INTRODUCTION

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Cardiac hypertrophy commonly develops in response to hemodynamic pressure overload (1). The participation of Ras in the development of hypertrophy is suggested by the observations that hypertrophic stimuli activate Ras, dominant-negative Ras mutants inhibit the hypertrophic response, and overexpression of constitutively active Ha-Ras^V12 elicits hypertrophic growth (2-4). Downstream effectors of Ras, particularly kinases of the Raf-MEK-ERK and MEKK1-JNKK-JNK pathways, may also participate in these growth processes (4).

Hypertrophic and failing hearts feature reduced contractile calcium transients with prolonged decay, myofibrillar disarray, and reduced expression of SERCA2, an enzyme responsible for reuptake of calcium into the sarcoplasmic reticulum (1, 5, 6). Exposure of cultured cardiac myocytes to PKC-activating phorbol esters also results in prolonged calcium transients and reduced SERCA2 (7, 8), and PKC is elevated in hypertrophic hearts (9). The PKC effectors responsible for the altered regulation of [Ca²⁺]_i have not been identified, but an intriguing possibility may be Raf, which can be activated by either PKC or Ras (4, 10). To address this possibility, the present study was undertaken to assess the effects of Ras, PKC, and related ERK activators on cardiac [Ca²⁺]_i.

	EXPERIMENTAL PROCEDURES

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Cell Culture-- Neonatal rat ventricular myocytes were dissociated and transfected via electroporation as described previously (11). To prepare myocytes for indo-1 measurements, 9 × 10⁶ cells/transfection were electroporated and split into two 35-mm wells containing fibronectin-coated glass coverslips for plating. Cells to be analyzed for morphology were plated as described previously (11). After 18-20 h in serum-containing plating medium, the cells were rinsed and refed with serum-free maintenance medium (Dulbecco's modified Eagle's medium supplemented with 1 nM T3, 5 µg/ml transferrin, 1 µg/ml insulin, and 0.1 ng/ml selenium). Unless otherwise stated, cells were kept in maintenance medium for 48 h prior to analysis.

Test Expression Constructs and Transfections-- pDCR Ha-Ras^V12 (activated Ha-Ras), pDCR Ha-Ras^V12S35, and pDCR Ha-Ras^V12C40 were obtained from D. Bar-Sagi (SUNY, Stony Brook, NY). RSV-Raf-1 BXB (codes for activated Raf-1 kinase) and RSV-C4B (codes for the amino terminus of Raf-1 kinase) were from U. Rapp (University of Wurzburg, Wurzburg, Germany). pCMV5 MEKK_COOH (codes for activated MEKK-1) was from G. Johnson (University of Colorado, Denver, CO). pEXV3-MAPKK1-Ala-217 (referred to as "dnMEK") was obtained from S. Fuller (Imperial College, London, UK) and codes for a mutant MAPKK1 (synonymous with MEK1), which can act in a dominant-negative fashion because it is poorly phosphorylated by Raf (12). PKAC was from M. Muramatsu (DNAX Research Institute, Palo Alto, CA). These constructs utilize the highly effective CMV,¹ SV40, or Rous sarcoma virus promoters to drive expression of the regulatory protein. The amount of DNA used per transfection depended upon the constructs. The transfection markers pGreen Lantern-1 (Life Technologies, Inc.) and CMV--galactosidase were used at 4 µg or 9 µg/transfection, respectively. Within each experiment, the DNA amounts per transfection were equalized with empty vector DNA such as pCEP. Although transfection efficiency is relatively low with this cell type (averaging approximately 5%), the use of -galactosidase and GFP allowed analysis of individual transfected myocytes for morphology, cell structures, and calcium transients.

