quantitative analysis of total cellular protein and DNA content, cells grown on 35-mm dishes were washed twice in HBSS, and 0.2 N perchloric acid (1 ml) was added. The precipitated macromolecules 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 µl of 0.3 N 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 (31). Data are the means of duplicate or triplicate wells from each treatment group for each cell isolation and are expressed as micrograms per dish. For quantitative analysis of -MHC and -MHC content, cells were washed twice in HBSS and lysed in 250 µl of sample buffer [62.5 mM Tris · HCl, pH 6.8, containing 8% (wt/vol) SDS, 5% (vol/vol) 2-mercaptoethanol, and 10% (wt/vol) glycerol]. The concentrations of -MHC and -MHC isoenzymes were assessed by SDS-PAGE and silver staining (31). MHC band intensity was quantified by laser densitometry and compared with the band intensity of purified MHC standards (0-300 ng). The positions of the -MHC and -MHC bands were confirmed by electrophoresis of -MHC and -MHC protein standards obtained from normal and hypothyroid adult rat hearts, respectively, and by Western blotting with an anti-MHC antibody that cross-reacts equally with both isoenzymes (data not shown). Results are the means of duplicate wells from each treatment group for each cell isolation and are expressed as micrograms per dish.

Pulse-chase biosynthetic labeling experiments. MHC degradation in control, PE-treated, and verapamil-treated cultures was assessed in pulse-chase biosynthetic labeling experiments, as previously described (3, 32). Cells in 35-mm dishes were incubated (24 h, 37°C) in myocyte growth medium supplemented with 8 µCi/ml [³⁵S]methionine. At the end of the pulse-labeling period, cells were rapidly rinsed twice in HBSS and either harvested by addition of 500 µl SDS sample buffer or chased for 24 h in growth medium supplemented with 2 mM unlabeled methionine, or methionine-supplemented growth medium containing PE (50 µM), verapamil (10 µM), or their combination. Cell samples were then separated by SDS-PAGE on 180-mm-long, 0.7-mm-thick, 7-17% vertical gradient SDS-polyacrylamide gels. In each experiment, a constant fraction of the total protein of each culture dish was applied to individual gel lanes. This ensured that for all pulse-chase experiments, the amount of radioactivity in MHC declined by decay rather than by simple dilution. After electrophoresis, gels were autoradiographed with fluorographic enhancement. Dried gels were exposed to unflashed Kodak XAR-5 film for varying time periods (2-4 days) at 80°C. Individual MHC bands on the autoradiographs were scanned three times, and the average area beneath the MHC peak was computed by autointegration. Linearity of detection of radioactivity by fluorography was assessed as previously described (32). The fractional rate of MHC degradation (MHC K_d, %/h) for each condition was estimated by the following formula where ln(MHC AU)₀ and ln(MHC AU)₂₄ are the natural logarithms of the average absorbance [in arbitrary absorbance units (AU)] of the MHC bands at times 0 and 24 h of the chase. MHC K_d values were converted to apparent half-lives (t_1/2; in h) according to the following formula

MAPK Western blots. Myocytes plated onto 100-mm dishes were maintained in myocyte growth medium for 48 h. Cells were then switched to fresh growth medium or growth medium containing verapamil (10 µM). After an additional 1 h, myocytes were stimulated (5 min) with phorbol 12-myristate 13-acetate (PMA, 200 nM) or PE (50 µM) in the presence or absence of verapamil. Thereafter, cells were scraped into 0.9 ml of MAPK extraction buffer [10 mM HEPES, pH 7.4, containing 50 mM sodium pyrophosphate, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 50 mM sodium fluoride, 0.1 mM sodium vanadate, 0.01% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride]. Aliquots of the cell extracts (25 µg of total protein) were separated by SDS-PAGE and transferred to nitrocellulose membrane by electroblotting. The blots were probed with a mixture of polyclonal antibodies directed toward ERK1 (44 kDa) and ERK2 (42 kDa). Primary antibody binding was detected by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody using the enhanced chemiluminescence kit from Amersham.

