A Role for the p38 Mitogen-activated Protein Kinase Pathway in Myocardial Cell Growth, Sarcomeric Organization, and Cardiac-specific Gene Expression

Primary ventricular myocytes were prepared from 1–4-d-old neonatal rats as previously described (Sprenkle et al., 1995; Thuerauf and Glembotski, 1997). After the enzymatic dissociation of ventricular tissue, the cells were plated onto uncoated plastic dishes in DME/F-12 (1:1)/10% FBS for 1 h during which time most of the fibroblasts adhered to the dish. The recovered cells were then transfected (see below), plated on fibronectin-coated plastic dishes or glass slides (3 × 10⁵ cells/mm²), and then maintained for about 18 h in DME/F-12 (1:1)/10% FBS. The cultures were then washed briefly with medium, refed with serum-free DME/F-12 (1:1), maintained for 48 h, and then either extracted for reporter enzyme assay (see below) or fixed and then analyzed by immunocytofluorescence or staining with fluorescent phallicidin.

After preplating (see above), myocardial cells were resuspended at a density of 30 million cells/ml minimal medium (DMEM:F12 [GIBCO BRL, Gaithersburg, MD] containing 1 μg/ml BSA) and transfections were carried out as described previously (Sprenkle et al., 1995; Thuerauf and Glembotski, 1997). Briefly, for each transfection, 300 μl, or 9 million cells, were mixed with 15–30 μg of either ANP-3003GL (Sprenkle et al., 1995), BNP-2501GL (Thuerauf and Glembotski, 1997), α-SkA-394GL (MacLellan et al., 1994), or pG5E1bLuc (Raingeaud et al., 1996); 3–9 μg of cytomegalovirus–β-galactosidase (used as a normalization reporter except where the test construct is known to activate CMV promoter activity [Gillespie-Brown et al., 1995; Paradis et al., 1996; Post et al., 1996]); and in some experiments, 15–45 μg of an activated Ras, Rac, Raf-1, JNKKK, MKK6, activating transcription factor-2 (ATF2)/Gal4, or MEF2C/Gal4 expression construct (see below). The levels of plasmid used in each culture within an experiment were equalized using empty vector DNA, such as pCEP. Each 300-μl aliquot was then electroporated in a Bio-Rad (Hercules, CA) Gene Pulser at 700 V, 25 μF, 100 Ω in a 0.2-cm-gap cuvette, a protocol that allows for the selective transfection of only cardiac myocytes (Sprenkle et al., 1995). This procedure results in an ∼30% viability (Sprenkle et al., 1995); Accordingly, the 3 million viable cells were plated into fibronectin-coated 35-mm wells, at 1 × 10⁶ cells/well, into 24-mm wells at 0.5 × 10⁶ cells/well, or into four-chamber Lab Tek chamber slides at 0.3 × 10⁶ cells/2 cm² well.

Transfected cells were maintained in DMEM:F12 supplemented with 10% FBS for ∼16 h after electroporation. The cells were then washed thoroughly and the medium was replaced with minimal medium. Unless otherwise stated, 24 h later, the medium was again replaced with minimal medium ± 50 μM phenylephrine with 1 μM propranolol added to block β-adrenergic receptors. Luciferase and β-galactosidase assays were performed as described (Sprenkle et al., 1995). Luciferase activity was measured for 30 s on a Bio Orbit 1251 Luminometer (Pharmacia Biotech. Inc., Piscataway, NJ). Data are expressed as “Relative Luciferase (Rel Luc)” = arbitrary integrated luciferase units/β-galactosidase units, representative of at least three independent experiments performed with two different plasmid preparations, and represent the mean and SEM of triplicate 35- or 24-mm wells.

To assess the effects of various signaling proteins, the following constructs were used: pDCR H-Ras^V12 (codes for activated Ha-Ras; from D. Bar-Sagi, State University of New York at Stony Brook, NY), pDCR Rac^V12 (codes for activated Rac; from M. Cobb and J. Frost, University of Texas Southwestern Medical Center, Dallas, TX), RSV-Raf-1 BXB (codes for activated Raf-1 kinase; from U. Rapp, University of Wurzburg, Wurzburg, Germany), pCMV5 MEKK_COOH (codes for activated MEKK-1; from G. Johnson, University of Colorado, Denver, CO), pcDNA3 MKK6 (Glu) (codes for activated MKK6, or p38/MAPKK; from R. Davis, University of Massachusetts, Worcester MA), ATF2/Gal4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain; from R. Davis), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA– binding domain; from J. Han, The Scripps Research Institute, La Jolla, CA), MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4 DNA–binding domain; from J. Han), pG5E1bLuc (codes for 5X Gal4 sites cloned upstream of a prolactin promoter driving luciferase expression; from R. Davis). Preliminary experiments using different concentrations of each construct verified that optimal doses were chosen.