Plasmid Construction-- Rat SERCA2 cDNA (nucleotides 267/3726, numbered relative to the start codon) in pBluescript was digested with ApaI and re-ligated, deleting 254 base pairs of the 5' noncoding sequence SERCA2 (13/3726). Next, the plasmid was linearized with DraIII at nucleotide 3076 of SERCA2 (located in the 3' noncoding region of the SERCA2 cDNA), and the overhang was filled with T4 polymerase, generating a blunt end. The plasmid was subsequently digested with KpnI, and the SERCA2 encoding fragment was inserted into the adenoviral shuttle vector, pACCMV.pLpA, at the KpnI/blunted HindIII sites. The resulting vector (pACCMV.pLpA-SERCA2) contains SERCA2(13/3076) inserted between the CMV promoter and the SV40 polyadenylation sequence.

Immunocytofluorescence-- Cardiac myocytes were fixed and stained for -galactosidase, actin, and SERCA2 as published (7, 11). An fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody was utilized to visualize the anti-SERCA2 primary antibody. Actin was visualized using rhodamine phalloidin (Molecular Probes), according to the manufacturer's instructions.

Indo-1 Measurements-- Cells were loaded with indo-1 as described previously (13). Indo-1 measurements were made in air-compatible Dulbecco's modified Eagle's medium at room temperature at 20 Hz. GFP-expressing myocytes, corresponding to myocytes that had taken up the GFP expression vector during the transfection procedure, were identified by monitoring fluorescence with an excitation wavelength of 475 nm. Excitation was then switched to 355 nm (optimal for indo-1), and indo-1 fluorescence was recorded from the cytoplasmic region of the myocyte. In the absence of indo-1, GFP-expressing myocytes were nonfluorescent at the excitation wavelength of 355 nm, indicating that GFP does not interfere with measurement of indo-1. Although indo-1 fluorescence is related to free calcium concentration via well established equilibrium relationships, exact calibration of the signal in vivo is problematic (14); the results are thus reported as the ratio between the fluorescence emission at 405 and 485, or as "normalized" transients, where the ratios at diastole and at the peak systole of the transient equal 0 and 100%, respectively.

ERK Assays-- Myocytes were transfected with the test constructs and a hemaglutinin-tagged ERK2 expression vector (Cobb, University of Texas Southwestern Medical Center, Dallas, TX). Cultures were extracted, immunoprecipitated for the hemaglutinin epitope tag, and assayed for ERK activity in vitro as described (11).

	RESULTS

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Effects of Ha-Ras^V12 on Cell Morphology, SERCA2, and [Ca²⁺]_i-- Control myocytes (cotransfected with empty expression vector) were mildly stellate or elongate (Fig. 1A) and averaged 841 ± 115 µm² (mean ± S.E., n = 13) in surface area. Ha-Ras^V12 transfection (16 µg/electroporation) resulted in an approximate 6-fold increase in cell area (to 4984 ± 412, n = 25) and the cells exhibited a highly irregular, sprawling morphology (Fig. 1B). Although Ha-Ras^V12-transfected myocytes sometimes stained strongly for actin, the actin filaments were not striated (Fig. 1C). The absence of striations in the Ha-Ras^V12-transfected myocytes does not represent a limitation of the detection technique, because striated myofibrils were easily identified in neighboring, nontransfected myocytes (Fig. 1C). Also, this staining procedure visualizes striations in myocytes subjected to pacing of contractions, phenylephrine, or transfection with the p38 activator MKK6 (11, 15).