ANF promoter activity. An expression plasmid consisting of 3003 bp of upstream regulatory sequences of the rat ANF gene linked to the gene encoding firefly luciferase was used to analyze ANF transcription in control, PE-treated, and verapamil-treated myocytes using both the calcium phosphate method and adenoviral-assisted transfection (17). 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. A constitutively active Rous sarcoma virus long terminal repeat ligated to the bacterial -galactosidase reporter gene plasmid (pRSV-lacZ, ATCC) was cotransfected to normalize for DNA transfer efficiency. For the calcium phosphate method, myocytes (grown on 60-mm dishes) were incubated with DNA-calcium phosphate solution (containing 10 µg of p3003ANF-luc and 2 µg of pRSV-lacZ) at 37°C for 6 h. Cells were then washed and maintained in growth medium or in growth media supplemented with verapamil, PE, or their combination. After an additional 48 h of culture, the cells were assayed for luciferase and -galactosidase activities as previously described (25). Relative light units were measured using an enhanced luciferase assay kit (Analytical Luminescence Laboratory, Ann Arbor, MI) and a luminometer (Berthold, model LB9501).

ANF mRNA analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (7) after 48 h of culture under control conditions or after treatment with PE, verapamil, or their combination. 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 µg) was separated by denaturing agarose gel electrophoresis, subjected to alkali pretreatment, transferred to nylon membranes by capillary action, and cross-linked by ultraviolet irradiation. ANF mRNA levels were detected by hybridization to a ³²P-labeled, 786-residue ANF cDNA probe (20). The Northern blots were also hybridized with a ³²P-labeled 24-base oligodeoxyribonucleotide probe specific for rat 18S rRNA (31).

Data analysis. Results were expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. One-way repeated measures ANOVA, Friedman repeated measures ANOVA on ranks, or Kruskal-Wallis one-way ANOVA on ranks followed by the Student-Newman-Keuls test was used for the statistical comparison of multiple groups, as appropriate. Data were analyzed using the SigmaStat Statistical Software Package, version 1.0 (Jandel Scientific, San Rafael, CA).

	RESULTS

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PE induces [Ca²⁺]_i transients in low-density NRVM cultures. As seen in Fig. 1, NRVM plated at low density and maintained in DMEM-medium 199 for 48 h were either quiescent (Fig. 1A) or displayed only occasional [Ca²⁺]_i transients and contractions (Fig. 1B). However, within 30 min of PE exposure, [Ca²⁺]_i oscillations were induced in >80% of the quiescent myocytes. In the minority of myocytes that displayed occasional spontaneous [Ca²⁺]_i transients, PE increased the frequency of [Ca²⁺]_i oscillations. Each [Ca²⁺]_i transient was accompanied by cell contraction, as assessed by visual inspection. Pretreatment with verapamil (10 µM) suppressed the spontaneous [Ca²⁺]_i transients and also suppressed the PE-induced [Ca²⁺]_i oscillations (Fig. 1C), indicating that both were highly dependent on Ca²⁺ influx via voltage-gated L-type Ca²⁺ channels. Both the frequency and amplitude of the contractile activity appeared to increase after prolonged exposure to PE. After 24-h exposure, virtually all of the NRVM were contracting at a rate of 1-2 Hz, whereas NRVM maintained in medium containing verapamil or PE plus verapamil remained quiescent.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Phenylephrine (PE) stimulates intracellular Ca2+ concentration ([Ca2+]i) transients in low-density neonatal rat ventricular myocyte (NRVM) cultures. In A, [Ca2+]i was recorded from a single fura 2-loaded, quiescent NRVM superfused in Krebs buffer, followed by Krebs buffer containing 50 µM PE (shaded bar). In this cell, no spontaneous [Ca2+]i transients were noted under control conditions (0-5 min). However, [Ca2+]i transients were observed within 1 min after PE exposure. Sampling rate (1.5 Hz) did not allow for resolution of very rapid [Ca2+]i transients that occurred during bursts of activity. In B, a similar recording was obtained from a single fura 2-loaded myocyte that displayed occasional spontaneous [Ca2+]i transients. PE increased frequency of [Ca2+]i oscillations. In C, a single fura 2-loaded NRVM was first pretreated with verapamil (10 µM, 1 h) and then superfused with Krebs buffer containing 50 µM PE + 10 µM verapamil. No [Ca2+]i transients were generated by PE exposure.