To study the effects of activating the MAPK pathways on cell size and sarcomeric organization, myocardial cells were cotransfected with a test expression construct or an empty vector control and CMV–β-galactosidase. Double-staining experiments demonstrated a cotransfection efficiency of ∼85%. After treatment for 48 h with or without PE, cultures were fixed, as described (McDonough and Glembotski, 1992). Transfected cardiac myocytes were identified by immunostaining for β-galactosidase using a Texas red–conjugated anti–mouse IgG. Since the β-galactosidase is cytosolic, staining was uniform throughout the myocardial cells, facilitating the determination of cell area (see below). The same samples were also stained with BODIPY-conjugated phallicidin, an actin-specific stain, to evaluate sarcomeric organization, as described (McDonough and Glembotski, 1992).

To study the effects of the test expression constructs on endogenous cardiac gene expression, myocardial cells were cotransfected with a test expression construct or an empty vector control and ANP-3003GL. After fixation, the cells were immunostained for luciferase using a rabbit anti– luciferase antiserum, and they were immunostained for ANP using a mouse monoclonal antibody to rat ANP (Glembotski et al., 1987) and visualized using differential fluorescence. Generally, positive ANP staining was visualized using a Texas red–conjugated anti–mouse IgG, and positive luciferase staining was visualized using an FITC-conjugated anti–rabbit IgG.

Transfected myocytes that immunostained positively for β-galactosidase were microscopically visualized under fluorescent illumination and photographed. The photographic images were then digitally acquired using a scanner (model ES-1200C; Epson America, Inc., Torrance, CA) attached to a Apple Power Mac 8500 (Cupertino, CA). The area in pixels of each digitized image was determined using NIH Image software and compared to a standard image possessing an area of 1 μm². This enabled the designation of area, in square micrometers, to each cell image; between 20 and 50 images of different cells derived from each treatment were analyzed. The values reported are the mean areas, in square micrometers ± standard error.

Transfected myocytes were identified by positive β-galactosidase immunostaining and observed using a Texas red– compatible filter. The cells were then viewed after phalloidin staining using an FITC-compatible filter and scored positively for sarcomeric organization if the myofilament alignment resembled that in cells treated with PE. Approximately 50–100 cells from each of three cultures per treatment were assessed. Generally, treating CMV–β-galactosidase/pCEP-transfected cells with PE (positive control) or without (negative control) resulted in ∼25 and 2% of the β-galactosidase–positive cells scoring for sarcomeric organization, respectively. In experimental cultures transfected with CMV–β-galactosidase and a test construct, the number of cells scoring positive for sarcomeres was normalized to (divided by) the number of cells scoring positive for sarcomeres obtained with PE, which gave maximal values, and the results are displayed as percentages of maximal values.

To test the effects of treatments on MAPK activity levels, hemagglutinin (HA)-tagged forms of ERK1 (pCEP4 HA-wt-ERK1 from M. Cobb), JNK (SRa-HA-JNK from G. Johnson) or p38 (pCEP4 HA-wt-p38Hog1 from M. Cobb) were cotransfected with the test constructs. After the appropriate times, cultures were extracted in a buffer containing 10 mM Tris, pH 7.6, 1% Triton X-100, 0.05 M NaCl, 5 mM EDTA, 2 mM o-vanadate, and 20 μg/ml aprotinin. After brief centrifugation, extracts were incubated for 2 h at 4°C with HA monoclonal antibody (12CA5; Boehringer-Mannheim Corp., Indiannapolis, IN), bound to protein A–Sepharose (Pharmacia Biotech, Inc.) and immune-complex kinase assays were carried out using the appropriate substrates, as described (Derijard et al., 1994; Post et al., 1996). Briefly, reactions were initiated by the addition of 1 μg of the appropriate substrate, MBP for ERK, GST–c-Jun for JNK, Phas-I for p38, and 6 μM [γ-³²P]ATP (5,000 Ci/mmol) in a final volume of 30 μl of kinase buffer (20 mM Hepes, pH 7.4, 20 mM MgCl₂, 20 mM β-glycerophosphate, 2 mM DTT, 20 μM ATP). After 30 min at 25°C, the reactions were terminated by the addition of Laemmli sample buffer, and the phosphorylation level of substrate proteins was evaluated by SDS-PAGE followed by autoradiography and phosphorimage analyses.