View larger version (93K): [in this window] [in a new window]   Fig. 1.   Effects of Ha-RasV12 on cell morphology and SERCA2 expression. A, control myocytes cotransfected with empty expression vector and GFP. Three separate myocytes are visualized. B, myocytes cotransfected with Ha-RasV12 and GFP. Four separate myocytes are visualized. C, Ha-RasV12-transfected myocyte stained for actin. Note that the transfected myocyte (arrow) occupies most of this field of view. Neighboring nontransfected myocytes are labeled with arrowheads. D, the same field as in C, viewed for GFP. E, SERCA2 expression in control myocytes. Cells transfected with control vector and the CMV--galactosidase were stained for -galactosidase and SERCA2 and visualized with a combination of Texas Red and fluorescein isothiocyanate-compatible optics; the transfected myocyte is red (arrow), whereas the nontransfected myocytes (arrowheads) are green. F, the same field as in E, illuminated to selectively visualize SERCA2. G, SERCA2 expression in Ha-RasV12-transfected myocytes. For G, the cells were illuminated and labeled as in E. H, the same field as in G, illuminated as in F. Original magnifications were 400× for A, B, E, F, G, and H and 1000× for C and D. Calibration bars represent 50 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effects of ERK effectors on [Ca2+]i. Myocytes were transfected with the indicated constructs and maintained for 48 h in serum-free medium prior to analysis. Traces obtained from individual myocytes paced at 0.3 Hz were aligned (electrical stimuli at 0.2 s) and averaged to yield a summary trace for each condition (for the number of cells examined for each condition refer to Table I). A, effects of Ha-RasV12 on [Ca2+]i. Calcium transients measured in cells transfected with control vector (16 µg/transfection) or Ha-RasV12. B, the transients in A normalized to their respective maxima and minima. C, effects of Ha-RasV12S35 and Ha-RasV12C40 on [Ca2+]i (16 µg/transfection). D, the transients in C normalized. E, effects of BXB-Raf on [Ca2+]i; myocytes were transfected with either 16 µg of control vector or 16 µg of BXBRaf. F, The transients in E normalized.

Effects of Ha-Ras^V12S35 and Ha-Ras^V12C40 on ERK Activation and [Ca²⁺]_i-- Raf and phosphatidylinositol 3-OH kinase (PI 3-kinase) are among the best known Ras effectors in mammalian cells. To distinguish between these pathways, Ha-Ras^V12 mutants have been identified that selectively couple to Raf (Ha-Ras^V12S35) or PI 3-kinase (Ha-Ras^V12C40) (16, 17). As a first step toward evaluating the role of these effector pathways in the growth and [Ca²⁺]_i responses to Ha-Ras^V12, the differential coupling of Ha-Ras^V12S35 and Ha-Ras^V12C40 to the Raf-MEK-ERK pathway was verified; to do this, myocytes were cotransfected with hemaglutinin-ERK2 along with Ha-Ras^V12, Ha-Ras^V12S35, or Ha-Ras^V12C40, and in vitro kinase assays were performed on hemaglutinin-ERK2-specific immunoprecipitations. Cotransfection with Ha-Ras^V12 resulted in a strong activation of ERK (17-fold), whereas transfection with Ha-Ras^V12S35 yielded a moderate activation (2.5-fold). In contrast, transfection with Ha-Ras^V12C40 did not activate ERK, at all (0.8-fold). Thus, although Ha-Ras^V12S35 specifically activates the Raf-MEK-ERK pathway, it is a less effective stimulator of ERK activation than Ha-Ras^V12; this result is consistent with other reports (17), and should be considered when interpreting the effects of these Ras mutants on cell growth and [Ca²⁺]_i.

Effects of BXB-Raf and MEKK1_COOH on [Ca²⁺]_i-- Because Raf links Ras to ERK activation, it was of interest to test the effects of constitutively active Raf (BXB-Raf) on [Ca²⁺]_i. BXB-Raf transfection results in substantial activation of ERK and cell enlargement but does not increase myofilament expression or organization (11). BXB-Raf-transfected myocytes exhibited normal R_dia but displayed reduced R_sys, increased t_peak, and prolonged decay (p < 0.05 for all parameters, data pooled from three separate cell preparations; Fig. 2, E and F, and Table I). The alterations of transient kinetics by BXB-Raf are consistent with the results obtained with Ha-Ras^V12 and Ha-Ras^V12S35 and show striking similarities to the contractile defects induced by pressure overload hypertrophy (5, 6).