PE-induced [Ca²⁺]_i transients and contractile activity are necessary for hypertrophic growth. We next examined whether PE stimulated myocyte growth and whether PE-induced [Ca²⁺]_i transients and contractile activity were required for expression of specific aspects of the hypertrophic phenotype. NRVM were treated with PE (50 µM), verapamil (10 µM), or their combination for 48-72 h. Of note, this concentration of verapamil was the minimum concentration of the drug required to completely inhibit spontaneous contractile activity over a 24-h period without affecting cell viability, as assessed by visual inspection for cell detachment.

View larger version (142K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effects of PE and verapamil on NRVM cell size. Low-density NRVM cultures were treated (48 h) with serum-free medium alone (C) or medium supplemented with PE (50 µM), verapamil (V; 10 µM) or PE and verapamil (PE + V). Verapamil was added to inhibit both basal and PE-induced [Ca2+]i transients and contractile activity. In A, live cells were visualized by Hoffman modulation contrast imaging (all fields viewed with a ×40 objective). In B, cell area measurements were obtained by fura 2 loading, fluorescence microscopy, and image analysis using Image-1 software. Data are means ± SE from 120 cells in each group. Data were compared by ANOVA on ranks followed by Student-Neuman-Keuls test. * P < 0.05 vs. control. + P < 0.05 vs. verapamil.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Effects of PE and verapamil on myosin heavy chain (MHC) isoenzyme content of NRVMs. Low-density NRVM cultures (375,000 cells/35-mm dish) were treated (48 h) with serum-free medium alone (C) or medium supplemented with PE (50 µM), verapamil (V; 10 µM), or PE and verapamil (PE + V) to inhibit PE-induced [Ca2+]i transients and contractile activity. Cells were then scraped from dishes in 500 µl of SDS sample buffer, and protein samples were separated by SDS-PAGE. Each lane was loaded with 25 µl of cell extract, except for lane 2 (PE), which was loaded with 10 µl. Gels were then silver stained and scanned by laser densitometry. Positions of -MHC and -MHC bands were confirmed by electrophoresis of known -MHC and -MHC standards obtained from normal and hypothyroid rat hearts, respectively.

View larger version (144K): [in this window] [in a new window]   Fig. 4.   Verapamil blocks PE-induced myofibrillar assembly in NRVM. Low-density NRVM cultures were maintained (48 h) in serum-free DMEM-medium 199 alone (C) or in medium supplemented with verapamil (V; 10 µM), PE (50 µM), or their combination (PE + V). Cells were then fixed, permeabilized, stained with FITC-phalloidin, and viewed under a scanning laser confocal microscope (×60 objective, zoom factor 4.0). As is evident from figure, PE-stimulated sarcomeric assembly was highly dependent on Ca2+ influx through voltage-gated L-type Ca2+ channels.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   MHC protein half-life in PE-treated NRVM cultures. Low-density NRVM cultures were biosynthetically labeled (24 h) with [35S]methionine and chased (24 h) in serum-free medium alone (C) or medium supplemented with PE (50 µM), verapamil (V; 10 µM), or their combination (PE + V). Total cell protein was then separated by SDS-PAGE and autoradiography with fluorographic enhancement. MHC band intensity was quantified by laser densitometry, and fractional rate of MHC degradation (MHC Kd, %/h) for each condition was estimated by following formula: MHC Kd = 100 · [ln(MHC AU)0  ln(MHC AU)24]/24, where ln(MHC AU)0 and ln(MHC AU)24 are natural logarithms of average absorbance (in arbitrary absorbance units) of MHC bands at times 0 and 24 h of chase. MHC Kd values were converted to apparent half-lives (in h) according to following formula: MHC t1/2 = 100 · [ln(2)/MHC Kd]. Data are means ± SE for 6 myocyte isolations. Data were compared by repeated measures ANOVA on ranks followed by Student-Neuman-Keuls test. * P < 0.05 vs. control. + P < 0.05 vs. verapamil.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Other Ca2+ channel blockers prevent PE-induced NRVM hypertrophy. Low-density NRVM cultures were treated (48 h) with serum-free medium alone (C) or medium supplemented with verapamil (V; 10 µM), diltiazem (D; 10 µM), or nifedipine (N; 10 µM) in presence or absence of PE (50 µM). To account for variability in total protein-to-DNA ratio from experiment to experiment, individual values were expressed as a percentage of control cells from each myocyte isolation. Data are means ± SE for 4-20 myocyte isolations. Mean ± SE for control group was 52.3 ± 2.8 µg total protein/µg DNA. Data were compared by repeated measures ANOVA on ranks followed by Student-Neuman-Keuls test. * P < 0.05 vs. control. + P < 0.05 vs. PE.  P < 0.05 vs. each drug treatment in absence of PE.

