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. 1997 Oct 6;139(1):115-27.
doi: 10.1083/jcb.139.1.115.

A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression

D Zechner  1 , D J Thuerauf, D S Hanford, P M McDonough, C C Glembotski
Affiliations

A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression

D Zechner et al. J Cell Biol. .

Abstract

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

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Figures

Figure 1
Figure 1
Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and either HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (∼3 × 106 cells each) were extracted and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of each panel). The relative density of each band was determined using Molecular Dynamics Image Quant software (Sunnyvale, CA). Each treatment was carried out on two identical cultures, and the average of the band density for each treatment was then normalized to the maximal value obtained in each experiment. Shown is the percentage of the maximum; the average variation between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.
Figure 2
Figure 2
Effects of Ras, Rac, and MAP kinase pathway expression constructs on cardiac-specific promoter activities in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac V12), Raf (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and either an ANP (ANP-3003GL), BNP (BNP-2501GL), or α-SkA (α-SkA-394GL) promoter/luciferase reporter construct. These reporter constructs contain either the full-length, 3,003 bp of rat ANP 5′-flanking sequence, the full-length, 2,501 bp of rat BNP 5′-flanking sequence, or −394 bp of the rat α-SkA 5′-flanking sequence driving the expression of a luciferase reporter. All cultures were also transfected with CMV-β-galactosidase. After a 48-h incubation in serum-free media, the cultures were extracted, and luciferase and β-galactosidase enzyme activities were assessed, as described in the Materials and Methods. Values for luciferase enzyme units obtained with each treatment were normalized to the maxima. Values are means ± SE, n = 3 cultures. In this experiment, luciferase values were not normalized to β-galactosidase since one of the constructs, MEKKCOOH, is a strong inducer of CMV-driven reporter expression, and it is believed that such normalization can be misleading, as previously reported (Gillespie-Brown et al., 1995; Paradis et al., 1996; Post et al., 1996).
Figure 3
Figure 3
Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf (Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV–β-galactosidase (A–E′) or ANP-3003GL (F–J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the same media containing 10 μM of the α1-adrenergic receptor agonist, phenylephrine (PE) + 1 μM propranolol (the latter to block potential binding to β-adrenergic receptors), cultures were fixed in paraformaldehyde. (A–E) β-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red–conjugated second antibody and photographed using a rhodamine-compatible filter. (A′–E′) Actin organization in the same β-galactosidase–positive cells shown in A–E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F–J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red–conjugated second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 μM.
Figure 4
Figure 4
Morphometric analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size and sarcomeric organization in myocardial cells. (A) Photographic images of myocardial cells transfected and treated as described in the legend to Fig. 3, A–E, were digitized and the areas (μm2) of ∼20–50 cells from each treatment were determined using NIH Image software, as described in the Materials and Methods. Shown are the mean area values for each treatment ± SE (n = 3 cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A′–E′, was evaluated using BODIPY-phalloidin to stain actin. Upon visually inspecting 50–100 transfected (i.e., β-galactosidase–positive) cells per treatment using a fluorescence microscope, cells were scored for possessing organized sarcomeres (i.e., appearing similar to cells shown in Fig. 3, B′ or E′). The number of cells transfected with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that had been transfected with the empty vector control; the results are shown as a percentage of these maximal values. Generally, ∼25% of the cells transfected with the empty vector control and then treated with PE displayed highly organized sarcomeres. Shown are the mean area values for each treatment ± SE (n = 3 cultures).
Figure 5
Figure 5
Effects of SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells were cotransfected with MKK6 (Glu) or an empty vector control (pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL, or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. Cultures were then maintained for 48 h with or without SB 203580 (20 μM) or with or without DMSO (vehicle control) and with or without PE (10 μM) + propranolol (1 μM), as shown and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to pCEP + PE. Values are means ± SE, n = 3 cultures.
Figure 6
Figure 6
Effects of PE on the activities of p38, JNK, and ERK in myocardial cells. Myocardial cells were treated for 30 min with or without PE (10 μM) + propranolol (1 μM) and then extracted and subjected to SDS-PAGE followed by Western analyses using antibodies (New England Biolabs, Inc., Beverly, MA) that detect p38, JNK, or ERK only when activated by dual phosphorylation on Thr180 and Tyr182 (following the manufacturer's protocols). Developed blots were then analyzed using a Molecular Dynamics PhosphorImager. Each bar represents the mean blot intensity of three identically treated cultures ± SE.
Figure 7
Figure 7
Fluorescent microscopic analyses of the effects of SB 203580 on size and sarcomeric organization in myocardial cells. Myocardial cells were cotransfected with MKK6 (Glu) or an empty vector control (pCEP and pCEP + PE) and CMV–β-galactosidase and then plated on glass slides, as described in the legend for Fig. 3. After maintenance for 48 h in serum-free control medium with or without SB 203580 (20 μM) or DMSO (vehicle control) and with or without PE (10 μM) + propranolol (1 μM), as shown, cultures were fixed in paraformaldehyde and immunostained for β-galactosidase expression (Gal) (A– F), and the same cultures were stained for actin with BODIPY-phalloidin (Phall) (A′–F′). Bar, 50 μm.
Figure 8
Figure 8
Morphometric analyses of the effects of SB 203580 on size and sarcomeric organization in myocardial cells. (A) Photographic images of β-galactosidase–positive myocardial cells transfected and treated as described in the legend to Fig. 3 were digitized, and the areas were determined as described in the legend to Fig. 4 A. Shown are the mean area values for each treatment ± SE (n = 3 cultures). The SB 203580 vehicle, DMSO, was included in all controls and had a slight inhibitory effect itself on the ability of PE and the test constructs to increase cell area. (B) The myofilament structure was evaluated using BODIPY-phalloidin to stain actin, as described in the legend to Fig. 4 B. Shown are the mean area values for each treatment ± SE (n = 3 cultures).
Figure 9
Figure 9
Effects of PE or MKK6 (Glu) on MEF2C-dependent luciferase production in myocardial cells. Myocardial cells were transfected with pG5E1bLuc and either Gal4 (codes for Gal4 DNA–binding domain only), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA–binding domain), or MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4 DNA–binding domain). All cells were also transfected with CMV–β-galactosidase for normalization purposes. (A) Cultures were maintained with or without PE (10 μM) + propranolol (1 μM), as shown, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained in control media for 24 h, and then extracted and assayed for luciferase and β-galactosidase reporter activities, as described in Materials and Methods. Luciferase enzyme units were normalized to β-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/ Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.
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