In each experiment, two identically treated cultures (1.5 × 10⁶ cells/35-mm dish) were used for each treatment, and after densitometric analyses of the exposed phosphorimage plates, values for each treatment were averaged.

To characterize the effects of overexpressing various signaling proteins on each of the three MAPK family members in the cardiac context, myocardial cells were cotransfected with constructs encoding gain-of-function forms of Ras, Rac, Raf-1 kinase, JNKK kinase, or p38 kinase, and constructs encoding HA-tagged p38, JNK, or ERK. In the cardiac myocytes, Ras V12 served as a poor activator of either p38 or JNK, but as expected, it was a strong activator of ERK (Fig. 1). Rac V12 had no effect on p38 or ERK in the cardiac cells; however, it strongly activated JNK, consistent with its hypothesized ability to serve as an upstream activator of MEKK1 (Lange-Carter et al., 1993; Derijard et al., 1994). Raf BXB, which encodes an active form of Raf-1 kinase (Bruder et al., 1992; Kolch et al., 1993), served primarily as an ERK activator, while MEKK_COOH, a truncated active form of MEKK1 (Lange-Carter et al., 1993), mildly activated p38 by about fourfold, moderately activated JNK by 8–10-fold, as expected, but more strongly stimulated ERK by about 25-fold (Fig. 1). The ability of MEKK_COOH to activate both JNK and ERK is consistent with results in other cell types (Minden et al., 1994). Importantly, however, MKK6 (Glu), an activated form of the p38 kinase, MKK6 (Raingeaud et al., 1996), potently activated p38 in the cardiac myocytes by about 16-fold, with no effect on either ERK or JNK (Fig. 1). A kinase-dead form of MKK6, MKK6 (K82A) (Raingeaud et al., 1996), did not activate p38 in the cardiac cells (not shown). These results verify the utility of constructs encoding activated forms of Raf, MEKK1, and MKK6 as stimulators of the MAPK pathways, and in particular, they clearly show that MKK6 serves as a very selective p38 activator in cardiac myocytes.

The abilities of the various expression constructs to activate three cardiac genes (ANP, BNP, and α-SkA) that serve as hallmarks of the hypertrophic growth program were tested using ANP-3003GL, BNP-2501GL, or α-SkA– 394GL. These reporter constructs possess 3,003, 2,501, or 394 bp of the ANP, BNP, or α-SkA 5′-flanking sequences, respectively. As expected from previous studies (Thorburn et al., 1993; MacLellan et al., 1994; Thuerauf and Glembotski, 1997), Ras V12 served as a strong activator of both natriuretic peptide (NP) promoters, fostering up to 50-fold activation of luciferase expression (Fig. 2). The Rac V12 construct also activated these promoters, but less strongly than Ras, ∼10-fold; this may reflect the differential efficacies of ERK and JNK as inducers of the cardiac genes studied. Although Raf BXB and MEKK_COOH stimulated NP and α-SkA promoter activities by up to 20-fold, most notable were the effects of the p38-activating construct, MKK6 (Glu), which stimulated up to 130-fold (Fig. 2). These findings suggest that while each of the MAPK pathways can stimulate cardiac natriuretic peptide and α-SkA gene expression, the p38 pathway as stimulated with MKK6 (Glu) confers the strongest induction of the three genes studied.

Further studies were undertaken to compare the effects of Raf BXB, MEKK_COOH, and MKK6 (Glu) with the gold standard, PE, on other features of the program, such as cell size and sarcomeric organization. Compared to cells maintained in control media (Fig. 3, A and A′), the PE-treated cells were much larger (Fig. 3,B), displaying an approximately two- to three-fold increase in area (Fig. 4,A), and they possessed a high degree of sarcomeric organization (Fig. 3,B′). In general, the PE-treated cultures possessed about 10-fold more myocytes displaying organized sarcomeres than the control cultures (Fig. 4,B). PE-treated cultures also displayed significantly increased levels of endogenous ANP expression, observed as the prototypical perinuclear staining found often in hypertrophic cardiac myocytes (Fig. 3, F [control] vs. G [PE-treated]). Interestingly, cultures transfected with Raf BXB or MEKK_COOH displayed increases in size (Figs. 3, C [BXB] and D [MEKK_COOH], and 4 A), and while the usual shape of the Raf BXB–treated cells was similar to PE-treated cells, the MEKK_COOH-treated cells were almost always very long and thin. Moreover, while either Raf BXB (Fig. 3,H [BXB]) or MEKK_COOH (Fig. 3,I [MEKK-1]) fostered the induction of endogenous ANP expression, neither construct supported sarcomeric organization (Fig. 3,C′ [BXB] and 3 D′ [MEKK-1]; also see Fig. 4 B). These results suggested that neither ERK (Raf BXB) alone nor ERK and JNK (MEKK_COOH) were sufficient to confer all the features of the hypertrophic phenotype.