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Diltiazem, nifedipine, and 2,3-butanedione monoxime block PE-induced MHC accumulation in NRVMs. Low-density NRVM cultures were treated (48 h) with serum-free medium alone (C) or medium supplemented with PE (50 µM) or PE and diltiazem (D; 10 µM), nifedipine (N; 10 µM), and 2,3-butanedione monoxime (B; 7.5 mM) to inhibit PE-induced [Ca2+]i transients and/or contractile activity. Cells were then scraped from dishes in 250 µl of SDS sample buffer, and protein samples were separated by SDS-PAGE. Each lane was loaded with 25 µl cell extract. Gels were then silver stained and scanned by laser densitometry.

Mechanical activity is required for PE-induced NRVM hypertrophy. 2,3-Butanedione monoxime (BDM), an inhibitor of actin-myosin cross-bridge cycling, was then used to determine whether mechanical activity, rather than [Ca²⁺]_i transients per se, was required to elicit PE-induced NRVM hypertrophy. Previous studies from our laboratory have demonstrated that in NRVM, acute or chronic exposure to 7.5 mM BDM only modestly reduced the amplitude of [Ca²⁺]_i transients but markedly reduced cell shortening (3, 27). [Higher concentrations of BDM have been shown to promote voltage-dependent inactivation of L-type Ca²⁺ channels (11).] Therefore, myocytes were treated (48 h) with PE (50 µM), 7.5 mM BDM, or their combination, and the resulting cell extracts were analyzed for total protein, DNA, and MHC content. As seen in Fig. 8, PE increased total protein-to-DNA ratio in control, but not in BDM-treated cells. Similarly, BDM prevented PE-induced -MHC and -MHC accumulation (Fig. 7).

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Inhibition of mechanical activity prevents PE-induced NRVM hypertrophy. Low-density NRVM cultures were treated (48 h) with serum-free medium alone (C) or medium supplemented with PE (50 µM), 2,3-butanedione monoxime (B; 7.5 mM), or their combination (PE + B). Data are means ± SE for 6 myocyte isolations. Data were compared by repeated measures ANOVA on ranks followed by Student-Neuman-Keuls test. * P < 0.05 vs. control. + P < 0.05 vs. 2,3-butanedione monoxime.

Phenylephrine activates MAPK independently of [Ca²⁺]_i transients and contractile activity. In contrast to their general effects on cellular growth and myofibrillar assembly, [Ca²⁺]_i transients and contractile activity were not required for PE-induced activation of MAPK. As seen in Fig. 9, both PMA (200 nM) and PE (50 µM) activated ERK1 and ERK2. MAPK activation (as assessed by the upward shift in apparent molecular weight of both ERK1 and ERK2) occurred within 5 min of exposure to either agonist in control cells, as well as NRVM pretreated (1 h) with verapamil (10 µM) to prevent [Ca²⁺]_i transients and beating.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   [Ca2+]i transients and contractile activity are not required for mitogen-activated protein kinase activation by PE. Low-density NRVM cultures were maintained in myocyte growth medium for 48 h. Cells were then switched to fresh growth medium or growth medium containing verapamil (10 µM). After an additional 1 h, myocytes were stimulated (5 min) with phorbol 12-myristate 13-acetate (PMA; 200 nM) or PE (50 µM). Total protein homogenates (25 µg) were separated by SDS-PAGE and transferred to nitrocellulose membrane. Blots were probed with a mixture of polyclonal antibodies directed toward ERK1 (44 kDa) and ERK2 (42 kDa). Apparent molecular masses are indicated to left of bands. Activation of ERK1 and ERK2 is represented by upward shift in molecular mass, designated as p44 and p42, respectively.