dnMEK Inhibits the Effects of Ras, Raf, and Protein Kinase C on [Ca²⁺]_i-- As a further test of the involvement of the MEK-ERK pathway in the regulation of cardiac [Ca²⁺]_i by Ras and Raf, the effects of cotransfection with dnMEK on [Ca²⁺]_i were tested. Cotransfection of dnMEK with Ha-Ras^V12 strongly corrected the effect of Ha-Ras^V12 on R_sys, returning R_sys to near normal values, and partially inhibited the effect of Ha-Ras^V12 on transient decay (Fig. 3, A and B, and Table I). The ability of dnMEK to antagonize the effects of Ha-Ras^V12 is consistent with the hypothesis that Ha-Ras^V12 alters [Ca²⁺]_i via the Raf-MEK-ERK pathway. Consistent with the relative position of Raf and MEK in the ERK activation cascade, cotransfection of dnMEK also strongly inhibited the effects of BXB-Raf on [Ca²⁺]_i (Fig. 3, C and D), returning the transients to normal kinetics.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effects of dnMEK on [Ca2+]i. Data were obtained and averaged as described in the legend to Fig. 2. A, dnMEK versus Ras. Myocytes were transfected with 1 µg of Ha-RasV12, 45 µg of dnMEK, the combination of Ha-RasV12 + dnMEK, or 46 µg of control vector and maintained for 48 h in serum-free medium prior to analysis. B, the transients in A normalized. C, dnMEK versus Raf. Myocytes were transfected with 61 µg of control vector, 16 µg of BXB-Raf, 45 µg of dnMEK, or the combination of BXBRaf + dnMEK and maintained for 48 h in serum-free medium prior to analysis. D, the transients in C normalized. E, dnMEK versus PKC. Myocytes were transfected with 32 µg of control vector or 32 µg of dnMEK and then exposed to serum-free medium or medium + 300 nM of PKC-activating phorbol ester (PMA) for 72 h prior to analysis of indo-1 fluorescence. F, the transients in E normalized.

Expression of SERCA2 Inhibits the Effects of Ha-Ras^V12 and BXB-Raf Transfection-- Because transfection with Ha-Ras^V12 leads to down-regulation of SERCA2 expression and alteration of [Ca²⁺]_i, it was of interest to monitor [Ca²⁺]_i handling in myocytes cotransfected with expression vectors for both Ha-Ras^V12 and for SERCA2. Coexpression of SERCA2 with Ha-Ras^V12 resulted in partial but not complete restoration of the calcium transients (Fig. 4, A and B, and Table I). For example, cotransfection of SERCA2 did not counteract the effect of Ha-Ras^V12 on R_dia but significantly counteracted the effects of Ha-Ras^V12 on R_sys and t_peak; SERCA2 also exhibited a trend toward correcting the effect of Ha-Ras^V12 on t_decay. SERCA2 transfection, by itself, had no effect on cell morphology and did not alter the effect of Ha-Ras^V12 on cell morphology. Cotransfection with SERCA2 also strongly corrected the effect of BXB-Raf on [Ca²⁺]_i (Fig. 4, C and D, and Table I). These results are consistent with the hypothesis that the Ras-Raf-MEK-ERK pathway may alter [Ca²⁺]_i, in part, by regulating SERCA2 expression. However, the inability of SERCA2 cotransfection to completely restore the alterations in [Ca²⁺]_i induced by Ha-Ras^V12 suggest that Ha-Ras^V12 activity might also alter [Ca²⁺]_i by additional pathways that are independent of SERCA2.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effects of SERCA2 expression on [Ca2+]i. Data were obtained as in Fig. 2. A, myocytes were transfected with 48 µg of control vector, 16 µg of Ha-RasV12, 32 µg of the SERCA2 expression plasmid, or the combination of Ha-RasV12 and SERCA2 and maintained in serum-free medium for 48 h prior to analysis. B, the transients in A normalized. C, myocytes were transfected with 32 µg of control vector, 16 µg of BXB-Raf, 16 µg of the SERCA2 expression plasmid, or the combination of BXB-Raf and SERCA2 and maintained in serum-free medium for 72 h prior to analysis. D, The transients in C normalized.