When myocardial cells were transfected with MKK6 (Glu), they were on average four times larger than control cells (Figs. 3, E and 4,A), and notably, they displayed sarcomeric organization that was visually similar to that observed upon PE treatment (Figs. 3,E′ and 4 B). Consistent with the high degree of sarcomeric ordering was the finding that cells transfected with MKK6 (Glu) displayed spontaneous contractile activity. Moreover, like PE-treated cells, MKK6 (Glu)–transfected myocardial cells possessed significantly elevated levels of endogenous ANP (Fig. 3,J), consistent with the ability of MKK6 (Glu) to strongly activate NP promoter activities (Fig. 2). Thus, it was apparent that MKK6 (Glu), a selective p38 activator in the cardiac myocytes, was able to mimic the three hallmark features of the hypertrophic growth program.

To demonstrate that the effects of MKK6 (Glu) and PE on myocardial cell growth and gene expression involved p38, cultures were treated with the highly specific p38 inhibitor, SB 203580 (Young et al., 1993). At 20 μM, SB 203580 has been shown to block p38/MAPK, while concentrations as high as 100 μM have been shown to have no effect on 20 other protein kinases tested, including ERK and JNK (Cuenda et al., 1995). In the present study, SB 203580 (20 μM) blocked PE and MKK6 (Glu)–inducible NP promoter activity by between 40 and 70% (Fig. 5, A and B) and decreased MKK6 (Glu)–activated α-SkA by >90% (not shown). Additionally, myocardial cell p38 was shown to be activated by PE, as was ERK; however, JNK was not stimulated under these conditions (Fig. 6). Taken together, these results confirmed a central role for p38 in MKK6 (Glu) induction of cardiac gene expression and strongly suggest that the ability of PE to induce NP expression is at least partly due to the MKK6/p38 pathway.

The stress-activated MAPK pathways, especially p38, are well–known stimulators of ATF2 (Gupta et al., 1995; Raingeaud et al., 1996). ATF2 can dimerize with other ATF family members (e.g., cAMP response element–binding protein [CREB] or ATF-1), Rb, NF-κB, or c-jun and enhance transcription through cAMP response elements, AP-1 sites, or NF-κB sites. Accordingly, the abilities of MKK6 (Glu) or PE to activate ATF2-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase, a construct encoding a fusion protein composed of the ATF2 transactivation domain and the GAL4 DNA–binding domain, and either MKK6 (Glu) or pCEP. Transfection with MKK6 (Glu) or treatment pCEP-transfected cells with PE enhanced ATF2-mediated luciferase production (Fig. 5,C). Moreover, SB 203580 served as a potent inhibitor of MKK6 (Glu)–activated ATF2, consistent with a major requirement for p38. Interestingly, PE-activated ATF2 was only partially blocked by SB 203580 (Fig. 5,C), as was PE-activated BNP transcription (Fig. 5 B), suggesting that the ability of this agonist to activate cardiac gene expression is partly dependent on p38, or a very similar kinase.

The SB 203580 compound also had potent inhibitory effects on myocardial cell size and sarcomeric organization induced by PE or MKK6 (Glu) (Fig. 7). PE- or MKK6 (Glu)–mediated sarcomeric organization and increases in cell size were reduced by >90% by SB 203580 (Fig. 8). These results are consistent with a central role for a p38-like pathway in myocardial cell growth conferred by PE or by MKK6 (Glu).