Effects of [Ca²⁺]_i transients, contractile activity, and PE on ANF gene expression. We also examined the role of [Ca²⁺]_i transients and contractile activity in PE-induced stimulation of ANF promoter activity. As seen in Fig. 10A in which the CaPO₄ method was used in transient transfection experiments, normalized luciferase activity in PE-treated myocytes was 246 ± 19%, where expression in control cells for each of six experiments was normalized to 100%. Addition of verapamil to the serum-free culture medium significantly reduced basal ANF promoter activity to 20 ± 1% of control cells. However, PE was still capable of stimulating ANF promoter activity even in the presence of the Ca²⁺ channel blocking agent (38 ± 2% of control cells, or nearly double the level of expression as compared with cells treated with verapamil).

View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 10.   Effects of [Ca2+]i transients, contractile activity, and PE on atrial natriuretic factor promoter activity. In A, NRVM were transiently transfected with p3003ANF-luc and pRSV-lacZ using CaPO4 method. Cells were then maintained in control medium (C) or medium supplemented with verapamil (V; 10 µM), PE (50 µM), or their combination. After an additional 48 h of culture, cell extracts were assayed for luciferase and -galactosidase (-gal) activities. Data were expressed as luciferase/-galactosidase activity where enzyme activity in control cells was normalized to 100%. Normalized luciferase activity was 4.4 ± 1.1 × 107 relative light units in control cells. Data are means ± SE from 6 different cell isolations. In B, transfections with p3003ANF-luc and pRSV-lacZ were also performed with replication-deficient adenovirus Ad5dl312. Cells were then maintained in control medium (C) or medium supplemented with verapamil (10 µM), PE (50 µM), or their combination. After an additional 24 h of culture, cell extracts were assayed for luciferase and -galactosidase activities, as described above. Normalized luciferase activity was 1.3 ± 0.7 × 105 relative light units in control cells. Data are means ± SE from 4 different cell isolations. Data were compared by repeated measures ANOVA on ranks followed by Student-Neuman-Keuls test. * P < 0.05 vs. control. + P < 0.05 vs. verapamil.

View larger version (71K): [in this window] [in a new window]   Fig. 11.   Effects of [Ca2+]i transients, contractile activity, and PE on atrial natriuretic factor (ANF) mRNA levels. NRVM were maintained (48 h) in control medium (C) or medium supplemented with PE (50 µM), verapamil (V; 10 µM), or their combination (PE + V). Total RNA (10 µg) was size fractionated, transferred to nitrocellulose, and sequentially probed with 32P-labeled cDNA and oligodeoxyribonucleic acid probes specific for rat ANF mRNA and 18S rRNA, respectively. As is evident from figure, PE increased ANF mRNA levels in both control and verapamil-treated NRVM. Similar results were obtained in 2 other experiments.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Cultured NRVM have been extensively used as a model system with which to define the signal transduction pathways that cause hypertrophic growth of cardiac muscle in response to both neurohormonal and mechanical stimuli. With respect to the growth-promoting effects of mechanical load, several studies have shown that NRVM undergo hypertrophy in response to either externally applied or intrinsically generated mechanical load in the absence of exogenous growth factors. However, there are several potential mechanisms wherein neurohormonal and mechanical signaling pathways may interact to elicit specific aspects of the hypertrophic phenotype. For instance, isolated NRVM plated at low density onto a collagen-coated substratum exhibit little spontaneous contractile activity. However, Simpson (38) showed that the catecholamines norepinephrine (NE) and epinephrine (E) stimulated contractile activity in quiescent, low-density NRVM cultures maintained in serum-free medium. The effects of NE and E on contractile frequency appeared to be due to the ability of the drugs to simultaneously stimulate both ₁- and ₁-adrenoreceptors. ₁-Adrenergic stimulation alone reportedly produced enlarged cells that did not beat, whereas combined ₁- and ₁-adrenergic stimulation slowly induced contractile activity over a 24-h period even when protein synthesis and hypertrophy were inhibited with cycloheximide (39). In contrast, Kimura et al. (16) showed that NE evoked a positive chronotropic response within 1 min of exposure to the drug. This rapid induction of beating was dependent only on activation of ₁-adrenoreceptors, since contractile activity was induced by exposure to NE in the presence of the -receptor antagonist propranolol. Our present results depicted in Fig. 1 are consistent with the observations of Kimura et al. (16) and indicate that in cultured NRVM, ₁-adrenoreceptor stimulation triggers [Ca²⁺]_i transients that are highly dependent on voltage-gated L-type Ca²⁺ channels. It is also apparent from Fig. 1 that PE does not appreciably stimulate inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellular stores, since no [Ca²⁺]_i increase was detected when Ca²⁺ influx was blocked with verapamil.