	DISCUSSION

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In the present study cultured neonatal rat ventricular myocytes were transiently transfected with effectors of the Ras-Raf-MEK-ERK and related pathways, along with "transfection marker" constructs allowing the transfected myocytes to be identified and analyzed for cell size and morphology. Additionally, a novel technique was developed in which GFP was used as a transfection marker for living myocytes; this provided the opportunity to analyze the effects of the second messenger effectors on [Ca²⁺]_i regulation, a physiological parameter of extreme importance to cardiac function in health and disease. The data obtained are generally consistent with earlier reports demonstrating that microinjection of Ha-Ras^V12 into cardiac myocytes elicits a strong growth response and hypertrophic gene expression (2), and the cardiac-targeted expression of Ha-Ras^V12 in transgenic mice increases heart size concomitant with a decrease in myofibrillar organization (3, 23). Indeed, in the present study, transfection with Ha-Ras^V12 elicited an extreme increase in cell size along with loss of the contractile apparatus, a morphological phenotype resembling hypertrophic cardiomyopathy (24, 25).

Transfection with Ha-Ras^V12 also altered expression of SERCA2, a calcium-regulatory enzyme involved in calcium reuptake during the decay phase of the contractile calcium transients, which is also commonly down-regulated in whole animal models of pressure overload hypertrophy and failure (1, 5, 6). Indo-1 measurements of [Ca²⁺]_i further revealed that Ha-Ras^V12 transfection severely compromised the magnitude of the contractile calcium transients and prolonged the decay phase of the transients, a calcium mobilization phenotype that is consistent with the effect of Ha-Ras^V12 to diminish SERCA2.

To investigate the potential effector pathways via which Ha-Ras^V12 alters [Ca²⁺]_i, expression plasmids encoding Ras mutants that selectively couple to Raf-MEK-ERK (Ha-Ras^V12S35) and PI 3-kinase (Ha-Ras^V12C40) pathways were employed. Ha-Ras^V12S35 altered [Ca²⁺]_i and cell size in a manner more consistent with Ha-Ras^V12 than did Ha-Ras^V12C40, suggesting that the Raf-MEK-ERK pathway is more involved in the growth and calcium disregulation responses to Ha-Ras^V12 than PI 3-kinase. The effect of Ha-Ras^V12S35 on [Ca²⁺]_i and cell size was less than that of Ha-Ras^V12, however, indicating that Ras effectors other than Raf may also play a role in these responses. Another factor to be considered, though, is the degree to which Ha-Ras^V12 and Ha-Ras^V12S35 activate ERK. Although Ha-Ras^V12S35 is selective for the ERK pathway, it is not as effective a stimulator of ERK as Ha-Ras^V12. Therefore the lesser effect of Ha-Ras^V12S35 on [Ca²⁺]_i compared with Ha-Ras^V12 might relate to the lesser stimulatory effect of Ha-Ras^V12S35 on ERK activity.

Transfection with a constitutively active Raf construct (BXB-Raf) also strongly modified [Ca²⁺]_i, generally reducing the magnitude of the transients, slowing the time to reach the peak magnitude, and strongly prolonging transient decay, which are alterations in [Ca²⁺]_i handling commonly observed in myocytes isolated from hypertrophic and failing hearts (5, 6). Notably, the effects of both Ha-Ras^V12 and BXB-Raf were strongly antagonized by cotransfection with dnMEK, further implicating the Raf-MEK-ERK pathway as a possible route for the disregulation of cardiac [Ca²⁺]_i. On the other hand, cotransfection with dnMEK did not fully correct the effects of Ha-Ras^V12 on [Ca²⁺]_i; this suggests that Ras effectors other than Raf may also modify [Ca²⁺]_i, a hypothesis that will be the subject of further investigation.