Recent studies have established that the transcription factor, MEF2C, serves as a specific substrate for p38, such that phosphorylation on serine 387 confers MEF2C-mediated transcriptional activation in RAW 264.1 cells (Han et al., 1997). Accordingly, the abilities of PE or MKK6 (Glu) to activate MEF2C-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA–binding sites cloned upstream of luciferase and a construct encoding a fusion protein comprised of the MEF2C transactivation domain and the GAL4 DNA–binding domain. Using this system, the activation of MEF2C after phosphorylation on serine 387 can be studied in the cardiac myocytes without interference from any endogenous MEF2 family members. Treatment with PE or transfection with MKK6 (Glu)–enhanced MEF2C-mediated luciferase production (Fig. 9, MEF2C/Gal4). When cells were transfected with an altered MEF2C/Gal4 chimera, in which serine 387 was mutated to alanine, neither PE nor MKK6 (Glu) conferred luciferase induction (Fig. 9, MEF2C-S/Gal4). These results indicate that like MKK6 (Glu), PE can activate MEF2C in cardiac myocytes, an event that requires phosphorylation at serine 387 by p38. This result further supports the notion that in part, PE enhances myocardial cell growth, sarcomeric organization, and the related gene expression through a pathway involving p38 or a very similar kinase.

Under certain conditions, cardiac myocytes undergo nonmitotic, hypertrophic growth that is typified by dramatic increases in cell size, high degrees of sarcomeric organization, and enhanced expression of certain cardiac-specific genes. Several results from this study indicate that MKK6-activated p38 is sufficient to confer the three main features of this unique hypertrophic growth program. First, MKK6 (Glu), which amongst the MAPKs induces only p38 (Fig. 1 and Raingeaud et al., 1996), conferred sarcomeric organization, increased cell size, and increased cardiac gene expression in a manner similar to the well-characterized α₁-adrenergic receptor agonist, PE. Second, when induced by MKK6, all three features of the growth program could be blocked by the p38-specific inhibitor, SB 203580. Interestingly, PE-enhanced sarcomeric organization, cell size, and, to some extent, cardiac gene induction were also blocked by SB 203580, indicating that the p38 pathway probably plays an important role in α₁-adrenergic receptor signaling in myocardial cells. However, since the effects of PE on BNP transcription and ATF2-enhanced transcription were only partially blocked by SB 203580, it appears that while p38 may be central to some features of the hypertrophic response, it may play only a partial role in mediating other aspects of the growth program.

To our knowledge, this is the first report to document that the activation of the p38/MAPK pathway can mimic the morphological changes and gene inductive effects of growth factor treatment in any cell type. Thus, while p38/ MAPK is known as being a stress-activated kinase, it can apparently contribute to cell growth in a manner that may represent a compensatory response to stress. Although such a role for p38 contrasts with earlier findings that p38 induces apoptosis (Xia et al., 1995), it is consistent with recent studies indicating that this stress kinase can promote survival in certain cell types (Juo et al., 1997); in this respect, p38 appears to function in a cell-specific manner.

The mechanism by which MKK6-mediated p38 activation could lead to myocardial cell hypertrophic growth remains to be elucidated; however, recent work has revealed several downstream p38 targets that could be involved. For example, p38 phosphorylates and activates several transcription factors, such as ATF2 and Elk-1 (Gupta et al., 1995; Livingstone et al., 1995; Raingeaud et al., 1995, 1996), which could augment the expression of cardiac-specific genes induced during hypertrophy. Many of these inducible genes are known to possess relevant cis-acting sequences, including serum response elements, cAMP response elements, AP-1 sites, and NF-κB sites. Most recently, it has been demonstrated that the muscle cell–enriched transcription factor, MEF2C, a MADS box protein known to bind to A/T-rich regions of muscle-specific genes and known to be required for proper growth and development of cardiac muscle (Edmondson et al., 1994; Olson and Srivastava, 1996), serves as a substrate for p38 but not ERK or JNK (Han et al., 1997). In that report, the p38-specific phosphorylation was shown to lead to the activation of MEF2C as a transcription factor. Both the rat ANP and BNP 5′-flanking regions, as well as regulatory regions of other genes induced during the hypertrophic growth program (e.g., α-skeletal actin and β-myosin heavy chain genes), contain A/T-rich regions that are required for transcriptional induction and could bind MEF2C or related proteins (MacLellan et al., 1994; Thuerauf et al., 1994; Karns et al., 1995; Sprenkle et al., 1995). Thus, it is possible that p38 could phosphorylate and activate transcription factors that augment the expression of genes that participate in the cardiac growth program.