PE and other G_q-coupled agonists have been shown to also trigger a variety of signaling events associated with the hypertrophic phenotype. These phenotypic alterations include the tyrosine phosphorylation of several signaling molecules including ERK1 and ERK2, induction of c-fos and other immediate/early genes, transcriptional activation of the secondary response genes -MHC, -skeletal actin and ANF, increased protein synthesis, and the assembly of newly synthesized myofibrillar proteins into sarcomeres. However, the role of [Ca²⁺]_i transients and mechanical activity in these signaling events remains controversial. In the present study, we have attempted to dissociate features of PE-stimulated myocyte hypertrophy that are dependent on [Ca²⁺]_i transients and contractile activity from those that do not require this activity.

As reviewed by Chien et al. (6), induction of the myocyte hypertrophic phenotype by a variety of extracellular signals requires both transcriptional activation and enhanced assembly of individual contractile protein subunits into sarcomeres. Our results clearly indicate that [Ca²⁺]_i transients and mechanical activity are critical for the alterations in cellular architecture that are typical of PE-induced NRVM hypertrophy. Despite a careful examination of several signal transduction pathways, the intracellular mechanisms responsible for PE-induced sarcomeric protein assembly remain largely unknown. Initial studies implicated protein kinase C (PKC) activation in sarcomeric protein assembly. Dunmon et al. (9) showed that PKC activation of low-density NRVM cultures with either PE or the phorbol ester PMA not only induced immediate early gene expression and stimulated nuclear gene transcription, but also caused the appearance within the cytoplasm of organized sarcomeres. Other protein kinases, including Raf-1, MAPK kinase (MAPKK or MEK), MAPK, and S6 kinase were also activated by PE treatment or PMA (for review, see Ref. 2). However, activation of PKC with PMA did not stimulate myofibrillar assembly when Ca²⁺ influx was blocked by the L-type Ca²⁺ channel blocker verapamil, despite a marked degree of PKC activation/translocation (32, 35; unpublished data). Furthermore, the slow assembly of myosin light chain-2 into sarcomeres in response to PE treatment was not dependent on activation of MAPK (41), despite the fact that this kinase has been implicated in the transcriptional activation of numerous cellular genes essential for growth. As indicated in Fig. 4 of the present study, PE-induced sarcomeric assembly required the induction of [Ca²⁺]_i transients and contractile activity. These results support previous studies which indicate an important role for intrinsic and/or externally applied mechanical load in the induction and maintenance of myofibrillar protein assembly (3, 8, 31, 35-37). As demonstrated here and in previous studies, the assembly of myofibrillar proteins such as MHC and actin into functional sarcomeres also profoundly reduced the susceptibility of these proteins to intracellular degradation, thereby contributing to contractile protein accumulation and cellular hypertrophy (3, 8, 32, 35-37).