Importantly, transfection with dnMEK also inhibited the effect of the PKC-activating phorbol ester, PMA, on [Ca²⁺]_i. Additionally, cotransfection with a dominant-negative Raf construct (C4BRaf) inhibited the ability of a constitutively active protein kinase C construct (PKAC) to alter [Ca²⁺]_i. These data indicate that the Raf-MEK-ERK cascade represents a point of convergence via which hypertrophic stimuli that activate Ras and/or PKC lead to diminishment and prolongation of the cardiac contractile calcium transients. PKC might activate Raf by directly phosphorylating it (10, 26), or, alternatively, it has been proposed that PKC can activate Ras (27), which would also subsequently lead to Raf and ERK activation. PMA is also known to have hypertrophic effects on cardiac myocytes (28) and to stimulate the disorganization and breakdown of the contractile apparatus in cultured adult rat myocytes (29) resulting in a morphological phenotype similar to the effects of Ha-Ras^V12 transfection.

Previous studies have demonstrated two mechanisms via which ERK activation might regulate SERCA2 expression. Ha-Ras^V12 transfection leads to inhibition of the rat SERCA2 promoter (30), suggesting a control of SERCA2 expression at the transcriptional level. Additionally, SERCA2 mRNA stability is reduced by phorbol esters (8). PMA, in conjunction with ionomycin, also down-regulates other SERCA isoforms in lymphocytes (31), suggesting that the regulation of SERCA by PKC might be a general strategy for [Ca²⁺]_i regulation in both cardiac and noncardiac cell types. The observation in the present study that cotransfection with SERCA2 tends to compensate for the effects of Ha-Ras^V12 and BXBRaf on [Ca²⁺]_i is consistent with previous studies, demonstrating that infection of cultured neonatal ventricular myocytes with a SERCA2-expressing adenovirus acts to correct the effects of PMA on [Ca²⁺]_i (32).

SERCA2 down-regulation may not be the only means by which the [Ca²⁺]_i is altered in hypertrophic myocytes. For example, prolongation of relaxation occurs in pressure overload rats prior to the reduction in SERCA2 levels (33), and calcium transients may be reduced in cardiac myocytes obtained from hypertrophic hearts under conditions in which the SR is well loaded with calcium (34). It has been suggested that impaired calcium-induced calcium release occurs in hypertrophic myocytes because of increased distance between voltage-dependent calcium channels and ryanodine receptors at the T-tubule-SR dyads (34). Because Ha-Ras^V12 transfection down-regulates myofibrillar expression and the SR is closely associated with myofibrils (35), there is a strong possibility Ha-Ras^V12 transfection might alter SR structure and function. Further analysis of cellular ultrastructure will be needed to resolve this question.

Because excessive ERK activation may prove deleterious to the maintenance of the cardiac phenotype, it is interesting to consider the role of the related MAPKs JNK and p38 in cardiac gene expression. Recent results suggest that JNK activity in cultured neonatal ventricular myocytes may be regulated by [Ca²⁺]_i and contractile activity (19) and that contractile activity and JNK activation represent positive regulators of cell growth and the contractile apparatus (15, 36). Transfection with constitutively active upstream regulators of p38 also dramatically increases cell growth, myofibril expression, and organization (11, 37) in a manner consistent with the compensatory phase of cardiac hypertrophy. Thus, MAPK family members may participate in the development and maintenance of the cardiac phenotype, and altered activity of these enzymes may lead to the development of both compensatory or potentially pathological changes in morphology and physiology. The striking ability of dnMEK to inhibit the effects of both Ha-Ras^V12 and PMA on cardiac [Ca²⁺]_i suggests that blockade of the Raf-MEK-ERK cascade via gene transfer or other techniques could conceivably be an effective therapeutic strategy in treating the contractile defects associated with cardiac hypertrophy and failure.