Alternatively, p38 may activate other downstream kinases that serve as the final steps in the signaling program. For example, it is well known that p38 can phosphorylate and activate MAP kinase–activated protein kinases (MAPKAPs)-1, -2, and -3 (MAPKAP-3 a.k.a. 3pK) (Stokoe et al., 1992a; Young et al., 1993; Rouse et al., 1994; English et al., 1995; Ludwig et al., 1996; McLaughlin et al., 1996; Sithanandam et al., 1996; Tan et al., 1996). In response to p38 activation by growth factors, MAPKAPs have been shown to phosphorylate and activate selected transcription factors, such as CREB and ATF-1, usually at protein kinase A/CaMK consensus sequences (Tan et al., 1996). Thus, it is possible that via the MAPKAPs, myocardial cell p38 stimulation could culminate with the activation of transcription factors often thought of as being downstream of non-MAPKs, e.g., protein kinase A or CaMK.

In addition to altering the function of transcription factors, p38-mediated MAPKAP/3pK activation could modulate other pathways that might favor cell survival, and in cardiac myocytes, these pathways could contribute to the development of myocardial cell-specific features, such as myofilament organization. For example, MAPKAP-2 has been shown to be activated during ischemic preconditioning of isolated rat hearts (Bogoyevitch et al., 1996; Maulik et al., 1996). Such preconditioning is known to serve as a myocardial stress adaptation, resulting in enhanced protection from ischemia-induced myocardial cell death (Murry et al., 1986; Parratt, 1994; Cumming et al., 1996; Gottlieb et al., 1996). In part, it is believed that this cardioprotection is derived from the induction and activation of heat-shock proteins (hsp's) 27 and 70 (Marber et al., 1993; Mestril et al., 1994; Parratt, 1994), both of which are known to protect cells from apoptosis (Mehlen et al., 1996; Samali and Cotter, 1996; Sharma et al., 1996). Interestingly, MAPKAP-2 and -3 have been shown to phosphorylate hsp 27, a modification known to enhance its protective properties (Ahlers et al., 1994; Huot et al., 1995). Moreover, after phosphorylation induced by either heat-shock or mitogen stimulation, hsp 27 has been shown to bind to and stabilize actin filaments in mouse fibroblasts (Lavoie et al., 1993). Such hsp 27–mediated filament stabilization in cardiac myocytes could be a major contributor to the striking sarcomeric organization observed upon MKK6-mediated p38 activation. Intracellular signaling pathways leading to hsp activation and/or phosphorylation in cardiac myocytes could conceivably extend back to α₁-adrenergic receptors. Indeed, adrenergic receptor stimulation has been shown to activate hsp's in a variety of cell types; most notable is the finding that α₁-adrenergic agonists activate hsp 70 in rat aortic cells (Chin et al., 1996) and in rat cardiac cells (Meng et al., 1996).

In summary, our results demonstrate a role for MKK6 and p38 in myocardial cell hypertrophic growth and gene expression. This is consistent with the view that the hypertrophic growth program represents a compensatory response of the myocardium to stress. In a physiological context, the mediators of the hypertrophic response are often hemodynamic stresses, such as increases in blood pressure or volume. Accordingly, increases in myocardial cell size and contractile function afforded by such growth could be viewed as cellular adaptations designed to counteract a physiological stress. Indeed, induction of the cardiac natriuretic peptide genes, which encode hormones that decrease blood pressure and volume, represent an endocrine compensatory response by the myocardium. The findings that the p38 pathway can mediate all three primary features of the myocardial cell growth program represent a major advancement in our understanding of the signals that regulate this important induction process. Future studies aimed at determining how other signaling pathways, perhaps even the other MAPK pathways, complement the p38 pathway and how p38 itself contributes to myocyte growth, sarcomeric organization, and cardiac gene induction, should reveal new roles for this interesting stress-kinase pathway in the heart.

This work was supported in part by National Institutes of Health Grants NS/HL-25073 (C.C. Glembotski), HL-46345 (C.C. Glembotski), HL-56861 (C.C. Glembotski) and HL-54030 (P.M. McDonough). This work was done during the tenure of a predoctoral research fellowship from the American Heart Association, California Affiliate, awarded to D.S. Hanford.

α-SkA

α-skeletal actin

ANP and BNP

A- and B-type cardiac natriuretic peptides

ATF

activating transcription factor

CMV

cytomegalovirus

ERK

extracellular signal–regulated kinase

HA

hemagglutinin

hsp

heat-shock protein

JNK

NH₂-terminal kinase

MAPK

mitogen-activated protein kinase

MAPKAP

MAPK-activated protein kinase

PE

phenylephrine