One mechanism whereby PE-induced [Ca²⁺]_i transients and mechanical activity may stimulate sarcomeric assembly in cultured NRVM is by the load-dependent formation of focal adhesions and costameres. In a recent study, Sharp et al. (34) demonstrated that both intrinsic and externally applied mechanical load stabilized the cell-surface distribution of ₁-integrin, the transmembrane cell surface receptor which mediates cell attachment to the extracellular matrix. Inhibition of the spontaneous contractile activity of high-density NRVM with nifedipine caused the rapid disruption of focal adhesions and the loss of ₁-integrin from the cell surface, whereas application of 5% static stretch partially prevented these changes. Restoration of contractile activity (by removal of nifedipine from the culture medium) caused the reaccumulation of ₁-integrin on the cell surface and the reformation of focal adhesions which temporally corresponded with the reassembly of myofibrillar proteins into sarcomeres. Thus [Ca²⁺]_i transients and actin-myosin cross-bridge formation (even in the absence of other agonists) were sufficient for sarcomeric assembly (3, 24, 31). Nevertheless, nonsarcomeric, filamentous actin appeared to accumulate in PE-stimulated NRVM even in the presence of verapamil (Fig. 4), which may explain why the PE-stimulated increase in cell surface area was relatively resistant to blockade of Ca²⁺ influx.

In contrast to the clear dependence of sarcomeric assembly on PE-stimulated [Ca²⁺]_i transients and mechanical activity, we found that MAPK activation and ANF gene expression were less dependent on these signals. Our results should be evaluated in light of prior studies by Sadoshima et al. (29), which suggested that ANG II-mediated activation of MAPK was highly dependent on [Ca²⁺]_i. Their conclusion was based on the finding that pretreatment of NRVM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM blocked the ANG II-mediated increase in resting [Ca²⁺]_i and also completely prevented agonist-induced MAPK activation. However, it should be pointed out that ANG II did not stimulate [Ca²⁺]_i oscillations in their low-density cultured heart cells. Furthermore, the concentration of BAPTA-AM used to prevent the ANG II-induced increase in [Ca²⁺]_i actually lowered resting levels of [Ca²⁺]_i. As indicated in Figs. 1 and 10 of this report, verapamil treatment did not significantly lower resting [Ca²⁺]_i or block MAPK activation but clearly suppressed phenylephrine-induced [Ca²⁺]_i transients and beating. These results are in agreement with previous studies that have indicated that MAPK activation occurs by both Ca²⁺-dependent and Ca²⁺-independent pathways (5, 19).

Although verapamil treatment decreased basal ANF promoter activity (Fig. 10) and ANF mRNA levels (Fig. 11), ₁-adrenergic stimulation was still capable of augmenting ANF gene expression in the absence of [Ca²⁺]_i transients and contractile activity. Our results confirm and extend previous studies by Sei et al. (33), who demonstrated that ₁-adrenergic stimulation significantly increased ANF mRNA levels in both control and nifedipine-treated myocytes. Thus the analysis of ANF promoter activity by transient transfection, as well as by quantitative Northern blotting, revealed [Ca²⁺]_i/contraction-dependent and [Ca²⁺]_i/contraction-independent components of PE-stimulated ANF gene expression. Furthermore, our present finding of decreased basal ANF transcription in verapamil-treated cells suppports previous studies from this laboratory using spontaneously contracting, high-density NRVM (10). Those studies demonstrated that contractile arrest (produced with verapamil, BDM, or K⁺ depolarization) markedly reduced basal ANF promoter activity, mRNA levels, and protein secretion (10).

In summary, ₁-adrenergic stimulation induced [Ca²⁺]_i transients and contractile activity that were required for MHC accumulation and sarcomeric assembly in cultured NRVM. The intracellular mechanisms responsible for generating [Ca²⁺]_i oscillations in response to ₁-adrenergic stimulation in these cultured heart cells requires further investigation, but the results of the present report highlight the importance of both mechanical and neurohormonal factors (and their potential interactions) in eliciting specific aspects of the hypertrophic phenotype.